Renal Transplant Pathology

Renal Transplant Pathology

Volker Nickeleit

Michael Mengel

Robert B. Colvin


Renal transplantation provides a cost-effective therapy worldwide that improves survival and quality of life for patients with end-stage renal disease (1). Over the last 5 years, an average of 16,000 patients per year received renal transplants in the United States, but over 100,000 patients wait for a donor (2). While overall survival is excellent, a substantial fraction of patients experience episodes of graft dysfunction, for which management is based primarily on renal biopsy findings (3). These biopsies provide urgent and perplexing challenges for the pathologist, because there is little time for consultation, several diseases can impinge on the graft simultaneously, and a wide range of potent therapy is possible, whose appropriate selection rests firmly on the accuracy of the diagnosis. We hope that this chapter will provide a practical resource to pathologists and clinicians trying to solve clinical dilemmas and to investigators seeking innovative solutions to prevent graft loss.

Brief Historical Background

The first public demonstration of a successful renal transplant was by Emerich Ullmann, on March 7, 1902, in the lecture hall of the Society of Physicians in Vienna (4). He showed a dog with an autotransplant in the neck that produced visible urine for 5 days from the ureter in the skin; 12 days later, he reported his findings in the medical literature (5). In 1902, Dr. Ullmann also attempted the first kidney transplant (from a pig) to a patient, but this was technically unsuccessful (6). Alexis Carrel, working in Lyons, France, developed the end-to-end vascular suture techniques in 1902 that are widely used in transplantation and, for this and his subsequent work on organ preservation at the Rockefeller Institute in New York, received the Nobel Prize in 1912 (7). In 1906, Mathieu Jaboulay, also from Lyons, used Carrel’s technique to transplant a xenograft kidney (pig or goat) to the limbs of two patients with chronic renal failure; both failed within an hour (8). Three years later, Ernest Unger in Berlin transplanted a monkey kidney to a girl dying of renal failure; no urine was produced, and Unger concluded that the biochemical barrier was insoluble (9). Working in some obscurity, Dr. Yu Yu Voronoy, in 1936 in Kherson, Ukraine, transplanted the first human kidney. The donor died from a head injury, and the recipient had acute renal failure from mercuric chloride poisoning. The kidney was ABO incompatible (B to O), and the kidney never worked, but the vessels were patent at autopsy 2 days later (10).

In 1945, Drs. David Hume, Charles Hufnagel, and Ernest Landsteiner at the Peter Bent Brigham Hospital and Harvard Medical School in Boston transplanted a human cadaver kidney to the axilla of a young woman comatose from acute renal failure due to septicemia (11). The kidney worked for several days and was then removed after the woman regained consciousness; her own kidneys then made a full recovery. In 1951-1953, Dr. Hume continued this approach, transplanting kidneys into the thighs of nine patients without immunosuppression; one graft functioned for 6 months (12). In 1952, N. Oeconomos and J. Hamburger performed the first living kidney transplant. A mother donated a kidney to her son, whose congenital single kidney had to be removed due to a traffic accident injury. The kidney functioned without any immunosuppression for 21 days before developing anuria (13).

Mastery of the surgical aspects encouraged the surgeons to begin transplanting kidneys from identical twins, who do not require immunosuppression. The first such operation was performed on December 23, 1954, by Drs. Hume, Joseph Murray, and Hartwell Harrison (14). The recipient survived 8 years, succumbing to recurrent disease, the major risk in twin recipients.

Broad clinical application awaited the definition of the underlying immunologic events and the means to thwart immunologic rejection. The need for skin graft treatment of war burn wounds motivated the scientific efforts of the young Peter Medawar to ponder “why it was not possible to graft skin from one human being to another, and what could be done about it.” Medawar did indeed do something about it, showing in 1953 that injection of lymphoid cells in neonatal mice sometimes established a lifelong, specific tolerance to subsequent transplanted skin from the same donor, for which he received the Nobel Prize in 1960 (15). The discovery of the immunosuppressive ability of 6-mercaptopurine by Robert Schwartz and William Damesheck in Boston in 1959 (16) was soon applied in humans in 1960. Gertrude Elion and George Hitchings (Nobel Prize 1988) at Burroughs Wellcome discovered azathioprine that Roy Calne in Cambridge proved beneficial and less toxic in dog kidney grafts (17). Joseph
Murray (Nobel Prize 1992) first tried azathioprine in humans and added corticosteroids to the regimen (14). The improved results obtained by combination of azathioprine with corticosteroids ushered in the era of clinical renal transplantation in the early 1960s, through the successful studies of Thomas Starzl (18) and Murray et al. (14). Innovative therapies, such as antithymocyte globulin (ATG or ALG) (1970s), cyclosporine and anti-CD3 monoclonal antibody (OKT3) (1980s), mycophenolate, tacrolimus (1990s), and others, have markedly increased success. Recent clinical trials with protocols to induce mixed or complete chimerism have shown promise of achieving Medawar’s goal of specific tolerance without immunosuppression (19,20,21).

The pathologic literature on renal grafts began with the photomicrographs of canine allograft rejection, published by Carl Williamson of the Mayo Clinic in 1926. He illustrated a “marked lymphocytic infiltration” and “intense glomerulitis” in the dog and attributed graft loss to “atypical glomerular nephritis” (22). He noted that recipients responded differently to autografts and allografts and hoped that “in the future it may be possible to work out a satisfactory way of determining the reaction of the recipient’s blood serum or tissues.” Subsequent work reported in 1953 by William Dempster in London (23) and Morten Simonsen in Denmark (24) showed that canine grafts are infiltrated by pyroninophilic mononuclear cells, which they concluded were donor-derived plasma cells and their precursors, a “response of the renal mesenchyma to the recipients’ individual-specific antibodies and antigens” (25). The infiltrating cells were later shown to be of recipient origin using radiolabeled cells (26). Simonsen illustrated an example of endarteritis in a small artery in a dog, but did not appreciate its distinctiveness, interpreting the lesions, which also had fibrinoid necrosis, as “periarteritis nodosa” (24).

The first renal transplant biopsy in a patient was in 1952, when a living donor kidney developed anuria on day 21. The slides were recently discovered at the Necker Hospital and, upon review, show a combination of T-cell- and antibody-mediated rejection (Fig. 29.1) (27). Appreciation of the general pathology of human renal transplants began in the 1960s, particularly by Gustav Dammin at Harvard and Kendrick Porter at St. Mary’s Hospital in London. Among the early discoveries were the descriptions of endarteritis in acute rejection by Dammin, which he attributed correctly to recipient mononuclear cells (28), chronic transplant arteriopathy (29) and chronic transplant glomerulopathy by Porter (30,31), the relationship of the arteriopathy to anti-human leukocyte antigen (HLA) antibodies by Paul Russell et al. (32), and the pathology of hyperacute rejection and its relationship to humoral antibodies by Kissmeyer-Nielsen et al. (33). The recurrence of glomerulonephritis in transplants was first described in isografts by Richard Glassock, Dammin, and colleagues (34). Perhaps most important to pathologists, Priscilla Kincaid-Smith demonstrated the value of the renal biopsy in clinical management, concluding that “the renal biopsy provides a clear-cut diagnosis of rejection and indicates which patients should receive prompt treatment for rejection” (35). Our goal is to make this always true!

Standard Immunosuppression

Calcineurin inhibitors (CNIs, cyclosporine or tacrolimus) are the mainstay of most standard protocols, usually with corticosteroids (prednisolone or prednisone) and mycophenolate mofetil (MMF) or azathioprine (“triple therapy”). The drugs are tapered in the initial 3 to 6 months to baseline maintenance levels, which in adults are typically approximately 100 ng/mL of cyclosporine and 5 to 15 ng/mL of tacrolimus at the trough level, depending on the other drugs in the regimen and the immunologic risk (36). ATG can substitute for CNIs in
patients with delayed graft function (DGF). For treatment of acute rejection episodes, the usual first defense is a short course (2 to 3 days) of high-dose steroids orally (prednisolone) or intravenously (methylprednisolone), followed if necessary by rescue with ATG. Additional FDA-approved drugs include rapamycin (sirolimus, a blocker of IL-2 signaling and cell proliferation) and monoclonal antibodies to the IL-2 receptor (basiliximab [Simulect]), CD52 (daclizumab, CAMPATH1) and CD20 (rituximab [Rituxan]), and inhibitors of costimulatory signals (belatacept). All of these drugs have the potential for complications related to immunosuppression, and some cause nephrotoxicity, especially CNI.

FIGURE 29.2 Kaplan-Meier plot of kidney graft failure by year of transplantation from 1989 to 2005 for 140,900 standard criteria deceased donor kidneys, censored for death due to other causes (38). Each colored line is a single year cohort from transplant year 1989 on the bottom to 2005 on the top. The half-life is indicated by the horizontal line at 50%. Most of the improved survival during this time period occurred in the first year posttransplant (shown in the insert). The slopes after 1 year are close to parallel, indicating little or no change in the rate of late graft loss. (Reprinted with permission from Lamb KE, Lodhi S, Meier-Kriesche HU. Long-term renal allograft survival in the United States: A critical reappraisal. Am J Transplant 2011;11(3):450-62; copyright 2011, John Wiley and Sons.)

FIGURE 29.3 Graft survival according to the HLA match and type of donor. Survival of HLA-identical sibling grafts (HLA-id) is better than for related grafts with one HLA haplotype match (1-Haplo), demonstrating the importance of complete HLA matching. However, living unrelated grafts (Unrel LD) have better survival than deceased donor grafts (DD), indicating the state of the graft itself is also important. Living unrelated grafts have similar survival as one HLA haplotype-matched living grafts. Figure provided by Michael Cecka (University of California at Los Angeles) based on data from the Renal Transplant Registry of the Organ Procurement and Transplantation Network and United Network of Organ Sharing as of March 2011. N, Number of patients and T½, calculated half-life in years.


Diagnostic Value

Renal biopsies remain the gold standard to determine the cause of graft dysfunction, which occurs in about 30% of recipients early after transplant and at a rate of 2% to 4% per year after the first year (3,52). Biopsies are particularly useful to guide treatment in ambiguous clinical situations and are used in combination with other diagnostic tests, including imaging and laboratory tests. Biopsies best distinguish acute rejection, acute tubular necrosis, infections such as polyomavirus nephropathy (PVN), thrombotic microangiopathy (TMA), recurrence of original disease, CNI toxicity, and chronic rejection (53,54). Biopsy findings change the clinical diagnosis in an average of 36% of patients (27% to 46%) and therapy in 59%, with no obvious diminishing value in the last 20 years (53,54,55,56,57,58,59,60) (Table 29.1). Biopsy results change therapy in both the early and late (greater than 1 year) posttransplant periods with approximately equal frequency (58,59). Most importantly, biopsy findings lead to reduced immunosuppression in 22% (19% to 39%) of patients.

Sensitivity and Specificity

The sensitivity of the biopsy depends on the size, number, and content of the cores. In a study of 130 biopsies with multiple cores, acute rejection was found in only one of two cores in 10% of the cases (61). Thus, the sensitivity of a single core is approximately 90%. Similarly, 10% of 79 paired biopsy cores had one core that was insufficient for the diagnosis of rejection (62). The sensitivity
of “n” biopsy cores can be calculated as 1-(1-sensitivity of a single core)n. Accordingly, if one core has a sensitivity of 90%, two cores have a predicted sensitivity of 99%, substantiating the conventional wisdom that recommends two cores.

The specificity of the biopsy is impossible to measure because no higher standard for comparison is available. The short-term clinical course or response to therapy is not the final arbiter, because rejection may be occult or delayed. One study showed a specificity of 87% compared with a blinded retrospective clinical review (63). The results that show the biopsy results correlate with the clinical course in 80% to 89% of cases are also reassuring (56,61). Molecular testing for gene expression may increase the specificity and sensitivity of the biopsy and is a subject of active investigation.


Most renal biopsies are done with ultrasound-guided biopsy “guns” and 16- to 18-gauge needles (64,65). These have an excellent record of safety in experienced hands. None of the large series in adults reported any deaths due to biopsy (0/5026) and few graft losses (1/3996, 0.03%) (56,64,65,66,67,68). A multicenter audited series of 2127 protocol biopsies reported no patient deaths and one potentially avoidable graft loss (68). Pediatric transplant biopsies have a similar low complication rate: 0/212 biopsies from 19 centers led to death or graft loss, and only one required surgical exploration for bleeding (69). The types of complication are the same as from biopsies of native kidneys, namely, hematuria, ureteral obstruction from clots, hemorrhage, shock, and arteriovenous fistula. Follow-up showed 75% of the fistulas spontaneously closed, and none had an impact on renal function requiring intervention (70).

Ultrasound guidance increases the probability of obtaining cortex from 75% to 91%; guidance by on-site examination with a dissecting microscope increases adequacy to 100% (71). Transfemoral vein renal biopsies offer an alternative method for patients who are deemed unsuitable for percutaneous biopsy. These yield adequate tissue in 51% of the biopsies (10 or more glomeruli) with rare major complications (1/58 causing obstruction from hematuria) (72). A 16-gauge needle appears to be the best compromise between tissue yield and complications. Among 1171 protocol biopsies in adults with an automated 16- or 18-gauge needle, no graft losses occurred (73). The 16-gauge needle had no worse major complication rate (73,74) and a better yield of tissue than the 18-gauge needle (76% vs. 53% yielded greater than 7 glomeruli and ≥1 artery) (73). The 16-gauge needle had a higher hematoma rate than did the 18-gauge needle in children (75).

Diagnostic Approach to Biopsies

Typically, two cores are divided for light and immunofluorescence microscopy, with most of both cores going for light microscopy. Most biopsies taken after 1 year are processed for electron microscopy. Electron microscopy is important if glomerular disease or chronic rejection is suspected. We prepare about 15 sections stained with hematoxylin and eosin (H&E) (three levels) and five for trichrome and five cut at 2 to 4 µm and stained with periodic acid-Schiff reagent (total of five slides); Jones silver stain is an alternative. Elastin stains are recommended for evaluation of intimal fibrosis. Each section is carefully examined for (a) the nature and degree of the interstitial cellular infiltrate (e.g., activated mononuclear cells, edema, intracapillary cells); (b) arterial and arteriolar lesions (e.g., endarteritis, myocyte necrosis, thrombi, nodular hyaline); (c) tubular injury, inflammation (tubulitis), and viral inclusion bodies; and (d) glomerular lesions. Further levels are obtained if no diagnosis is evident. We recommend that all biopsies be assessed for C4d, as well as IgG, IgA, IgM, C3, lambda, kappa, albumin, and fibrin by immunofluorescence microscopy or by immunohistochemistry (IHC) when frozen sections are not available. Frozen sections for light microscopy are of limited value, but can be prepared in urgent situations; the diagnostic accuracy was reported to be 89% compared with permanent sections (76). Rapid (2 to 4 hours) permanent sections are an alternative used at our centers and provide quite satisfactory preparations.


The recommended requirements are at least 10 nonsclerotic glomeruli and 2 arteries (with two or more medial layers) (61,77). However, the adequacy of the biopsy sample depends entirely on the lesions seen. One artery with endarteritis is sufficient for the diagnosis of acute rejection, even if no glomerulus is present; similarly, immunofluorescence or electron microscopy of one glomerulus is adequate to diagnose membranous glomerulonephritis. In contrast, a large portion of cortex with 10 glomeruli and a minimal infiltrate does not exclude rejection. A normal medulla also does not rule out rejection (78), because medulla has a lower sensitivity for rejection than cortex (77%) (79). However, when a prominent mononuclear infiltrate and tubulitis are present in the medulla, rejection is highly likely, provided infection, obstruction, and drug allergy are excluded (79). The minimal adequacy for C4d scoring is for viable cortical or medullary tissue. No glomeruli are required. This means that if tissue is sparse, the portion with medulla can be used for C4d staining. Necrotic and scarred areas are not sufficient, since they are commonly negative, even in samples positive elsewhere.

Diagnostic Classification

Jean Hamburger emphasized that graft rejection could not be attributed merely to different intensities of a single type of immune response (80). This has proved to be true, with many different immunologic mechanisms of injury to each of the cellular targets in the graft. The principal alloreactive initiators of graft rejection are T cells and antibodies, which trigger a variety of secondary mediators (e.g., activation of macrophages, complement). The ideal diagnostic classification should be based on pathogenesis, have therapeutic relevance, and be reproducible. The classification in Table 29.2 is our current attempt to meet these criteria.

Banff Criteria and Scoring System

Several grading systems have been proposed over the years to codify renal allograft rejection. At the present time, the most widely used system is called the “Banff working schema” (“Banff” for short). Banff started as a collaborative effort of investigators meeting in Banff, Canada, with the leadership of Kim Solez, Philip Halloran, and Lorraine Racusen, to achieve a consensus that would be acceptable to the FDA for drug trials and useful for routine diagnostic use (77). This system has gone through a number of significant revisions and modifications over the years since it was published in 1993. The most important of these were the incorporation of the NIH Cooperative Clinical Trials in Transplantation (CCTT) criteria (61) in 1999, which separated the category of endarteritis (81), and
the addition of AMR in 2003 (82). Banff scores the individual elements of the biopsy by light microscopy and uses these to classify rejection (Table 29.3). Combinations of individual scores are then used to define various categories of acute and chronic rejection. While many details are still being refined, Banff has had a beneficial effect in the standardization of definitions for publications and provides a stimulus for consensus development and translational research.

TABLE 29.2 Pathologic classification of renal allograft diseases


Immunologic rejection

  1. T-cell-mediated rejectiona

    1. Acute

      1. Tubulointerstitial (Banff type I and borderline/suspicious)

      2. Endarteritis (Banff type II)

      3. Arterial transmural inflammation or fibrinoid necrosis (Banff type III)

    2. Chronic

      1. Tubulointerstitial inflammation and fibrosis

      2. Transplant arteriopathy

  2. Antibody-mediated rejectiona

    1. Hyperacute

    2. Acuted

      1. Acute tubular injury (Banff type I)

      2. Capillaritis (Banff type II)

      3. Arterial fibrinoid necrosis (Banff type III)

    3. Chronic

      1. Transplant glomerulopathy

      2. Multilamination PTC basement membranes

      3. Transplant arteriopathy

    4. Variants

      1. Smoldering/indolent (mononuclear capillaritis)

      2. C4d deposition without evidence of active rejection

      3. C4d-negative (mostly chronic or smoldering)


Auto/alloantibody-mediated diseases

  1. De novo membranous glomerulonephritis

  2. Anti-GBM disease in Alport recipients

  3. Anti-nephrin disease in recipients with congenital nephrosis (Finnish type)

  4. Anti-TBM disease in TBM antigen-deficient recipients

  5. Anti-angiotensin II type 1 receptor antibody syndrome


Nonalloimmune injury

  1. Acute ischemic injury (ATN)

  2. Drug toxicity

    1. CNI toxicity (cyclosporine, tacrolimus)

    2. mTOR inhibitor toxicity (rapamycin, sirolimus)

    3. Antiviral drug tubular toxicity (foscarnet, adefovir, tenofovir)

    4. Acute allergic tubulointerstitial nephritis

  3. Infection

    1. Viralb

      1. Polyomavirus

      2. Adenovirus

      3. Cytomegalovirus

      4. Herpes simplex

    2. Bacterial/fungalb

      1. Acute/chronic pyelonephritis

      2. Tuberculosis

      3. Malakoplakia

  4. Major vessel disease

    1. Arterial/venous thrombosis

    2. Arterial dissection

    3. Arterial stenosis

    4. Atheromatous emboli

  5. Pelvis/ureter

    1. Urine leak

    2. Obstruction

  6. De novo glomerular diseaseb: focal segmental glomerulosclerosis, diabetic nephropathy

  7. Neoplasia: Post-transplant lymphoproliferative disease

  8. Idiopathic: interstitial fibrosis and tubular atrophy, not otherwise classified (IF/TA, NOS)c


Recurrent primary diseaseb

  1. Immunologic: IgA nephropathy, lupus nephritis, anti-GBM disease

  2. Metabolic: amyloidosis, diabetes, oxalosis

  3. Other: dense deposit disease, focal segmental glomerulosclerosis

a Often, T-cell and AMR concur. Many features, such as transplant glomerulopathy or the multilamination of PTC basement membranes, can be induced by antibodies and/or T cells.

b Partial list.

c Formerly termed chronic allograft nephropathy, or CAN.

d Banff 2013 includes endarteritis and thrombotic microangiopathy as potential manifestations of acute AM or mixed TCMR and AMR.

The criteria for diagnosis of rejection are not absolute, but based on clinicopathologic correlations in patients on standard immunosuppressive therapy. Drugs have the ability to modify rejection, for example, a decrease in the intensity of infiltrate and edema with cyclosporine or a decrease in eosinophils with steroids. Some of the newer drugs may have other effects. For example, CAMPATH1, which profoundly depletes T and B cells, can lead to episodes that clinically behave like rejection, yet do not meet the criteria of Banff, due to sparse mononuclear infiltrates (83). To the extent that our criteria correspond to mechanisms of the rejection process itself (tubulitis, endarteritis, capillaritis), the criteria will be robust. Nonetheless, we have to be prepared to identify novel features and mechanisms of rejection that might occur with new drugs.


Grafts typically had a previous “life,” and they often show some signs of hypertension-induced so-called arterionephrosclerosis and, occasionally, even other renal diseases. Both procurement and zero-hour implantation biopsies designate tissue samples obtained at the time of grafting to assess donor disease.
Procurement biopsies are primarily collected to give information on the organ suitability for transplantation, and implantation biopsies provide insight into preexisting diseases relevant for comparative analyses posttransplantation; thus, the diagnostic implications of both biopsy types are similar. Since procurement biopsies are often evaluated on a rush basis by general surgical pathologists during off-hours in the frozen section laboratory and recorded under the donor’s name, the analysis is commonly rudimentary, and the results are often unavailable for subsequent graft management under the “new” recipient’s name. In some cases, both procurement and zero-hour implantation biopsies are collected. In general, guidelines for the interpretation and recording of donor biopsies have not been definitively established. Often, Banff scoring criteria are used, which can, unfortunately, easily lead to subsequent confusion during the evaluation of diagnostic graft biopsies since the scoring results of “old preexisting donor lesions” versus “new de novo posttransplant changes” are not distinguished from each other using ci, ct, and cv scores.

TABLE 29.3 Banff scores of individual features


Banff term

Banff Score





Interstitial inflammation (% of nonfibrotic cortex)a






Total inflammation (% all cortex)






Tubulitis (maximum mononuclear cells/tubule)b






Arterial inflammation (% lumen endarteritis)c





Transmural or necrosis

Glomerulitis (% glomeruli involved)d






Capillaritis (cells per cortical PTCe; requires >10% of PTC to be affected for scoring)






C4d deposition in PTC (% positive)f






Interstitial fibrosis (% of cortex)






Tubular atrophy (% cortex)






Arterial intimal thickening (% narrowing lumen of most severely affected artery)g






Transplant glomerulopathy (% of capillaries with duplication in most severely affected glomerulus)h






Arteriolar hyalinosis (number with focal or circumferential hyaline)



1 focal

>1 focal

1 circumferential

Mesangial matrix increase (% affected glomeruli)i






a Excludes perivascular, nodular, and subcapsular infiltrates.

b Excludes atrophic tubules; for longitudinal sections, count per 10 epithelial nuclei.

c Mononuclear cells in intima (v1,2) or media (v3).

d Threshold for number of cells/glomerulus not defined.

e PTC, peritubular capillary; note whether cells are only mononuclear cells or include neutrophils.

f Note whether frozen or fixed tissue used. Requires at least 5 high-power fields of sample.

g Note if lesions are characteristic of chronic rejection or fibroelastosis.

h cg1 with duplication in >1 glomerular capillary loop in one glomerulus; cg1a changes seen by EM only, cg1b changes seen by light microscopy (according to 2013 updates to the Banff scoring scheme) (481).

i Increase defined as >2 mesangial cells in width in at least 2 glomerular lobules.

Procurement/Harvest Biopsies

The pathologist may be asked to advise whether a particular kidney from a deceased donor is suitable for transplantation, sometimes in the middle of the night. The most common questions are (a) the degree of scarring in the “marginal” donor, (b) the presence of active renal disease, and (c) the clinical significance of incidentally discovered neoplasms.

The question of donor vascular disease (hypertensive and age-related arterionephrosclerosis) arises more often now due to increasing use of older donors (84). The utilization of procurement biopsies increases with donor age: 5% at age 20, to 20% at age 45, 40% at age 55, and 60% at age 65 (85). Most studies show a correlation of glomerulosclerosis (86), interstitial fibrosis (87), and intimal fibrosis (88,89) with donor age. Up to 40 years of age, 54% of deceased donor biopsies are normal, while only 7% of donor kidneys 40 years or older are normal (90). However, even septuagenarians can have kidneys with relatively minor glomerulosclerosis that varies from 1.5 to 23% (91). Among a group of “marginal” donors aged 60 to 75 years, 57% had less than 10% glomerulosclerosis (89). Thus, age alone is only an imperfect predictor of the overall degree of nephrosclerosis and the suitability of an organ for transplantation.

Most examinations of procurement biopsies are limited to frozen sections of subcapsular wedges, which have numerous pitfalls in the interpretation. Conventional frozen sections (with the exception of biopsies carefully frozen in precooled isopentane) have prominent artificial interstitial spaces, which can be
mistaken for fibrosis or edema. Glomerular cellularity cannot be reliably assessed, although thrombi and crescents can be identified. The minimum number of glomeruli needed to correlate with outcome was found to be 25, and the minimum number required to obtain consistent results from paired biopsies was 15 (92). In our opinion, at least 25 glomeruli should be studied, from as deep in the cortex as is feasible. If a scar is sampled, as indicated by clusters of globally sclerotic glomeruli, this area should be noted, but it should be treated separately in the analysis to avoid overstating the percent sclerosis. Most importantly, a wedge biopsy is not representative, since it includes mostly outer cortex, the zone where glomerulosclerosis and fibrosis due to vascular disease is most severe. Intimal fibrosis, in contrast, most prominently affects arcuate and larger-caliber arteries and therefore is underrepresented in a wedge biopsy (93,94,95). If needle core biopsy samples are obtained on isolated/procured kidneys by the surgeons in the operating room with a so-called biopsy gun, then the tissue cores may show predominately renal medulla thereby limiting the diagnostic yield considerably. This problem can be avoided by “shooting” tangentially rather than perpendicularly into the “naked procured” organ. Good results were also reported using skin punch biopsy tools (94). In general, procurement biopsies are examined with H&E stains only; serial step sections and special stains (including trichrome incubation for the evaluation of sclerosis) are usually not performed, thereby further limiting the diagnostic yield.

Thus far, no study has established an absolute, validated threshold of glomerular sclerosis, fibrosis, or arteriosclerosis beyond which a donor kidney must not be used. It is clear that some abnormalities do not measurably affect long-term prognosis, in part due to other more severe causes of graft loss (e.g., rejection, cardiovascular disease, infection). Glomerulosclerosis as an indicator for organ suitability has been evaluated in many studies with contradictory results. A seminal report showed that allografts with good function at 6 months had less global glomerulosclerosis in the donor biopsy than did those with poor function (2% vs. 20%) (86). A threshold of less than 20% glomerulosclerosis characterized the group with a lower rate of DGF (33% vs. 87%) and graft loss (7% vs. 38%), and as a result, the proposed 20% cutoff has had some subsequent support. Graft survival was strikingly diminished in recipients of grafts with greater than 20% glomerulosclerosis, compared with those having 0% (35% vs. 80% 5-year graft survival rate) (96). However, a large study of 387 donor biopsies found that donor glomerulosclerosis was not an independent predictor of outcome if age was included in a multivariate analysis (97). In a large, well-analyzed UNOS study of 3444 deceased donor kidney biopsies, glomerulosclerosis greater than 20% predicted decreased graft survival only when associated with decreased creatinine clearance in the donor and, even then, only to a minor degree (3.4% more graft loss) (98). According to this result, glomerulosclerosis should not be the sole criterion for discarding donor kidneys. A recent well-conducted single-center series from Baltimore analyzed 371 donor biopsies, mainly collected from an “expanded donor organ pool” with relatively long ischemia times, kidneys that had been declined by other transplant centers. In this cohort of mostly “marginal donor organs,” five histologic features (global glomerulosclerosis, periglomerular fibrosis, arteriosclerosis, arteriolosclerosis, and scar formation) were weighted and incorporated into a cumulative chronic histologic scoring index. Overall, graft survival was 90% at 1 year, and at 5 years, it ranged from 53% in organs with high cumulative indices up to 90% in those with low indices (99). Thus, in this study, more than 50% of organs with relatively marked sclerosis and chronic injury functioned 5 years postgrafting and kept patients off dialysis. Data from Baltimore underscore that even marginal donor organs can be beneficial for some recipients, particularly in “old for old” or dual organ transplantation programs (100,101,102,103). Another study found a high donor-recipient age ratio, that is, old donor organ into young recipient, to be associated with increased risk for subsequent allograft failure; the age of the donor organ (greater than 55 years with presumed higher degrees of chronic injury) was, however, not in and of itself an independent risk factor for poor long-term prognosis (104). Whether the recently proposed baseline “Leuven sum score” (donor age and glomerulosclerosis, interstitial fibrosis, tubular atrophy evaluated at time of procurement/transplantation), indeed, helps to better predict 5-year graft survival and allocation of donor organs remains to be seen (105,106,107). Thus chronic renal injury including the percentage of globally sclerosed glomeruli does not provide universal guidelines for the suitability of donor kidneys for transplantation.

At present, 19% of all kidneys recovered in the United States are discarded. The Organ Procurement and Transplantation Network reports that in 2011 in the United States, while there were 940,000 patients waitlisted for a kidney or kidney/pancreas transplant, only 17,600 transplants were performed. According to the National Kidney Foundation, the largest (and growing) reason for discard of donor organs is an abnormal biopsy finding that led to 42.8% of kidney discards during the period 2005-2009, up from 37.2% during 1995 to 1999. Compared to dialysis costs, each transplanted kidney saves tens of thousands of health care dollars over costs for dialysis and typically improves the recipient’s quality of life. There is great concern that rigid and arbitrarily set morphologic criteria for the evaluation of procurement biopsies result in needless discard of kidneys (100,108). In the future, great efforts will have to be made by transplant pathologists to develop optimal criteria for the evaluation of procurement biopsies and to use precious donor kidneys wisely (106,108).

With regard to tumors, among the pitfalls in the frozen section interpretation of a possible clear cell carcinoma are epithelioid angiolipoma, intrarenal adrenals (109), and cystic renal cell carcinoma with scant epithelial lining. The detection of a renal cell carcinoma is not necessarily an absolute contraindication against transplantation (110). Completely resected, well-differentiated (Fuhrman grade 1 to 2) renal cell carcinomas less than 1 cm in diameter have an estimated minimal risk of less than 0.1% for tumor transmission, tumors greater than 1 and ≤2.5 cm have a low risk of 0.1% to 1%, and those between 4 and 7 cm have an intermediate risk of 1% to10% (110). In a recent review, Nalesnik et al. identified 64 organ donors with confirmed renal cell carcinomas that resulted overall in 7 (11%) tumor transmissions in the organ recipients and 1 (1.6%) carcinoma-related recipient death (111). Organs from patients with metastatic disease (including invasive breast, neuroendocrine, and colon carcinomas; malignant melanoma; leukemia; sarcomas; and lung cancer [stages I to IV]) carry a high risk (greater than 10%) of disease transmission, and they should not be transplanted (110).

In some procurement biopsies, thrombi may be encountered. While massive generalized thrombosis of intrarenal
vessels constitutes a contraindication to renal transplantation, organs with focal fibrin thrombi limited to the microvasculature can have an excellent prognosis, although these grafts may experience an initial period of DGF. Glomerular thrombi are found in 3% to 7% of donor biopsies, particularly in those originating from patients with head trauma (97,112,113). Thrombi in less than 50% of glomeruli after reperfusion had no effect on graft outcome in one series (114), but in another series of nine donor kidneys with glomerular thrombi (of unspecified extent), three had primary nonfunction (PNF) (97). Thrombi disappeared in five recipients with subsequent biopsies as soon as 8 days postgrafting, and long-term outcome was unaffected even in cases with “severe” microthrombosis (113). It is likely that unaltered fibrinolysis in the recipient results in full restoration of blood flow postgrafting. TMAs, in contrast, affecting small arteries with intramural vascular injury, extravasation of fragmented red blood cells, intimal remodeling, and swelling are poor prognostic indicators and should preclude transplantation. Anecdotal reports and personal experience suggest that eclamptic kidneys can fully recover (112). It is not established whether an occasional cholesterol embolus is a contraindication. However, donor-derived atheroembolization is often multifocal and associated with a high graft failure rate (115,116).

Zero-Hour Implantation Biopsies

Zero-hour implantation biopsies are performed either prereperfusion or, more frequently, postreperfusion. In general, the same aspects concerning tissue sampling (wedge biopsy vs. needle core biopsy) apply as discussed above for “procurement biopsies.” Often, needle biopsies are obtained by the transplant surgeons with only minor complications (0.5% of biopsies resulted in minor bleeding episodes at the University of North Carolina). Implantation biopsies are best evaluated following standard protocols including the analysis of multiple step sections, special stains, and elastic tissue stains. In general, immunofluorescence and electron microscopic evaluations are least helpful.

Implantation biopsies are primarily intended to provide information on the overall condition of the transplanted organ for subsequent comparative biopsy analyses. They may also provide some prognostic information, although prediction of long-term graft function in an implantation biopsy is somewhat limited since many factors encountered down the road (such as rejection, hypertension-induced de novo arterionephrosclerosis, or recurrence of renal disease) may influence outcome (105,106,107,108). The presence of acute tubular injury in an implantation biopsy—if not extreme—only poorly predicts subsequent episodes of DGF.

Chronic, hypertension-induced donor-derived arterionephrosclerosis is common. It mainly affects arterioles, arteries, and, to a much lesser degree, the tubulointerstitial and glomerular compartments. We found evidence of arterionephrosclerosis in approximately 68% of our conventional donor pool organs; in 19% of the cases, there were moderate to severe changes at a mean donor age of 37 years. Unexpected moderate to severe arteriosclerosis was also found in 19% of organs of living donation, which is suggestive of clinically undiagnosed episodes of hypertension in the donor. Moderate to severe arteriosclerosis was correlated in our analyses with inferior graft function during the first 12 months posttransplantation; however, it was not associated with an increased risk of graft failure at 1 year. Similar findings were also made by others (117).

Several studies have attempted to correlate the presence of neutrophils in a postperfusion biopsy with outcome. Older reports found that neutrophils in both glomerular and peritubular capillaries (PTC) predicted hyperacute rejection: Neutrophils in PTC predicted acute rejection, and neutrophils in glomeruli correlated with cold ischemia time and subsequent graft loss (114). Experience in recent years seems to be different. In the experience of one of the authors (VN) in a cohort of conventional transplant recipients polyomorphonuclear leukocytes in the microvasculature (mainly in glomerular capillaries) are uncommon; if present, usually, only few polymorphonuclear leukocytes are detected that do not carry any diagnostic or prognostic significance. Also, rare platelet microthrombi are occasionally seen (finely granular and relatively pale staining material); they typically resolve rapidly during follow-up. In an implantation postperfusion biopsy, the presence of the complement degradation product C4d along PTC is uncommon, but when observed predicts subsequent antibody-mediated acute rejection in some presensitized ABO-compatible transplant recipients (118). The presence of intratubular casts or tubular degeneration correlates with increased ischemic time but not with DGF (88).

The zero-hour implantation biopsy also provides a unique view of other subclinical renal lesions in healthy individuals. These lesions might require more extensive morphologic and clinical workup. The most common glomerular finding is IgA deposition, which was present in 11% of 108 living donors (two had mesangial sclerosis, and the others were normal by light microscopy) (119) and in 9% of deceased donors (120). In a large series from Nanking, 24% of 342 donor kidneys had IgA deposits (121). This suggests either that subclinical IgAN is quite common or that mesangial IgA without proliferation is not a disease. Donor-derived IgA disappears within weeks to few months on follow-up biopsies (112,122,123,124), and graft survival is no different from those without IgA (121).

Rarely, kidneys with membranous glomerulonephritis have been transplanted; one survived at least 3 years (125), and another case showed signs of resolution and GBM remodeling at 20 months posttransplant (126). Other donor renal diseases that resolved without obvious ill effects include lupus nephritis (127), acute postinfectious glomerulonephritis (128), membranoproliferative glomerulonephritis (type I) (129), and hepatorenal syndrome (130). In one anecdotal report, a graft with 25% crescent formation showed good function during a 2-year follow-up, whereas the contralateral kidney with 90% crescents failed (131).

In an unpublished but instructive case from the University of North Carolina, very small, scattered, dense mononuclear inflammatory cell aggregates were noted in the interstitial compartment of a zero-hour implantation biopsy. These infiltrates proved to represent extranodal involvement of a small lymphocytic lymphoma and resulted in subsequent graft removal.

Molecular Studies

Organ donation (donor events, procurement, storage, reperfusion) induces a significant molecular disturbance in the donor tissue, which is more intense in deceased donors (132,133). Over recent years, attempts have been made to assess gene expression profiles in donor organs and to develop possible adjunct strategies to determine organ quality and predict outcome postgrafting.

At the transcriptional level, the response to renal injury manifests as a decreased expression of functional genes (e.g., solute carrier transcripts), up-regulation of cell cycle, repair and tissue remodeling transcripts, embryonic pathways like wnt and notch, and injury-associated genes (e.g., cytoprotective heat shock proteins, HMGB1) (134,135).

Donor kidneys with impaired renal function (GFR < 45 mL) at 1-year postgrafting showed a distinctively different gene expression profile when compared to a set of well-functioning organs (GFR > 45 mL) including the up-regulation of genes in the functional classes of immunity, signal transduction, and oxidative stress (136). Interestingly, microarray analysis in a small series of implantation biopsies also found altered gene expression in those donor organs procured by laparoscopic nephrectomy including pneumoperitoneum as compared to open nephrectomy, indicating that certain systemic, surgically induced stress factors might already alter the molecular profile in a donor organ (137). Larger series are needed to further validate this finding. In another study, microarray results of 87 consecutive time-zero biopsies taken postreperfusion in 42 deceased and 45 living donor kidneys were compared to clinical and histopathology-based scores. Unsupervised analysis separated the kidneys into three groups at risk for DGF: living donors, low-risk deceased donors, and high-risk deceased donors. Neither clinical nor histopathologic risk scores discriminated high-risk from low-risk deceased donors. In contrast, 1051 transcripts were differentially expressed in the two deceased donor risk groups; however, no transcript clearly separated grafts with delayed function from those with immediate function. Further analysis revealed a continuum starting from living donors, moving to low-risk deceased donors, and ending with high-risk deceased donors, that is, from best to poorest functioning kidneys (132). In a consecutive study, the same group analyzed microarray results from implantation biopsies of deceased donors only. Two definitions of early dysfunction were used: serum creatinine greater than 265 µmol/L at day 7 posttransplantation or need for dialysis in the first week. The strongest correlate with early dysfunction was the mean expression of a set of 30 injury-associated gene transcripts primarily expressed by tubular epithelial cells such as osteopontin M receptor, integrin beta 6, lipocalin 2, versican, cathepsin S, and cadherin 6 (135,138). Prediction of early dysfunction was best when the injury gene expression profile was combined with donor or recipient age, thereby presumably taking chronic injury such as donor-derived arteriosclerosis into account. Of note, the injury transcripts did not predict late graft function.

FIGURE 29.4 Schema of acute/active rejection. Acute rejection episodes can either be pure antibody- or pure cell-mediated events or also represent mixed rejection with varying degrees of antibody and cellular components.


Traditionally, rejection has been divided on clinical grounds and the temporal occurrence postgrafting into hyperacute, acute, and chronic episodes. In this context, hyperacute and acute rejection is considered an early event characterized by relatively rapid deterioration of graft function, whereas chronic rejection is a late event months or years after transplantation often associated with creeping functional deteriorating and proteinuria. Over the years, this simple point of view has caused much confusion. Many physicians interpreted acute/active rejection and chronic rejection as separate entities without much overlap. Chronic rejection was often viewed as “end-stage transplant disease” with marked scarring unresponsive to therapy. Nowadays, rejection episodes with acute/active and concurrent chronic features are defined as chronic active rejection category 2 or 4 in the “Banff” scheme (139). Of note, inactive/burnt-out chronic rejection remains unclassified in the Banff scheme (139).

In the following sections, we will separate rejection-induced changes for didactic purposes into acute/active and chronic, “cell” (= T-cell-mediated rejection, TCMR) and “antibody” mediated rejection (AMR). The reader should, however, be aware that these categories show vast overlap (Fig. 29.4). For example, active/acute rejection can show varying degrees of concurrent minor or major chronic rejection that can be induced by cellular- and/or antibody-mediated injury (140,141,142). Since grafts “had a preceding life,” all rejection episodes can be superimposed on preexisting donor disease, such as hypertension-induced arterionephrosclerosis, glomerulonephritides, etc. (108). Thus, the picture can be complex, and the analysis of renal allograft biopsies requires a high level of expertise; a true challenge for the transplant pathologist! In the future, our understanding will further evolve, and new immunohistochemical marker sets, electron microscopy, and molecular assays including gene expression profiles will be incorporated into the standard diagnostic interpretation of an allograft biopsy.


Acute T-cell-mediated rejection (i.e., acute TCMR, Banff category 4 types 1 to 3) is the form of rejection that develops most commonly in the first few months after transplantation with sharply decreasing frequencies thereafter. Acute TCMR can occur as early as 6 days and as late as decades posttransplantation (143,144). It can involve the tubulointerstitial compartment, arteries, and glomeruli individually or in various combinations thereby reflecting differences in clinical presentation and outcome. The classical clinical features of severe acute TCMR are an abrupt rise in serum creatinine that progresses over several days, a decline of urine output, weight gain, fever, malaise, graft tenderness, and swelling (these symptoms are often muted or lacking in compliant patients under modern immunosuppression). Hematuria (mainly due to hemorrhage into injured tubules or infarction) and proteinuria (possibly induced by severe transplant glomerulitis and diffuse acute tubular injury) are very unusual presentations in acute TCMR. Of note, although typical, allograft dysfunction is by no means a sine qua non for the diagnosis of acute TCMR that is defined by histologic changes in the allograft. Rejection episodes detected in allografts with stable function are referred to as “subclinical”—a term not meant to imply a benign long-term prognosis if left untreated (145). The primary risk factors for acute rejection, T-cell and/or antibody mediated, are the degree of histocompatibility between the donor and the recipient, the level of presensitization (previous graft, pregnancy, blood transfusions), immunosuppressive drug protocols, and, last not least, the level of patient compliance with daily therapy (85,146). Other factors of importance are recipient age, race, and sex. Current immunosuppression regimens with CNIs, steroids, and MMF have considerably reduced the frequency of acute cellular rejection (147). For the transplant period 2005-2009, only 11.6% of patients with cadaveric and 10% of patients with living donor kidneys experienced acute rejection (cell or antibody mediated) by 1 year postgrafting (148). In the so-called DeKaf study, 34% of early diagnostic biopsies (mean sampling 12 months postgrafting) showed acute TCMR and 19% of late biopsies (149).

Pathologic Findings

T cells, some of them reactive to donor histocompatibility antigens in the graft, affect the interstitium, tubules, vessels, and glomeruli, individually or in various combinations. The approximate relative frequencies of the different patterns of acute cellular rejection are 45% to 70% tubulointerstitial (Banff category 4, type 1), 30% to 55% vascular (Banff category 4, type 2 or 3), and 2% to 4% glomerular (not specifically used for categorization of rejection in the Banff scheme). Percentages show considerable center variations. Approximately 20% to 40% of acute TCMR episodes, dependent on the histologic type, show C4d positivity along PTC, that is, evidence of concurrent antibody-mediated injury (Fig. 29.4) (142,150,151,152). Mixed AMR and acute TCMR episodes are more severe (152) and constitute an independent risk factor for graft failure (140,153). Mixed acute cellular- and AMR episodes are not well categorized in the currently employed Banff scheme of allograft rejection (142).

Gross Pathology

Gross specimens from kidneys with severe acute rejection (mainly mixed TCMR and AMR) are swollen due to interstitial edema and hemorrhage (Fig. 29.5). The failed allografts can increase up to threefold in weight (154) and sometimes rupture. In cross section, the cortex is of normal thickness or expanded with a geographic, mottled appearance showing areas of normal parenchyma, yellowish pale zones of ischemic infarction, and adjacent hemorrhage/hemorrhagic infarction. In most severe cases, early after transplantation, interlobar arteries may contain thrombi (then usually associated with AMR). If veins are affected by thrombus formation at the anastomotic site, then the differential diagnosis includes other underlying conditions such as coagulopathies. Extrarenal graft tissue including the ureter and perirenal adipose tissue is usually also affected and can show necrosis, hemorrhage, and inflammation including extrarenal/extraparenchymal transplant endarteritis.

The so-called pale rejection of the early days of transplantation due to pure tubulointerstitial cellular rejection (Banff category 4, type 1) does not occur anymore with modern immunosuppression.

Light Microscopy

The usual major finding in acute TCMR is infiltration of activated T lymphocytes, macrophages, and also to a lesser degree B cells, plasma cells, polymorphonuclear and eosinophilic
leukocytes into a mildly edematous interstitium and into the tubules (so-called tubulointerstitial cellular rejection; Banff category 4, type I rejection). In some cases, plasma cells dominate, that is, the so-called plasma cell-rich cellular rejection. The infiltrate can in more severe TCMR episodes also (or occasionally solely) affect arteries (so-called transplant endarteritis; Banff category 4, type II rejection) and glomeruli (so-called transplant glomerulitis; not specifically categorized in the Banff classification scheme). Inflammation is accompanied by signs of injury of the target cells, such as endothelial swelling, tubular cell activation, or apoptosis. Acute cellular rejection is often patchy and typically affects the cortex. Rejection-induced inflammation in the medulla is only seen in pronounced cases as a “spillover effect.” In general, medullary inflammation might have many etiologies such as PVN, pyelonephritis, allergic interstitial nephritis, etc., and it is least specific for rejection-induced injury. Diffuse and pronounced interstitial edema or hemorrhage is only seen in most severe forms of acute TCMR involving arteries and glomeruli. Such cases often also show concurrent AMR and C4d positivity.

FIGURE 29.5 This transplant failed within few weeks postgrafting due to severe rejection with fibrinoid arterial wall necrosis (v3 lesion) and presumed mixed acute antibody- and cell-mediated rejection. Segments of hemorrhagic infarction in the cortex and medulla border on zones with normal-appearing renal parenchyma. Large arterial cross sections show some degree of arteriosclerosis but no gross evidence of thrombus formation.

TABLE 29.4 Banff types of acute T-cell-mediated rejection


Any tubulitis + infiltrate of 10%-25%, or

Any infiltrate of ≥10% + tubulitis of 1-4 cells/tubule

Type Ib

Tubulitis >4/tubule + infiltrate >25%

A: with 5-10 cells/tubule (t2), or

B: with >10 per tubule (t3)

Type II

Mononuclear cells under arterial endothelium

A: <25% luminal area or

B: ≥25% luminal area

Type III

Transmural arterial inflammation or fibrinoid arterial necrosis with accompanying tubulointerstitial lymphocytic inflammation

a All cases should be analyzed for C4d deposition. If C4d is present, an additional diagnosis of concurrent antibody-mediated rejection is made.

b Cases with types I-III rejection can be due to concurrent alloantibody-induced injury. To use as a category of TCMR requires C4d in PTC to be negative.

From Racusen LC, Colvin RB, Solez K, et al. Antibody-mediated rejection criteria – an addition to the Banff 97 classification of renal allograft rejection. Am J Transplant 2003;3(6):708-14.

FIGURE 29.6 Transplant glomerulitis. A: Acute cellular rejection with transplant glomerulitis 6 days posttransplantation. Peripheral glomerular capillaries are dilated and “occluded” by mononuclear cell elements and swollen endothelial cells (arrows). The biopsy also showed transplant endarteritis and tubulointerstitial cellular rejection. This case of acute TCMR was not associated with acute AMR. The acute TCMR episode responded fully to thymoglobulin therapy. B: In comparison, a case of acute AMR, C4d-positive with transplant glomerulitis. This case shows mainly intracapillary polymorphonuclear leukocytes and fibrin microthrombi (compare to figure A); this case did not show concurrent acute CR. (PAS, ×500.)

It is important to remember that minimal threshold levels for the diagnosis of C4d-negative tubulointerstitial cellular rejection (Banff category 4, type 1 rejection) are controversially debated (61,141,155,156). The Banff criteria for tubulointerstitial cellular rejection seem high (Table 29.4). The Banff category 3 of the so-called borderline changes suspicious for TCMR is problematic; it is occasionally used as a “waste basket” that may even contain unrecognized cases of severe rejection episodes including missed transplant endarteritis/type 2 rejection.


In a minority of cases with acute TCMR, approximately 5% to 10%, a distinct form of glomerular inflammation is seen, called transplant glomerulitis. It typically occurs in the first 1 to 4 months posttransplant (157,158,159,160,161) although it may as well arise years after grafting and is then often associated with signs of chronic rejection and transplant glomerulopathy (162,163). Transplant glomerulitis is often focal and segmental and less frequently diffuse and global. It is characterized by dilatation of glomerular capillaries “occluded” by three or more mononuclear cell elements, that is, monocytes, lymphocytes, and activated and swollen endothelial cells (Fig. 29.6A) (141,161). Rarely, mitotic figures may be detected and, in severe forms, also
mesangiolysis. Intracapillary polymorphonuclear leukocytes and small fibrin thrombi can occasionally be seen; these latter features are more prominent in C4d-positive cases with (concurrent) AMR (Fig. 29.6B). Fibrinoid tuft necrosis, crescent formation, or GBM duplications are not features of rejection-induced glomerulitis. Glomerular lesions are best appreciated in 2-µm PAS-stained sections or silver stains with a good nuclear counterstain (GBM duplication characterizes transplant glomerulopathy and chronic rejection that can be associated with transplant glomerulitis in chronic active rejection episodes). Transplant glomerulitis with endothelial cells as a target of the cellular immune response occurs in approximately 60% of cases combined with other acute rejection-induced vascular lesions, that is, transplant endarteritis (53,157,158,159,163,164,165,166). In one series of 12 patients, 92% had associated endarteritis (165). In exceptionally rare cases, glomeruli can be solely affected without rejection-induced changes in other renal compartments (157,161,163). We found that approximately 20% of biopsies with transplant glomerulitis are “pure” Banff category 4 cellular rejection episodes lacking concurrent acute AMR/C4d positivity that is detected in the remaining cases. The predominance of intraglomerular monocytes (glomerular monocyte/T-cell ratio greater than 1) is more typical of acute AMR-induced transplant glomerulitis (167,168). In nonhuman primates protracted transplant glomerulitis evolved into chronic rejection/transplant glomerulopathy within 6 weeks (164).

Scattered circulating intraglomerular mononuclear cells in nondilated capillary loops are present in relatively many biopsies with acute TCMR (169) (Fig. 29.7) (157,163,170,171). These circulating mononuclear cells are not considered diagnostic for transplant glomerulitis. Currently, attempts are under way to better define morphologic features characterizing transplant glomerulitis and the potential significance of intraglomerular monocytes (172,173,174).


In acute TCMR, T cells and macrophages invade tubules and insinuate between tubular epithelial cells inside the basement membrane, a process termed “tubulitis” (Fig. 29.8). This is best demonstrated in PAS- or trichrome-stained slides high-lighting the TBM. Tubulitis is usually recognized by increased numbers of small dark nuclei often arranged along the inner aspect of the tubular basement membrane and occasionally surrounded by small clear spaces/halos (Fig. 29.9). Normal tubular epithelial cells have larger and less dense nuclei than do lymphocytes and tend to be located more apical toward the tubular lumens. However, it can sometimes be difficult to distinguish infiltrating mononuclear cells from apoptotic or degenerating tubular epithelial cells; then, “halos” surrounding nuclei of lymphocytes can help in identifying tubular cross sections with tubulitis. Also, CD3 and CD68 stains may be combined with a PAS incubation to help demonstrate tubulitis. Mononuclear cell elements constituting tubulitis can undergo proliferation based on the expression of Ki-67/MIB-1. Tubulitis affects mostly distal tubular segments in the
cortex; proximal tubules are often spared and collecting ducts in the medulla hardly ever involved (175,176,177). Although plasma cells found in the so-called plasma cell-rich cellular rejection can be abundant in the interstitium (subtype of Banff category 4, type I rejection), they rarely invade tubules, and “plasma cell” tubulitis is uncommon. Occasionally scattered neutrophils can be seen in tubular lumens as a sign of acute injury. Small interstitial granulomas form adjacent to ruptured tubules and leakage of Tamm-Horsfall protein (uromodulin) into the interstitium (Fig. 29.10); this feature is nondiagnostic and can occur in many forms of tubular injury. Tubulitis in atrophic tubules, that is, less than 50% of the original diameter and markedly thickened TBM, is currently considered to be a nondiagnostic sign of parenchymal scarring; at present, this feature is not used to establish a diagnosis of acute TCMR (see Fig. 29.9C). However, this view may change in the future since there is increasing evidence that all tubulitis (in atrophic and nonatrophic tubules) and all interstitial inflammation (in scarred and nonscarred regions) is a sign of TCMR (see Fig. 29.9D) (149,178,179). The newly introduced Banff scoring entity of “total inflammatory (ti) score” may help assess the significance of tubulointerstitial inflammatory cell infiltrates (139). In some cases of acute TCMR, marked reactive atypia of tubular epithelial cells including multinucleation is seen. Such regenerative, ischemia-induced changes often occur in the setting of protracted severe rejection with marked edema, transplant endarteritis, and/or concurrent acute AMR/C4d positivity. Very pronounced tubular epithelial atypia is usually inconsistent with a diagnosis of “pure” tubulointerstitial cellular rejection, Banff category 4 type 1.

FIGURE 29.7 Several mononuclear cells are present in glomerular capillaries. This is below the threshold of transplant glomerulitis. (PAS stain, 400× original magnification.)

FIGURE 29.8 Acute tubulointerstitial cellular rejection with tubulitis. A: Activated mononuclear cells and edema are present in the interstitium. A mitotic figure is present in the infiltrate (arrow). (×40 PAS) B: A CD3 stain in another case shows abundant T cells in the interstitial and intratubular compartments/foci of tubulitis (arrows). (PAS stain, 40× original magnification. Immunoperoxidase ×200.)

FIGURE 29.9 Diagnostic and nondiagnostic tubulitis. A: Diagnostic tubulitis: Over 10 mononuclear cells are in one tubular cross section (arrow). Surrounding tubules have zero to two infiltrating cells. (H&E ×400.) B: Diagnostic tubulitis: extreme tubulitis (t3) in a longitudinal section. (PAS ×400.) C: Nondiagnostic tubulitis: Inflammation in severely atrophic tubules is currently considered to be a nondiagnostic observation. Mononuclear cells (arrows) are in small tubules with thickened basement membranes. (PAS ×400.) The presence of occasional mitotic figures in “atrophic” tubules belies their designation of atrophy. (Insert, original magnification ×600.) D: Likely diagnostic tubulitis: Tubulitis is present in these smaller tubules with simplified, clear cytoplasm. However, the tubular diameters do not seem to be markedly reduced, and the TBM is not severely thickened. This focus of tubulitis possibly represents active rejection. (H&E ×400.)


Acute TCMR has a pleomorphic interstitial infiltrate of mononuclear cells (lymphocytes, macrophages), varying degrees of CD20-expressing B cells, plasma cells, and, occasionally, scattered polymorphonuclear leukocytes in areas of severe tubular injury (180). Inflammation typically surrounds nonatrophic tubules in a finger-like fashion. Eosinophils can be focally
prominent and may indicate the presence of transplant endarteritis (Fig. 29.11). Inflammation in the setting of tubulointerstitial rejection (Banff category 4, type 1) is associated with relatively mild and often focal edema; marked diffuse edema usually indicates endothelial injury seen in cases with transplant endarteritis (Banff category 4, type II rejection) and/or concurrent acute AMR with C4d positivity. In acute TCMR, edematous regions often show dilatation of PTC and so-called PT capillaritis with intracapillary mononuclear cell elements (181). Polymorphonuclear PT capillaritis is rare and usually indicates acute AMR.

FIGURE 29.10 Severe tubulitis. A: Acute cellular rejection with tubular rupture and granuloma formation. (H&E ×400.) B: Partial dissolution and rupture of the tubular basement membrane can be appreciated in periodic acid-silver stains (arrows). (×400.)

The infiltrating mononuclear cells are predominately T cells and macrophages (see Fig. 29.8). The T cells are typically activated (lymphoblasts), with increased basophilic cytoplasm, occasional nucleoli, and very rare mitotic figures, indicative of increased synthetic and proliferative activity. Small lymphocytes with dense nuclear chromatin and little cytoplasm are also present. Macrophages can already be seen in early acute TCMR and can account for up to half of the inflammatory cell infiltrates (170,182). On occasion, they may even constitute the major cell type, in particular if T-cell-depleting agents such as CAMPATH1 are used (183). Low numbers of B cells (CD20+) can also be detected. Whether their presence in acute TCMR indicates unfavorable long-term prognosis as suggested by some authors is undetermined (184,185,186,187). Plasma cells can be prominent, especially in acute rejection episodes that occur months after transplantation (188,189,190,191,192), and they often mark “steroid unresponsiveness” (185). Plasma cells only rarely invade tubules/cause tubulitis, and plasma cell-rich rejection is often C4d/DSA-negative (Fig. 29.12). Mast cells are present in increased numbers in acute rejection, as judged by tryptase content, and correlate with edema (193).

FIGURE 29.11 Acute cellular rejection with abundant eosinophils. Eosinophils (arrow) are about 20% of the infiltrate in this field. (H&E ×400.)

In acute TCMR, scattered granulocytes can be present in the interstitium; they are typically located adjacent to severely injured tubules. When neutrophils are abundant, the possibility of an acute AMR or pyelonephritis should be considered. Eosinophils are present in about 30% of biopsies with acute rejection, but are rarely more than 2% to 3% of the infiltrate (see Fig 29.11) (194,195,196,197,198). Focally abundant eosinophils are associated with the presence of transplant endarteritis, that is, they can mark Banff category 4 type 2 rejection (199,200). Basophils comprise a minor component of the infiltrate; these cells can also can invade tubules and make up to 5% of the infiltrate (201).

The degree of interstitial inflammation and tubulitis does not correlate tightly with the severity of TCMR or the presence or absence of transplant endarteritis (196), that is, little inflammation does not necessarily equal minimal rejection. This observation is particularly important when interpreting small graft biopsies and considering a diagnosis of “borderline/suspicious for rejection” (Banff category 3).


Infiltration of mononuclear cells under enlarged and “activated” arterial endothelial cells (mainly arcuate caliber vessels or interlobular arteries, less often arterioles) is a typical lesion
of acute TCMR (Fig 29.13). Many terms have been used for this process, including endothelialitis, endotheliitis, endovasculitis, intimal arteritis, infiltrative and proliferative transplant vasculopathy, or endarteritis. We prefer endarteritis, because it emphasizes the type of vessel involved and the site of inflammation; also, more than the intima can be affected in some cases. The biologic and diagnostic significance of endarteritis was probably first noted and illustrated by Dammin in 1960 (28). The importance of this lesion has been emphasized for many years (202), and it is widely accepted as a feature of acute TCMR, particularly if transplant endarteritis is accompanied by tubulointerstitial cellular rejection (61,81,196). However, a considerable proportion of acute TCMR with transplant endarteritis also has concurrent acute AMR (142,150,151,153). On occasion, endarteritis can occur as an isolated event without tubulointerstitial changes. Since the endothelial cell layer is a key immunologic target in transplant endarteritis, the close association between endarteritis and glomerulitis is not surprising, and the detection of glomerulitis should always raise suspicion for transplant endarteritis.

FIGURE 29.12 Plasma cell-rich acute TCMR. A: Acute tubulointerstitial cellular rejection rich in plasma cells (Banff category 4, type 1; C4d-negative, no DSA, no transplant endarteritis, no polyomavirus nephropathy) 8 years posttransplantation. Note abundant plasma cell aggregates in the edematous interstitium accompanied by scattered eosinophilic leukocytes. Plasma cells are “hugging” tubules, but they do not invade, that is, they do not cause “plasma cell” tubulitis. (H&E, ×640 oil.) B: This case of acute TCMR (C4d-negative, no DSA, no polyomavirus nephropathy) rich in plasma cells shows plasma cell tubulitis with one plasma cell (arrow) located inside the basement membrane (arrowheads) between tubular epithelial cells (T, tubular cross section). Plasma cell tubulitis is relatively uncommon. (H&E ×400.)

FIGURE 29.13 A, B: Acute cellular rejection with transplant endarteritis (Banff category 4, type 2 rejection). A: Small artery with few subendothelial mononuclear cells (arrows). (H&E ×400.) B: Small artery with many subendothelial mononuclear cells. The endothelial layer is not clearly defined and is probably partially denuded. Note: no thrombus formation and no inflammation in the medial smooth muscle layer. (PAS ×400.)

Endarteritis has been reported in 18% to 56% of renal biopsies with acute TCMR (61,196,203,204,205,206). The prevalence of endarteritis in biopsies is affected by the sample size, the timing of the biopsy with respect to antirejection therapy, HLA matching, and the level of immunosuppression. Endarteritis tends to affect larger arteries preferentially (196,207). If biopsy samples are small and do not contain arcuate caliber vessels, then transplant endarteritis may remain undetected. This
becomes a major problem if endarteritis occurs as an isolated rejection event.

FIGURE 29.14 Acute cellular rejection with transplant endarteritis (Banff category 4, type 2 rejection). A: An afferent arteriole has a prominent mononuclear inflammatory cell infiltrate. This finding in a small-caliber artery is of similar significance as endarteritis affecting larger arteries; compare with B. (H&E ×400.) B: Arcuate size artery with endarteritis (arrows). (PAS ×200.)

In cases of endarteritis, endothelial cells are usually activated with basophilic cytoplasm and show lifting from the supporting elastic interna/stroma by infiltrating inflammatory cells (Figs. 29.13, 29.14, 29.15, 29.16) (164,196,208). Very rarely, endothelial cells are apoptotic or show mitotic figures. One inflammatory cell under the arterial endothelium is considered to be sufficient for the diagnosis of transplant endarteritis (see Fig. 29.15) (61,81) that is often patchy, that is, only few arteries are affected, and segmental, that is, not the entire intimal circumference is inflamed. The infiltrating cells are typically mononuclear cell elements, T lymphocytes, macrophages, and varying numbers of myofibroblasts that produce collagens and promote intimal sclerosis/chronic vascular rejection in protracted cases (Fig. 29.17) (164). Although mitotic figures are uncommon, the infiltrating cells show proliferative activity based on Ki-67/MIB-1 expression (Fig. 29.18D) (164). The inflamed subendothelial intimal zones contain early matrix proteins including fibrinogen and fibronectin and in persistent cases within 3 to 4 weeks also collagen types 1 and 3 (164). Eosinophils can on occasion be found in the inflamed intimal zones; CD20-expressing B cells and plasma cells are exceptionally rare. Despite the obvious endothelial injury, thrombosis is conspicuously absent.

FIGURE 29.15 Acute cellular rejection with transplant endarteritis (Banff category 4, type 2 rejection). A: Minimum endarteritis. A single subendothelial infiltrating inflammatory cell is evident; endothelial cells are activated but intact. (PAS ×400.) B: Mononuclear cells mainly stick to the endothelium (arrows) and are only focally underneath the endothelium (arrowhead). Only subendothelial mononuclear inflammatory cells are diagnostic for transplant endarteritis. (H&E ×300.)

Mononuclear inflammatory cells that are solely adherent to the luminal surface of endothelial cells are insufficient for rendering a diagnosis of Banff category 4 type 2 rejection/transplant endarteritis (see Fig. 29.15B). Since many donor organs show preexisting hypertension-induced arteriosclerosis, transplant endarteritis is not infrequently superimposed on varying degrees of donor-derived intimal fibroelastosis (Fig. 29.18) that can sometimes be confused with chronic vascular rejection. Usually, elastic tissue stains allow for easy detection since hypertension-induced arterial intimal fibroelastosis gives an intense staining reaction that is lacking in cases of “chronic vascular rejection” (also see below). In
transplant endarteritis, inflammation is typically limited to the intima/subendothelial zone sparing the medial smooth muscle layer. Transmural inflammation involving all layers of arterial walls including segmental fibrinoid necrosis can occur in severe cases of acute TCMR (Banff category 4 type 3 rejection) (Fig. 29.19); however, this feature is more often seen in biopsies with (concurrent) acute AMR and C4d positivity (150). Arteries located in or adjacent to the renal capsule can also be affected by endarteritis; however, diagnostic interpretation is challenging due to the altered blood flow in intracapsular vessels of allografts.

FIGURE 29.16 Acute cellular rejection with transplant endarteritis (Banff category 4, type 2 rejection). Small artery with reactive, enlarged, basophilic endothelial cell cytoplasm. An endothelial mitosis is present (arrow). Eosinophils are seen in the surrounding mixed mononuclear interstitial infiltrate. (H&E ×600.)

FIGURE 29.17 Acute cellular rejection with transplant endarteritis (Banff category 4, type 2 rejection). Seven days posttransplantation, this patient experienced diffuse tubulointerstitial cellular rejection, marked diffuse transplant glomerulitis, and marked transplant endarteritis (Banff type IIB rejection, C4d-negative). The patient was not sensitized, crossmatch and PRA negative with no DSA at the time of biopsy. Inflammation along the intimal layer of an interlobular artery (A) is rich in CD3 (B) and, to a lesser degree, also CD68 (C)-expressing cells that are mitotically active (Ki-67/MIB-1 positivity, D).

FIGURE 29.17 (Continued) Note: Even in cases of early transplant endarteritis, intimal inflammation contains scattered myofibroblasts expressing smooth muscle actin (SMA, arrows, E). Myofibroblasts can synthesize collagens and promote chronic vascular rejection in protracted/suboptimally treated rejection episodes. This patient responded fully to thymoglobulin therapy. ((A) H&E, (B-D) IHC on formalin-fixed and paraffin-embedded tissue, (B) anti-CD3, (C) anti-CD68, (D) Ki-67/MIB-1, (E) anti-alpha smooth muscle actin, ×600 oil. L, arterial lumen; M, medial smooth muscle layer.)

In acute TCMR, PT capillaries are often dilated and contain increased numbers of mononuclear cells even in the absence of concurrent AMR (82,181,209). Capillaritis reflects trafficking of inflammatory cells from the blood into the inflamed tubulointerstitial compartment and the fact that the capillary endothelium is a target of TCMR. Some evidence suggests that PT capillaritis with a predominance of monocytes (monocyte/T-cell ratio greater than1) is an indicator of acute AMR (167). On rare occasions, polymorphonuclear leukocytes
are the dominant cell type in PT capillaritis suggesting in our experience concurrent acute AMR.

Infiltration of mononuclear cells into the wall of veins or lymphatics is found in about 10% of biopsies with acute TCMR. This is a sign of inflammatory cell trafficking in areas of inflammation without direct diagnostic significance (210). Not surprisingly, these lesions can be found in inflammatory processes other than rejection.

FIGURE 29.18 Acute cellular rejection with transplant endarteritis (Banff category 4, type 2 rejection). Subendothelial mononuclear cells are present in an artery (= acute rejection) superimposed upon preexisting arteriosclerosis (donor disease). This pattern can be confused with chronic rejection; elastic tissue stains usually help making a distinction (strong staining in cases of hypertension-induced arteriosclerosis vs. chronic vascular rejection largely lacking elastic tissue accumulations). (H&E ×200.)

FIGURE 29.19 Acute cellular rejection with severe transplant endarteritis (Banff category 4, type 3 rejection). An interlobular artery has segmental transmural inflammation including destruction of the lamina elastica interna (at 1o′clock; v3 in the Banff scoring system). There was no evidence of DSA, and a C4d stain was negative confirming the diagnosis of a TCMR process. The rejection episode responded to therapy. (PAS ×600 oil.)


All transplanted tissues (i.e., renal parenchyma, renal pelvis, ureter, and extraparenchymal vessels/tissue) can be involved by acute rejection. Lymphocytes and plasma cells infiltrate the urothelium and ureteral walls (Fig. 29.20) (154,164,211,212). Small arteries in the ureter can be affected by endarteritis and fibrinoid necrosis (154,164,213). Of 26 ureters from irreversibly rejected kidneys, 80% had acute rejection-induced injury (211). Endarteritis in peripelvic adipose tissue can result in fat necrosis occasionally mimicking neoplastic growth in imaging studies (see Fig 29.51). The inflammatory reaction in extraparenchymal structures usually corresponds to the severity of rejection found in the renal cortex.

Immunofluorescence Microscopy

Little, if any, immunoglobulin deposition is found by immunofluorescence in acute cellular rejection. Extravascular fibrin is typically present in the edematous interstitium. C3 and non-diagnostic C4d deposits can be prominent along the TBM, mainly along tubular segments undergoing atrophy. By definition, in pure acute cellular rejection (Banff category 4), PTC in the cortex and medulla lack any linear C4d deposition; the presence of C4d indicates a component of concurrent AMR/mixed acute TCMR and AMR.


In transplant glomerulitis, fibrin and scant IgM and C3 deposits are found along the GBM (161). C4d is usually detected in mesangial regions (by IF only; no mesangial staining by IHC) and, on occasion, with a linear or granular staining pattern along the GBM. In the absence of PTC C4d staining, these isolated glomerular C4d deposits are nondiagnostic for AMR- or antibody-induced glomerular injury. MHC class II/HLA-DR is usually strongly expressed by infiltrating inflammatory cells in segments with glomerulitis.

FIGURE 29.20 A: Ureter with acute cellular rejection. At low power, dense lymphocytic submucosal infiltrates are seen. (H&E ×25.) B: Urothelial invasion of mononuclear cells in acute rejection, involving the calyceal mucosa. Lymphocytes and eosinophils are in the submucosa. (H&E ×400.)


In acute TCMR, MHC class II/HLA-DR antigens and intercellular adhesion molecules are expressed stimulated by the release of interferon-gamma in inflamed regions (Fig. 29.21) (142,214). The detection of MHC class II in the cytoplasm of tubular epithelial cells by IF on frozen tissue samples may be used as an adjunct marker to establish a diagnosis of acute TCMR, in particular if the so-called Banff borderline changes (Banff category 3) are seen by standard light microscopy in corresponding formalin-fixed tissue sections (141,142,156). Tubules, in particular those with some evidence of atrophy, show prominent linear staining along basement membranes for C3 and, to a lesser degree, also C4d, C5b-9, and IgM. C3 is largely derived from tubular cells, as judged by C3 allotype antibodies; donor-specific C3 mRNA can be detected in rejecting renal allografts by PCR (215). Proximal tubular cells in culture synthesize C3 in vitro in response to IL-2 (216). The origin of C4d and C5b-9 is less well understood.

FIGURE 29.21 In cases of acute TCMR, the release of interferon-gamma stimulates the expression of MHC class II (HLA-DR) in the cytoplasm of tubular epithelial cells best detected by immunofluorescence staining (T, tubular cross sections with MHC class expression; asterisk, tubules without class II). The detection of tubular MHC class II can serve as an adjunct diagnostic parameter to establish a diagnosis of acute TCMR, in particular if Banff borderline changes (Banff category 3) are present or to confirm the concurrence of AMR and acute TCMR. (Direct immunofluorescence microscopy on frozen tissue with an antibody directed against MHC class II, ×200.)


Fibrin is typically present in the edematous interstitium (217) and likely a promoter for the development of the so-called scleredema in protracted cases of acute TCMR that is increasing accumulation of matrix proteins including collagens in an edematous stroma. The fibrin deposition derives from leakage of PTC walls and activation of the clotting system, probably by cytokine induction of macrophage procoagulants (218).


Most investigators find no significant immunoglobulin deposits in the arterial vessels in acute TCMR with or without transplant endarteritis (208); fibrin deposition is sometimes seen in inflamed intimal segments and in vessels with fibrinoid necrosis. Infiltrating cells in foci of endarteritis strongly express MHC class II/HLA-DR. Diffuse PTC C4d deposits or the exceptional event of arterial IgG deposits is evidence for concurrent acute AMR (219). C4d, C3, and, to a lesser degree, IgM are typically seen in arteriolar hyalinosis as a nondiagnostic staining pattern.

Electron Microscopy

Electron microscopy is generally not performed for diagnostic purposes in acute rejection; however, it may be indicated if the glomeruli are notably involved.


Transplant glomerulitis typically shows dilated capillary lumens filled with activated and enlarged endothelial cells as well as other mononuclear cell elements, mainly monocytes.
Polymorphonuclear leukocytes, platelets, and fibrin strands are occasionally seen. Mitotic figures can be detected but are usually rare. Glomerular endothelial cells are reactive, with a marked increase in cytoplasmic organelles (ribosomes, mitochondria, endoplasmic reticulum) and nuclei show open chromatin and prominent nucleoli (Fig. 29.22). The endothelial cells typically lose their fenestrations and are often separated from the GBM by a widened lamina rara interna. Rudimentary thin subendothelial new lamina densa formation can occasionally be seen as an early ultrastructural sign of evolving transplant glomerulopathy (220) scored in the Banff 2013 update as “cg1a” (see Fig. 29.22D). The mesangium has loose matrix and sometimes monocytes. Podocytes primarily those overlying segments with glomerulitis usually show foot process effacement.


Lymphocytes in the tubules accumulate between the epithelium and the TBM, frequently surrounded by a clear zone (221). The tubular epithelial cells remain in contact with the basement membrane, whereas the lymphocytes are often separated from it by a thin layer of epithelial cytoplasm. Breaks in the TBM are rarely found by electron microscopy (Fig. 29.23) (175). Leakage of Tamm-Horsfall protein into the interstitium through fractured TBM segments can occur; these deposits contain 4-nm-thick filaments arranged in parallel clusters and sometimes herniating into vessels (222). The tubular epithelial cells in the vicinity of mononuclear cells often show signs of injury, including vacuolization (221), necrosis, or apoptosis (175). Tubular basement membranes, especially those with evidence of atrophy, are thickened and can contain nondiagnostic small electron-dense particles, mainly complement products.

FIGURE 29.22 Transplant glomerulitis. A: A lymphocyte (L) is in contact with an activated glomerular endothelial cell (E). B: An activated endothelial cell has lost fenestrations and is separated from the original basement membrane (arrow) by an expanded subendothelial space/lamina rara interna that contains loose matrix, cell processes, and debris.

FIGURE 29.22 (Continued) C: Platelets are numerous in a capillary loop that has denuded endothelium. D: Activated endothelial cells are overlying a widened lamina rara interna containing rudimentary new densa as a sign of early, ultrastructural evidence of GBM remodeling and duplication (arrows). Light microscopy in this case showed focal minimal glomerulitis without transplant glomerulopathy. Early ultrastructural GBM remodeling/minimal duplication as illustrated in D is scored in the 2013 update of the Banff classification scheme as “cg1a”. (Electron micrographs. (A) ×6360, (B) ×5225, (C) ×6000, (D) ×5500.)


The interstitium is expanded by edema and a mixed infiltrate of activated lymphocytes and macrophages. Granulocytes are occasionally encountered. The fibroblasts may appear active with fibrils, typical of myofibroblasts.


Mononuclear cells accumulate in the PTC lumens in areas of interstitial inflammation and edema, sometimes to the point of apparent capillary occlusion. The intracapillary cells mostly consist of lymphocytes and monocytes, which are sometimes in contact with the endothelium or emigrating through the capillary walls (223). The endothelium shows signs of activation, as judged by nuclear enlargement; increased ribosomes, endoplasmic reticulum, mitochondria, and Golgi apparatuses; and loss of fenestration (223). The endothelial hypertrophy has been compared with normal postcapillary venules that are not anatomically recognized in the kidney (223). Which of these
features are specifically related to TCMR versus AMR has not been determined. Multilaminations of PTC basement membranes are typically minor but can become prominent with 5 to 7 basement membrane layers seen in 10% to 20% of late acute TCMR rejection episodes occurring post-year 1 (224). Such pronounced multilaminations in cases of acute TCMR (C4dnegative) represent protracted endothelial injury and evidence of early chronic rejection (224). The endarteritis lesions have been little studied ultrastructurally, because of the difficulty in sampling these very focal lesions.

Molecular Studies

The molecular phenotype in renal allografts with acute TCMR, mainly tubulointerstitial cellular rejection, Banff category 4, type 1, primarily comprises transcripts expressed by various subsets of activated lymphocytes and other inflammatory cell elements: cytotoxic T lymphocytes (granzyme B, perforin, and Fas ligand), effector memory T cells, T helper cells, regulatory T cells (225,226,227,228,229,230,231,232,233,234,235,236), and macrophages (237,238). Also, increased levels of transcripts regulated by interferon-gamma (TGF-β, TNF-α, RANTES, MIP-1α, HLA class I and II molecules, CXCL9, CXCL10, and CXCL11) and for T-bet, a master transcription factor for T cells, are found (229,239,240,241,242). As expected, successful treatment of rejection is followed by a significant decrease of all molecular signals/transcripts (228,239).

Complement components are also expressed during rejection, such as C3 and C1q, but probably this phenomenon reflects a “nondiagnostic” rejection-induced injury response (229,243,244,245). Tissue injury results in down-regulation of numerous transcripts associated with various physiologic functions, such as solute carriers and membrane transporters with high constitutive expression levels in normal tubular epithelium (233,234,240,246).

Recently, the Edmonton group used their comprehensive collection of human renal allograft mRNA microarray data to construct a hypothetical cellular rejection classifier (239,247). This classifier assigns to each biopsy a probability score for the presence of cellular rejection according to the detected molecular expression profile. Interestingly, IFN-γ and inducible or cytotoxic T-cell-associated transcripts, for example, CXCL9, CXCL11, GBP1, and INDO, were most predictive in this algorithm (239). A comparison of molecular marker expression with histologic Banff categories revealed discordant diagnoses in 20% of biopsies. Potential explanations for the observed discrepancies include an inadequate molecular test performance, sampling differences, treatment prior to biopsy that suppresses all molecular signals while histologic lesions might still be present, presence of isolated histologic lesions not reflected by major shifts in the gene expression profile, etc. However, a concordance rate of 100% can hardly be expected comparing different tests, and the reported findings are very encouraging emphasizing the value of biopsy diagnoses. In biopsies with
Banff category 3 “borderline” changes, 33% of cases showed a molecular phenotype similar to TCMR, while 67% were nonrejection-like, underscoring on a molecular level the diagnostic heterogeneity of this group. The tightest association was between molecular phenotype and Banff total inflammatory (ti) score, indicating that the molecular phenotype is largely due to tubulointerstitial inflammation.

FIGURE 29.23 Acute cellular rejection. A tubule is invaded by a lymphocyte with a few dense cytotoxic granules (arrows) that are oriented toward the epithelial cells. The tubular basement membrane has fine laminations but does not appear disrupted. (Electron micrograph; ×5200.)

Suthanthiran et al. pioneered work on mRNA profiles from urinary cells that reflect events in the renal allograft (248). Elevated levels of mRNA for OX40, OX40L (costimulatory proteins), PD-1 (programmed death-associated protein), and Foxp3 (master regulatory protein in regulatory T cells) indicate acute rejection (249). More recently in a large cohort of 485 kidney graft recipients, Suthanthiran et al. analyzed 4300 urine samples and found a 3-gene signature of CD3- mRNA, IP-10 mRNA, and 18S rRNA levels to be diagnostic of acute TCMR (250). Similar efforts are made with RNA retrieved from peripheral blood leukocytes (251).

Elevated expression levels of both microRNAs (miRNA) and mRNAs were found in acutely rejecting grafts. Profiling urinary microRNA expression levels of stable transplant patients and patients with acute rejection revealed micro R-10b and micro R-210 to be down-regulated and micro R-10a to be up-regulated in acute rejection compared to the control cohort. Only micro R-210 differed between patients with acute rejection compared to stable transplant recipients with urinary tract infections and, thus, may potentially serve as a biomarker of acute rejection (252). These preliminary data suggest that microRNA expression patterns correlate with mRNA profiles. They might possibly be of diagnostic value due to their stability, especially in body fluids and after formalin fixation and paraffin embedding of tissue samples (253,254).

Etiology and Pathogenesis


The primary T-cell allograft response is to the antigens encoded in the MHC, present in all vertebrates. Genetic experiments in mice showed that one genetic locus (the MHC) was the major determinant of graft survival. Two chemically and functionally different classes (I and II) of histocompatibility molecules are encoded in the MHC. Disparity of either alone is sufficient to cause graft rejection in mice (255). The exquisite sensitivity of the immune system to these antigens has been elegantly demonstrated using Kbm mutant mice. Skin grafts from donors that differ from the recipient in only 1 to 3 amino acids in a single MHC molecule are promptly rejected (255,256). The human MHC (termed HLA for human leukocyte antigen) spans 4000 kb on the short arm of chromosome 6 and contains multiple genes (257). The dominance of HLA antigen mismatch in determining outcome is supported by the observation that grafts from an HLA-identical sibling survive longer on average than those from a non-HLA-identical sibling (see Fig. 29.3).

Class I MHC antigens consist of highly polymorphic transmembrane 45-kDa glycoprotein α-chains associated with monomorphic 12-kDa β2-microglobulin. About 100 alleles of the A, B, or C class I loci have been defined with alloantibodies and over 1000 with genetic probes (256). Class I antigens are widely distributed on all nucleated cells, but their concentration on the cell surface varies widely, even to the point of undetectability by standard immunohistochemical techniques (e.g., placental and Langerhans cells). In normal human tissues, the vascular endothelium (arteries, veins, capillaries) stains most intensely for class I antigens; the parenchymal cells are moderately positive (258).

Class II MHC antigens contain noncovalently associated transmembrane α- and β-chains of 25 to 28 and 29 to 34 kDa, with most of the polymorphism on the smaller β-chain. Three gene families (DP, DQ, DR), each with multiple α-and β-genes, have been identified. Class II antigens are more restricted in distribution than class I and vary by species and class II family. In humans, class II antigens are normally found on B cells, dendritic cells, capillary endothelium, monocytes, Langerhans cells, and activated T cells (258). DR but not DQ is demonstrable on capillary endothelium; both are on dendritic cells. Parenchymal cells normally have less intense staining for HLA-DR and no DQ or DP (157,259,260). Normal arterial endothelium has little or no detectable class II antigens.

The expression of MHC molecules is regulated on the cell surface, under the control of inflammatory mediators, such as the interferons and tumor necrosis factor (261,262).
IFN-γ, produced by antigen-activated T cells and NK cells, induces both class I and class II MHC antigens on epithelial and endothelial cells (263). Increased surface density of MHC molecules enhances the susceptibility to T-cell-mediated lysis and the ability to present antigen (264). However, induction of graft MHC molecules is not necessary to promote rejection. IFN-γ-deficient mice reject kidney allografts as quickly as normal mice without induction of MHC molecules (265). Paradoxically, class I- and class II-deficient grafts are also rejected efficiently, with MHC expression undetectable by immunoperoxidase techniques (266,267).

The normal function of MHC class I and II molecules is to “present” antigen to T cells. MHC molecules bind certain peptide antigens more avidly and thereby present these antigens more effectively. T cells are positively selected in the thymus for recognition of self-MHC molecules, so that they are normally “restricted” to antigen presented by self-MHC antigens. Cells with a high reactivity to self-MHC or no affinity for MHC are deleted. The T cells recognize an altered conformation of self-MHC molecules plus the associated antigen. Graft rejection thus occurs in part because the T cells recognize the foreign MHC antigens as if they were self-MHC molecules altered by association with some “X” antigen (the altered self-hypothesis) (268) or because of their intrinsic affinity for the MHC. These theories explain the high clonal frequency of T cells directly reactive to nonrelated individual cells and why the MHC is the “major” determinant of allograft survival (269).

Direct recognition of HLA antigens on graft cells can result in cell-mediated cytotoxicity and release of cytokines. T cells can also react with MHC alloantigens from the graft that are processed and presented by autologous dendritic cells or infiltrating macrophages, as with other protein antigens (270). This response is termed the “indirect” pathway to distinguish it from the “direct” recognition of antigens on the donor cell surface. Indirect responses cause rejection by the action of lymphokines or through activation of B cells or macrophages associated with dendritic cells.


Non-MHC histocompatibility antigens (“minor antigens”) are defined simply by their ability to elicit graft rejection and a genetic locus outside the MHC. These antigens are responsible for graft rejection between MHC-identical congenic mice and HLA-identical siblings. Their chemical nature and distribution are largely unknown. In the mouse, 30 to 50 minor loci are calculated to trigger skin graft rejection (271). Graft-infiltrating T cells can be isolated that recognize minor antigens on donor tubular epithelium (272). The vascular endothelium expresses polymorphic non-MHC histocompatibility antigens, which have not yet been well characterized at a molecular level.

Antigen Response by T Cells

Engagement of MHC-associated antigen by the specific T-cell receptor leads to expression of IL-2 receptor on the cell surface. Second (costimulatory) signals from the antigenpresenting cell are required to move the T cell to produce T-cell growth factors, such as IL-2, IL-4, or IL-15. Costimulatory signals, including CD86, CD40, and OX40L, are provided by “professional” antigen-presenting cells, such as dendritic cells and monocytes. These bind to CD28, CD40L (CD154), and OX40 on T cells, respectively. T cells that see antigen without the second signal are rendered anergic, that is, refractory to specific antigen stimulus in the future. The conditions that promote such an outcome are of considerable clinical interest as a potential strategy to produce tolerance.

Upon recognition of antigen on a cell surface with sufficient costimulatory signals, the T cells make two types of responses: secretion of cytokines and chemokines, which affect the behavior of nearby cells, and the development of cytotoxic effector functions that kill cells that express the relevant MHC. Delayed-type hypersensitivity to exogenous protein antigens is mediated by the former mechanism, while resistance to viral infection is dependent on the latter. These are the two nonex-clusive mechanisms proposed for acute graft rejection.

T cells are heterogeneous in function. CD4 cells, which recognize peptide antigens (typically antigens exogenous to the cell) presented by self-class II molecules, are critically important for graft rejection. Deficiency of CD4 cells prevents heart or skin graft rejection in mice (273). CD4 cells can be divided according to their cytokine production into Th1 and Th2 cells, as proposed by Mosmann et al. (274), although some cells have overlapping cytokine profiles. Th1 cells produce IFN-γ, IL-2, and TNF-α and express CXCR3 and CCR5 chemokine receptors. Th1 cells activate macrophages, mediate delayed hypersensitivity reactions, mediate cytotoxicity via Fas ligand/Fas, and are typically present in the infiltrate of acutely rejecting grafts. IFN-γ and TNF-α activate macrophages to produce nitric oxide, which causes vasodilation and edema, reactive oxygen species, and more TNF-α. TNF-α in turn induces apoptosis via activation of caspases. IL-12 produced by other cells (e.g., dendritic cells and macrophages) promote Th1 cell activity and inhibit Th2 cytokines. IL-10 and IL-13 produced by macrophages and other cells promote Th2 activity and inhibit production of Th1 cytokines.

Th2 cells synthesize IL4, IL-5, IL-10, and TNF-α and have the chemokine receptors CCR3, CCR4, and CCR8. These cells provide help for B cells and production of IgE and IgG4 antibodies (275). Th2 cytokines promote eosinophil production and infiltration. IL-4 and IL-13 stimulate the production of eotaxins (CCL11, CCL24, CCL26) by parenchymal cells, including endothelial cells (276). Th2 cells are sufficient to mediate cardiac allograft rejection in mice and promote a heavy infiltrate of eosinophils (277). It is likely that some of the variation in morphology of acute graft rejection is due to differences in the proportionate contribution of Th1 and Th2 cells in the infiltrate.

CD8 cells, which recognize peptide antigens presented by class I MHC molecules, produce IFN-γ and mediate direct cytotoxicity via granzymes A and B and perforin and by Fas ligand (278,279). Perforin forms a membrane channel that allows the granzymes into the target cell cytoplasm, where they activate Bid and caspases that trigger apoptosis (280). Expression of granzyme B, but not perforin, depends upon IL-12 and correlates strongly with cytolytic activity. Across an isolated class I mismatch, perforin deficiency prolongs survival of heart or skin grafts (281). However, fully mismatched recipients that lack either perforin or granzymes A and B reject kidneys with equal efficiency and develop tubulitis and endarteritis, arguing against a necessary role for either mediator (282).

A subpopulation of T cells, termed regulatory T cells (Treg), inhibits activation of effector T cells. Treg are antigen specific and can suppress memory CD8 cells, apparently via a CD30-dependent pathway (283). Activation of Treg function requires engagement of the TCR (i.e., is antigen specific), but can affect other cells through soluble mediators (antigen nonspecific). Most Treg are CD4+CD25+CD152+ and produce IL-10 and TGF-β, but not IL-2 or IL-4 (284,285). Differentiation into Treg is promoted by TGF-β and is mediated by the master transcription factor, Foxp3, which continues to be expressed in Treg cells and serves as a distinctive marker of these cells in tissues.


A burgeoning, complex, and confusing literature is emerging on the role of chemokines in transplantation. In acutely rejecting human kidney grafts, a variety of chemokines are produced (IP-10, RANTES, MIP-1α, MIP-1β, lymphotactin, MCP-1), and the infiltrating cells express several chemokine receptors (CXCR4, CXCR3, CCR5, CCR2, and others) (286,287,288,289,290,291). CCR5 is mostly in the diffuse infiltrate (287), while CXCR4 is in nodular aggregates of mononuclear cells (292). The pattern of expression suggests a predominance of Th1 over Th2 cells (i.e., CCR5 and not CCR3 or CCR8) (287). Tubules synthesize RANTES (293), IL-8 (294), CXC3L1 (292), and IL-6 (295) in acute rejection; none of these are detected in normal kidneys. These may be a response to local T-cell production of IL-17 (296). MCP-1, MIP-1α, MIP-1β, and RANTES are increased in the basolateral surface of tubules (289,293,297). Heparan sulfate in the TBM may provide sites for chemokines, such as CCL4, to bind and create gradients (298). MCP-1 and MIP-1β levels are higher in type 2 than in type 1 rejection (297).

Pathogenetic Mechanisms

The various components of the kidney are affected to differing degrees in individual episodes of acute rejection. While each of these targets is believed to be affected by T cells, macrophages, and cytokines, the pathogenesis may vary somewhat. IHC in situ hybridization, and laser capture microdissection are yielding new insights into the cells and molecules that participate in rejection.


Both CD8+ and CD4+ T-cell subsets are present in varying proportions, as well as a minor population of CD4+CD8+ cells (299,300). Typically, the CD8+ cells are enriched in the graft and are found permeating diffusely in the renal cortex (157,301,302). CD4+ cells are usually selectively concentrated in perivascular aggregates (157,301,303). T cells in the infiltrate express the CD45RO isoform of activated/memory cells (304). Most infiltrating CD3+ T cells express the usual α/β-TCR (305,306); in a minority of specimens, over 10% of T cells are γ/δ+ (306). A skewed distribution of TCR gene rearrangements has been reported, suggesting local selection of specificities (307,308,309,310,311).

Many infiltrating T cells express cytotoxic molecules, namely, perforin (312,313), FasL (314), granzymes A and B (315,316,317), and TIA-1/GMP-17 (317,318) (Fig 29.24). About 30% of the infiltrating cells are TIA-1/GMP-17+ (318); most of these express CD8, but 12% are CD4 (318). Thus, some cytotoxicity may be mediated by CD4+ T cells, consistent with the observation that donor-specific cytotoxic CD4+ T cells can be cultured from grafts (319). The infiltrating cells also express TNF-β (lymphotoxin) and TNF receptors (320).

FIGURE 29.24 Acute tubulointerstitial cellular rejection (Banff category 4, type 1). Lymphocytes in tubules express the cytotoxic granule associated protein TIA-1 (GMP17). (PAS combined with anti-TIA-1 antibody/immunoperoxidase technique ×400.)

Foxp3+ cells, presumably Tregs, are present in variable numbers in the infiltrate (321) (Fig. 29.25). Foxp3+ Treg cells are known to suppress cell-mediated reactions and serve to dampen T-cell-mediated graft rejection as recently shown in mice (322). Supporting this role in vivo, Foxp3+ cells infiltrate grafts in certain experimental conditions that promote tolerance (323) and specific depletion of Foxp3 cells triggers acute TCMR in the mouse (322). The presence of Foxp3+ cells in the graft has no correlation with the outcome of TCMR (321,324,325). However, Foxp3+ cells are more prevalent in borderline than TCMR biopsies (326). In stable grafts Foxp3+ infiltrates have been associated with donor hyporesponsiveness in vitro (327) and decreased Cr at 2-3 years (328). Grafts with subclinical rejection without Foxp3 cells had a significantly worse prognosis at 5 years than those with Foxp3+ cells, even among those with fibrosis (329). Further studies are warranted to assess their significance.

Activation of the infiltrating T cells has been demonstrated by the presence of IL-2 (320) and its receptor CD25 (182,330,331). In situ hybridization studies show cytokines are typically synthesized by a minority of the cells (presumably the specific alloreactive cells) (332). IFN-γ is synthesized in the graft (189,244,333) and is detected in lymphocytes scattered throughout the infiltrate (330). Other markers of activation expressed by the infiltrate are the transferrin receptor (334), CD38 (171), and CD69 (335). Some CD8 cells express CD152, and a few CD4 cells express CD40L.

Apoptosis of the infiltrating T cells can be demonstrated with the TdT-uridine nick end label (TUNEL) technique (318,336,337,338) and may occur at a frequency comparable to that in the normal thymus (1.8% of cells in a section) (318). Apoptosis probably occurs in infiltrating T cells as a result of activation-induced cell death and would thereby serve to limit the immune reaction (318).

FIGURE 29.25 Acute tubulointerstitial cellular rejection (Banff category 4, type 1). A: Cells in the infiltrate stain for Foxp3, a transcription factor for T regulatory cells. B: Foxp3 cells are present in the tubules (arrows). (Anti-Foxp3 immunoperoxidase, (A): ×200, (B) ×600.)

Macrophages expressing CD14 or CD68 sometimes rival T cells in abundance and may be the predominant leukocyte (302,339,340). Macrophages display markers of activation, including the tissue factor procoagulant-related antigen (182,341), receptors for VCAM-1 and fibronectin (CD49d, VLA-4), and Fas ligand (342). Macrophages expressing the tissue procoagulant-related antigen are associated with interstitial fibrin deposits (182). Endothelin, IL-6, and VEGF can also be detected in macrophages in rejection (295,343,344). Macrophages express costimulatory molecules CD80 and CD86, but not CD40 (331). Plasminogen activator inhibitor-1 (PAI-1) synthesis is detected in infiltrating cells, especially in hemorrhagic areas, perhaps serving a protective role (345). Urokinase plasminogen activator and its receptor are also produced by the infiltrating cells (346).

B cells can be present, even in early biopsies. The local synthesis of CXCL13 and expression of its receptor CXCR5 in B-cell clusters have been noted in patients who developed rejection in the first 9 days (347). About 2% to 30% of the infiltrating mononuclear cells have been reported to express CD57 (HNK-1) (157,182,259,302). Numerous CD56+ cells (also considered to be NK cells) have been reported (315), but using a panel of three “NK” antibodies (PEN 5, CD57, and CD56), positive mononuclear cells were rare (less than 1%) (318); similar results were noted by others (348).


Tubulitis is an important mechanism of graft rejection that involves both CD8+ and CD4+ cells (157). Intratubular T cells with cytotoxic granules accumulate selectively in the tubules, compared with the interstitial infiltrate (318) (see Fig. 29.24). These cells account for 65% of the mononuclear cells in tubules compared with about 30% of the interstitial cells. Lymphocytes expressing perforin mRNA and perforin protein are closely associated with tubular epithelial cells (349). Intratubular T cells express CD103 (αEβ7), the integrin that binds to E-cadherin, which is normally on the surface of tubular cells (350,351,352). CD103+ cells are found exclusively in the tubules (352). CD103 probably contributes to their concentration within tubules (318): Mouse T cells deficient in CD103 do not effectively mediate tubulitis or tubular injury (353). Surprisingly, cyclosporine increases the expression of CD103 in the infiltrate (353). CD4 cells with Foxp3 expression are relatively concentrated in the tubules (321). T cells proliferate once inside the tubule, as judged by the marker Ki-67 (MIB-1), which labels 15% of the intratubular lymphocytes (354).

Increased numbers of TUNEL+ tubular cells are present in acute rejection, compared with normal kidneys (318,336,338); CNI toxicity; or ATN (318). Tubular cells have increased Bax and p53 and less Bcl-2 (355). The degree of apoptosis correlates with the number of cytotoxic cells and macrophages in the infiltrate, consistent with a pathogenetic relationship (318). Apoptosis of murine tubular cells can be induced in vitro by IL-2 or IFN-γ, both present in acute rejection (356). Tubular epithelial cell proliferation can also be detected with Ki 67, which labels 1.5% ± 2.3% of the epithelial cells (354).

Tubular cells can process and present antigen to activated T cells in vivo and in vitro (357) and express MHC class II and the costimulatory molecules CD80 and CD86 in acute rejection (358). Increased tubular epithelial cell expression of HLA-DR is typical, but not by itself diagnostic, of acute cellular rejection since it can also be seen in cases such as pyelonephritis (170,259,334,359,360,361,362) (see Fig. 29.21). Increased tubular HLA-DR antigen expression correlates strongly with the presence of a T-cell infiltrate and presumably is a local response to IFN-γ produced by these cells (156,170,244,259). Proximal and distal tubules express the IFN-γ receptor, detectable with immunoperoxidase in acute rejection (320,363). Tubular synthesis of C3 (215) correlates with local IFN-γ production (244) and exposure to Il-17 (296). Tubules also synthesize TNF-α (364), TGF-β1, IL-15, osteopontin, and VEGF (344,351,363). Expression of osteopontin correlates with CD68+ cell infiltration and tubular cell regeneration (Ki 67+).

Several adhesion molecules are increased on tubular cells during acute rejection. ICAM-1 (CD54) is increased (365,366,367) and closely correlated with HLA-DR. VCAM-1 is increased, mostly on the basal surface of tubular cells, and correlates with the degree of T-cell infiltration (339) and CD25+ cells (366). Decreased staining for urokinase and antithrombin III occurs in proximal tubular cells (368). Tubules also produce urokinase plasminogen activator and its receptor (346).

Some molecules and cells in tubules have the potential to inhibit acute rejection. Protease inhibitor-9 (PI-9), an inhibitor of granzyme B, is synthesized by tubules in acute rejection, suggesting this may suppress tubular injury by cytotoxic T cells (316). IL-15 produced by tubular cells inhibits expression of perforin (351). Tubules also produce the complement inhibitor clusterin (369), which is colocalized in the TBM with the C5b-9 complex and vitronectin (370).


Endarteritis is a common and significant manifestation of cellular rejection, observed in all solid allograft organs. There is good support for a T-cell-mediated type of injury in transplant endarteritis. The cellular arteritis occurs in the absence of antibodies (B-cell knockout mice) (Fig. 29.26) (371). As further evidence, in humans, rejection with intimal infiltrates can usually be reversed with antibody preparations mainly targeting T cells (204,372,373). However, antibodies can elicit endarteritis in mice (374) and may co-stimulate with TCMR arterial inflammation in humans. The cells infiltrating the endothelium and intima are T cells and monocytes/histiocytes, but not B cells (157,164,208). Both CD8+ and CD4+ cells invade the intima in early grafts, but later, CD8+ cells predominate (157), suggesting that class I antigens are the primary target. Some of the T cells express a cytotoxic phenotype (318,375). TNF receptors are detectable in the endothelium of arteries (320). Apoptosis of vascular endothelial cells can be detected in sites of endarteritis (318,336), and increased numbers of endothelial cells appear in the circulation (376).

Normal arterial endothelial cells express class I antigens, weak ICAM-1, and little or no class II antigens, or VCAM-1. During acute rejection, the endothelium of arteries expresses increased HLA-DR (157,259) and ICAM-1 and VCAM-1 (365,367), and endothelial synthesis of ICAM-1 and VCAM-1 can be demonstrated by in situ hybridization (377). Endothelium also up-regulates ligands for L selectin (sialyl Lewis X and lactosamine) (378). The endothelium of arteries with acute cellular rejection shows a striking increase in PDGF A-chain mRNA and protein (379). In endarteritis, PDGF B chain, in contrast, is largely limited to the CD68+ inflammatory cells, probably macrophages, under the endothelium; it probably promotes myofibroblast proliferation. IL-6 is also increased in the media and endothelium of vessels in acute rejection (295). In transplant endarteritis, the endothelium expresses PAI-1 mRNA (345) and the urokinase plasminogen activator receptor (346); the arterial media shows increased urokinase plasminogen activator (346).

FIGURE 29.26 Endarteritis in a cardiac allograft in a mouse deficient in B cells. This shows that endarteritis lesions can occur without the participation of antibody (371). (Elastic tissue stain of a cryostat section, ×400.)

Normal PTC endothelial cells express prominent HLA-DR, HLA class I antigens, LFA-3 (CD58), PECAM-1 (CD31), factor VIII antigen, and low levels of ICAM-1 and ICAM-2 (348,366,380). During acute cellular rejection, the capillary pattern for HLA-DR and ICAM-1 antigens is lost, probably due to disruption and necrosis of the endothelium (348,380). This was confirmed using a monoclonal antibody that reacted selectively with donor class I (HLA-A2) in six cases (380). Endothelial cells also have decreased endothelin expression in rejection with endarteritis, but not in tubulointerstitial rejection (381).


In transplant glomerulitis, intraglomerular mononuclear cells are primarily CD8+ (157,382) accompanied by macrophages (see Fig. 29.6A). The lymphocytes have an activated phenotype, as judged by expression of CD25 and HLA-DR. The glomeruli have increased staining for HLA class I antigens (157). TNF-α protein is detectable in glomerular endothelial cells (364), which normally have TNF receptors (320). Thromboxane synthetase and IL-6 increase in glomeruli in rejection, probably due to intraglomerular macrophages (295,383).

It is not known why rejection often becomes focused on specific anatomic compartments. Transplant glomerulitis may only involve some glomeruli and occasionally spares the tubulointerstitial and arterial compartments completely.

Clinical Course, Prognosis, Therapy, and Clinicopathologic Correlations

The first-line treatment for acute cellular rejection, that is, rejection in the absence of C4d staining and/or circulating DSA, is bolus steroids for up to 3 days; this therapeutic approach works well in patients with T-cell-mediated tubulointerstitial rejection (i.e., Banff category 4, type 1). In patients who do not respond, mainly those with transplant endarteritis and glomerulitis, the standard rescue therapy is thymoglobulin. Antibody treatment is continued typically for 10 days. If the acute cellular rejection episode is concurrent with C4d positivity/AMR (mixed Banff category 2 and category 4 rejection), then often intravenous immunoglobulin (IVIG) or plasmapheresis is added to the therapeutic regimen. Future treatment options for antibody-mediated components of acute mixed rejection episodes may also include rituximab (an anti-CD20 antibody) or bortezomib (targeting activated plasma cells) (390).

Following treatment of acute cellular rejection with pulse steroids, or thymoglobulin, a marked decrease in the interstitial infiltrate occurs, although the intratubular cells/tubulitis may remain, along with edema. Also, endarteritis may persist (although at lower levels of activity) in posttreatment biopsies suggesting an inadequate response to therapy and smoldering ongoing rejection. However, systematic studies of “postrejection treatment” protocol biopsies are lacking, and our overall understanding of the “morphologic” response to antirejection therapy and the significance of residual inflammatory cell infiltrates is incomplete.

FIGURE 29.28 The Banff types of rejection correlate well with graft survival (206). Type I does not significantly affect prognosis, but both type II and especially type III do diminish graft survival. This study was done before C4d stains were widely used or routine posttransplantation DSA testing was performed. A recent single-center observation suggests that C4d/DSA negative type II rejection has a favorable prognosis (similar to type 1 rejection) compared to cases with a concurrent antibody-mediated rejection component (153). (Adapted from Bates WD, Davies DR, Welsh K, et al. An evaluation of the Banff classification of early renal allograft biopsies and correlation with outcome. Nephrol Dial Transplant 1999;14(10):2364-2369.)

Certain pathologic features of acute cellular rejection have prognostic significance either individually or in combination. The most important predictors of outcome are the arterial lesions. Endarteritis, which defines type 2 rejection, has an adverse effect on prognosis, compared with tubulointerstitial rejection without arterial involvement. Several studies have demonstrated decreased survival or reversibility of type 2 rejection. Bates and colleagues studied the outcome in 293 patients with biopsies (206). Those with type 2 rejection had a 75% one-year graft survival versus greater than 90% among those with type 1, suspicious, or no rejection. Endarteritis was the only determinate in the Banff classification to predict graft failure (hazard ratio of 1.85) (391), similar to that in another series, in which endarteritis doubled the rate of graft loss (31% vs. 15%) (392). A large multicenter trial found that endarteritis increased the risk of a clinically severe rejection sixfold (61). Graft survival at 1 year (71% to 75%) versus (51% to 58%) (204) is higher for type 1 than type 2 rejection, and steroid resistance is more often found in the latter (196,393,394). Those with ≥25% of the luminal area involved (Banff type 2B) have a worse response to antirejection therapy and a twofold increased risk of graft loss, compared with those with endarteritis involving less than 25% of the luminal area (395). This is likely in part due to the presence of myofibroblasts in the inflamed intimal zones that can, in protracted cases, cause intimal sclerosis, that is, rejection-induced chronic sclerosing transplant vasculopathy (164,396,397). Arteriolitis is associated with endarteritis and has a similar adverse effect on prognosis (207). Type 3 rejection (necrotizing or transmural arteritis) has much worse prognosis than intimal type 2 endarteritis (20% to 32% 1-year survival) (196,206,264,394,398), in particular cases mediated by antibodies.

It is important to remember that most historic reports on outcome do not separate between acute TCMR and mixed cellular-and AMR episodes (mixed Banff category 2 and category 4 rejection) due to the lack of C4d staining results and data on circulating DSA titers in previous years. Thus, in old studies, the “mixed rejection cases” were mostly classified as acute TCMR and received, based on current standards, suboptimal therapy. Consequently, all historic outcome studies are nowadays difficult to interpret and need reevaluation (Fig. 29.28). A good example of increased insight is the lack of adverse outcome of endarteritis if C4d is negative (151) or donor specific antibodies are not present (153).

Infarction is an ominous finding in graft biopsies, if surgical trauma including malperfusion through small accessory arteries can be excluded (399,400). It is often caused by severe
rejection episodes with arterial thrombus formation and the presence of DSA/C4d positivity. Occasionally, infarction can be associated with infections, especially CMV or productive adenovirus (see below) (401). Old infarcts are occasionally found in well-functioning grafts, dating from the time of transplantation; these are of no significance (402).

The intensity of the interstitial infiltrate or tubulitis for that matter has no correlation with the severity of the rejection episode (55,61,196,372,393,398,399,403,404). Of note, Banff category 4 type 2 rejection with transplant endarteritis can lack any significant interstitial inflammation. Consequently, Banff category 3 so-called borderline/suspicious for rejection is a very problematic and controversially debated entity. Many, but not all, of these “borderline” cases are, indeed, rejection. Two large studies have shown that 75% to 88% of patients with suspicious/borderline changes and graft dysfunction at time of biopsy functionally improved with increased immunosuppression (405,406), comparable to the overall response rate in type 1 rejection (86%) (405). Untreated “borderline cases” can progress to frank rejection during follow-up (406,407). If there is any evidence that favors rejection (e.g., marked interstitial edema incompatible with “borderline” rejection, peritubular capillaritis, glomerulitis, C4d positivity, a rise in creatinine), a diagnosis of rejection should be made and therapy initiated. We find in “borderline cases” that the tubular expression of MHC class II/HLA-DR by immunofluorescence microscopy can be helpful to establish a definitive diagnosis of “rejection” (156).

The intensity of the inflammatory cell infiltrate lacks prognostic significance and so does the expression profile of CD3+ or CD2+ cells in the interstitium (170,334,408). In some studies, greater numbers of CD3- and CD2-expressing cells even had a better outcome than those with focal infiltrates. Thus, grading the rejection on the basis of the extent of the infiltrate is of dubious value. The proportion of CD8+ cells correlates with a poorer response to immunosuppressive therapy in some (157,170,301) but not all studies (372). By multivariate analysis, type 1 tubulointerstitial rejection with diffuse cortical CD8+ infiltrates was associated with a 46-fold increase in risk of graft loss within 10 weeks (301). Expression of granzyme B by greater than 2% or CD40 by greater than 25% of the infiltrating cells has been associated with shorter allograft survival (317). The reason for this is not clear; the CD8+ cells may be relatively resistant to immunosuppressive drugs or mediate more severe injury. Eosinophil-rich infiltrates (greater than 2%) have been associated with graft loss (86% vs. 37%) (194). One explanation may be the strong association of eosinophils with transplant endarteritis/type 2 rejection. An increased number of interstitial macrophages has also been associated with an adverse outcome (335). Plasma cell-rich acute rejection has been reported to have a poorer prognosis in most (188,189,190,191), but not all, series (192). When three studies (188,191,409) are combined in a meta-analysis, plasma cell infiltrates in acute rejection affect prognosis only in the first 6 months (increasing graft loss from 23% to 53%); after 6 months, the outcomes of acute rejection with or without plasma cells are equally poor (graft loss in 67% to 68%) (409). Some tubulointerstitial cellular type 1 rejection episodes are “rich” in CD20-positive B-cell clusters shown in some (184,187,410) but not all (180,411) studies to indicate poor outcome. Future trials have to determine whether monoclonal anti-CD20 antibody therapy, such as rituximab, may be of beneficial therapeutic value in CD20-rich rejection episodes (412,413).

The prognostic significance of transplant glomerulitis in pure TCMR has not been settled. Most studies report a poor prognosis, for example, 67% graft loss (157,158,159,161,165), likely due to the tight association of transplant glomerulitis and transplant endarteritis/type 2 rejection.


It is clear that single, severe acute rejection episodes or, more frequently, smoldering and recurrent rejection lead to chronic alloinjury with increased fibrosis and tissue remodeling termed chronic rejection. Chronic rejection is characterized by: sclerosing transplant arteriopathy without elastosis, transplant glomerulopathy, severe multilamination of peritubular capillary basement membranes in three PTC (≥7 circumferential layers in one and ≥5 layers in the remaining capillaries as defined by Liapis et al. (224)), and, to some extent, also by interstitial fibrosis and tubular atrophy (IFTA). If these lesions are detected in a graft biopsy, in particular in combination, then a diagnosis of chronic rejection can be rendered, and descriptive diagnostic terms such as “interstitial fibrosis and tubular atrophy” or the now outdated term “chronic allograft nephropathy” can be avoided. Chronic rejection is often associated with other “chronic” lesions such as hypertension-induced arterionephrosclerosis (Fig. 29.29), preexisting donor disease, or possibly CNI toxicity making the diagnostic workup of a late graft biopsy challenging for the pathologist.

The etiology of chronic rejection-induced changes in late graft biopsies, particularly if significant activity is lacking, is often difficult to determine: Were the lesions induced by cellular and/or antibody-induced injury? Interestingly, Liapis and colleagues showed that especially mixed chronic active cellular and concurrent AMR induced severe “chronic” changes to the microvasculature with marked multilaminations of peritubular capillary basement membranes. This feature carried a positive predictive value for chronic TCMR of 17% and for chronic AMR of 49% (negative predictive values for chronic rejection of any type, i.e., absence of severe PTC multilaminations, approximately 90%). In Liapis’ series of 40 cases with transplant glomerulopathy, 18% were interpreted as secondary to chronic TCMR, 10% as secondary to pure AMR, and 32% as mixed TCMR and concurrent AMR (224). Thus, both cellular and/or antibody-induced tissue injury and fibrosis are common.

Chronic rejection-induced changes are currently only imperfectly reflected in the Banff classification scheme, and strict defining criteria have to be established in the future. Although Banff recognizes the entity of chronic active rejection in categories 2 and 4, “burnt-out” inactive chronic rejection remains unclassified. Careful chart review and correlation with historic data is required to reach the most appropriate diagnosis in late graft biopsies with chronic changes including evidence of chronic rejection.

One histologic feature of chronic rejection that is mainly but not exclusively driven by smoldering TCMR is so-called chronic active sclerosing transplant arteriopathy (see Fig. 29.29). It is characterized by intimal widening due to de novo accumulation of collagens I and III lacking elastosis and varying degrees of intimal inflammation with mononuclear inflammatory cells (ranging from absent to prominent). This peculiar form of intimal sclerosis can be most prominent at arterial branching points in arcuate
caliber vessels and is typically found as a sequela of transplant endarteritis (396). In sclerosing transplant arteriopathy, the intima usually contains varying numbers of myofibroblasts, occasional foam cells, and, in active disease stages, scattered, often clustered mononuclear inflammatory cells that can be most prominent along the inner elastic lamina. Potentially, even eosinophilic leukocytes play a role in the development of intimal sclerosis (414). Endothelial cells are often enlarged with reactive nuclei sometimes overlying an ill-defined ring of smooth muscle cells, that is, so-called neomedia formation (see Fig 29.29B and C). The inner elastic lamina usually remains intact without major breaks that are only prominent in cases with preceding transmural inflammation (Banff v3 lesions). The differential diagnosis of sclerosing arteriopathy includes TMAs (see Fig 29.27) and hypertension-induced arteriosclerosis characterized by marked arterial intimal fibroelastosis and a lack of intimal inflammation.

FIGURE 29.29 Chronic active TCMR with sclerosing transplant arteriopathy and lymphocytic infiltrates, C4d-negative, DSA-negative (Banff category 4 chronic TCMR). A: Five months after transplantation and 4.5 months after acute TCMR with transplant endarteritis (C4d-negative, DSA-negative; compare with Figure 29.15) that was superimposed on preexisting mild to moderate hypertension-induced donor-derived arteriosclerosis, a repeat biopsy shows an arcuate caliber artery with aggregates of inflammatory cells (I) in the widened intimal zone. Inflammation overlies donor-derived intimal elastosis (asterisk) and is located under a rim of chronic rejectioninduced de novo intimal sclerosis lacking elastosis (arrows). B, C: The subendothelial rim of chronic rejection-induced sclerosis shows on higher-power examination a vaguely organized pattern with a loose arrangement of myofibroblasts called “neomedia formation.” ((A) Elastic stain, ×200; (B) PAS, ×400; (C) trichrome, ×400; L, arterial lumen; M, medial smooth muscle layer.)

Since chronic tissue injury due to rejection, regardless whether T-cell and/or antibody mediated, shows specific changes in glomeruli, arteries, and PTC, detailed features are discussed for didactic purposes in the section “chronic antibody-mediated rejection” below.


AMR was first recognized in 1966 in the form of hyperacute rejection in patients with pretransplant DSA (33). In 1970, chronic rejection was linked to posttransplant DSA (32). Evidence of antibody binding to the graft was sparse, and the concept of acute and chronic AMR was not widely accepted until the 1990s. DSA, largely reactive to HLA antigens, are now recognized by pathologists and clinicians as a significant cause of early and late graft dysfunction and failure (415). The primary reasons for increased appreciation of AMR are the improved diagnosis afforded by detection of the complement fragment, C4d, and improved sensitivity and specificity of the solid-phase antibody assays. AMR arises in three major forms, hyperacute, acute, and chronic rejection (see Table 29.5). These and the known variants (C4d-negative AMR, smoldering AMR, and accommodation) are discussed in this section. General aspects of pathogenesis and diagnosis will be considered first; more specific aspects will be discussed with each category. The reader should bear in mind that AMR (acute or chronic) often coincides with TCMR (acute or chronic).



Class I and II HLA antigens are by far the most common targets of AMR, just as they are for TCMR. Production of HLA alloantibodies depends on exposure to HLA antigens from pregnancy, blood transfusion or transplantation; these antibodies are predominately IgG. Acute AMR is mediated by either class I or class II donor-reactive antibodies (DSA) (416,417,418), while the chronic form is largely associated with class II DSA (419,420,421,422). The microvasculature in the human, in contrast to the mouse, constitutively expresses class II MHC.

In addition, the nonclassical polymorphic MHC antigen MICA (MHC class I-related chain A) is a potential endothelial target. MICA is induced on the endothelium and other cells under conditions of cellular stress and is a ligand for the NK receptor NKG2D. The glomerular capillary wall is the main site of MICA localization in normal and rejecting kidneys, rather than PTC (423). Preexisting antibodies to MICA can be detected in 7% to 10% of renal allograft recipients unrelated to pregnancy or previous transplant and are associated with acute rejection and lower graft survival (424,425,426). However, the donor specificity and mechanism of rejection is not clear. Three cases of acute AMR have been attributed to MICA antibodies in the absence of HLA DSA (427,428).


ABO blood group antigens are the best characterized of the non-MHC polymorphic endothelial target in renal transplantation. Other antigens are potentially important, as evidenced by the occasional HLA-identical grafts that develop AMR (42,43) or hyperacute rejection (429,430) and the elution of alloantibodies from rejected HLA-identical kidneys (431). Antibodies to H-Y antigens have been detected in females with male kidney grafts and are associated with acute rejection and plasma cell infiltrates, although not C4d deposition (432). Most putative non-MHC antigens have not been characterized at the molecular level nor are approved diagnostic tests available (415).

Autoantigens may provide an additional target of AMR. Autoantibodies to angiotensin II type 1 receptor (AT1R) have been linked to malignant hypertension and graft dysfunction, fibrinoid necrosis of arteries, and acute rejection with C4d in 33% of the cases (433). AT1R antibodies do not affect vessels in native organs, suggesting a component of rejection is necessary. AT1R antibodies were detected in 86% (6/7) of patients with acute AMR and negative HLA DSA (434). Autoantibodies to glutathione S-transferase T1 have also been detected in acute AMR (428). De novo development of antibodies to unknown and probably nonpolymorphic antigen(s) on umbilical vein endothelial cells was associated with glomerulitis and capillaritis, but not C4d deposition (435). Autoantibodies to agrin, a proteoglycan component of the GBM, have been reported in patients with transplant glomerulopathy (436), as have autoantibodies to peroxisomal trans-2-enoyl-CoA-reductase (437). B cells derived from grafts with chronic AMR produce a variety of polyreactive autoantibodies (438), and protein microarray studies have revealed a wide variety of autoantibodies to normal renal urothelium and glomeruli in patients with allograft rejection (439). Their role, if any, is unproved.

The enigma of autoantibodies is how the graft would be selectively affected. A clue comes from experimental studies in rodents that proved endothelial damage in ischemic organs is mediated by natural IgM autoantibodies to externalized membrane components (440). Perhaps, the graft specificity of autoantibodies in humans is similarly due to increased expression or exposure of the antigen in tissue injured from other causes, for example, rejection or drug toxicity. However, studies exploring the role of these autoantibodies in nontransplanted but severely injured organ as a control (e.g., acute kidney failure) are lacking.

B Cells and Plasma Cells

High-affinity IgG alloantibody response requires CD4+ T-cell reactivity through the indirect pathway (441), particularly the T follicular helper cell (442). Activated T cells provide help for B-cell memory, isotype switching, and affinity maturation through various T-cell-derived cytokines and costimulatory factors that recognize receptors on B cells (such as CD80/CD86, CD40L, and ICOS). The B-cell response leads to the production of long-lived plasma cells, which migrate to the bone marrow and continue to produce antibodies indefinitely, without requiring T-cell help (443). It is not known whether antibodies specific for graft antigens are maintained owing to the longevity of plasma cells or to the continuous generation of new memory B, although alloantibody-secreting plasma cells are detectable in the bone marrow (444). B cells also provide antigen presentation and help for T cells, in part due to binding of antigen on B-cell surface immunoglobulin (445,446). IgM alloantibodies specific for MHC antigens and carbohydrate antigens (ABO) may not require T-cell help (447).


The testing for antibodies to MHC antigens has markedly evolved over the last decade from panels of living lymphocytes as targets by cytotoxicity or flow cytometry (panel reactive antibodies, PRA) to solid-phase assays with purified antigen on plates (ELISA) or fluorescent beads (Luminex) (415). The advantages of the solid phase are high sensitivity, high specificity, and lack of dependence on donor cells. Both class I and class II single-antigen beads are available. The disadvantages of the solid-phase assay are that the antigens are not on their natural surface and may not have their normal conformation (415). Indeed, normal individuals can have antibodies reacting to HLA-coated beads that do not react to living lymphocytes of the same HLA type (448). The solid-phase assays are FDA approved for determining the presence or absence of DSA, not for quantitation. Some of the factors that confound quantitation by using the mean fluorescence intensity (MFI) have been described and include variable coating of the antigens on the beads, batch differences, interlaboratory variation, saturation, and the difficulty in combining results across beads (415). Before transplantation, donor blood lymphocytes are tested with serum from the potential recipient for donor reactivity by cytotoxicity and/or flow cytometry (crossmatch). A “virtual” crossmatch is done with solid-phase beads with the putative donor HLA antigens. This can narrow down the list of potential recipients and reveal compatibility that would otherwise be missed (415).

Effector Mechanisms

There are three major pathways by which antibodies can affect the endothelium (Fig 29.30) (449).


Antibody to MHC acting alone in vitro on cultured endothelium promotes proliferation and activation of multiple signaling pathways (450). Studies by Reed et al. have shown that antibodies to MHC molecules on cultured endothelial cells elicit strong responses that include proliferation and activation of intracellular signaling pathways (451,452,453,454,455,456) and exocytosis of Weibel-Palade bodies with release of von Willebrand factor and P selectin (457). Among the changes noted was increased phosphorylation of ERK (pERK), indicative of increased ERK activity (458), a pathway that promotes cellular proliferation (459,460).

FIGURE 29.30 Three major pathways by which antibody can affect the endothelium. (1) Antibody to MHC acting alone in vitro on cultured endothelium promotes proliferation and activation of multiple signaling pathways. (2) Complement activation by DSA can attract inflammatory cells through C3a and C5a and cause lysis of endothelial cells (462) and expression of adhesion and procoagulant molecules. (3) DSA can potentially mediate endothelial injury and activation via Fc receptors on NK cells, monocytes, and granulocytes. (Reprinted from Farkash EA, Colvin RB. Pathology: Diagnostic challenges in chronic antibody-mediated rejection. Nat Rev Nephrol 2012;8(5):255-257.)


Complement activation by DSA at the surface of the endothelium can have profound effects via a variety of complement components. The pathways that lead to complement activation and C4d deposition are diagrammed in Figure 29.31 (461). Complement mediates acute graft injury by attracting inflammatory cells mainly through the chemoattractants C3a and C5a and by lysis of endothelial cells (462). C3a also promotes vasospasm through the release of PGE2 from macrophages, and C5a causes edema through the release of histamine from mast cells. Both cause endothelial cell release of IL-6, IL-8, IL-1α, and CCL5 and increased expression of adhesion molecules E-selectin, VCAM-1, and ICAM-1 (463,464). Membrane attack complex (MAC) causes lysis and apoptosis of endothelial cells, a process dependent on C6 (465,466). In sublytic concentrations, soluble MAC increases expression of adhesion molecules, such as E-selectin, ICAM-1, and VCAM-1 on cultured endothelial cells and synthesis and secretion of IL-8, MCP-1, CCL2, and CCL5 and triggers endothelial synthesis of tissue factor (467,468,469,470).


NK cells, monocytes, and granulocytes, via DSA and Fc receptors, can potentially mediate endothelial injury and activation (a process known as antibody-dependent cell-mediated cytotoxicity, ADCC) (471,472) and provide a third, relatively neglected, pathway of DSA-mediated graft injury. Complement-independent mechanisms may be relevant to the pathogenesis of AMR, particularly those cases with little or no C4d deposition. NK cells express the low-affinity FcγRIII (CD16), and monocytes express in addition the high-affinity FcγRI. Interaction of effector cells with target cells via Fc receptors and antibody can lead to lysis of the target and/or production of cytokines and chemokines such as IFN-γ, TNF-α, MCP-1, and others (473,474). Monocytes cultured with endothelial cells and DSA stimulate the production of MCP-1 and IL-6 (475). NK cells, via their Fc receptors, are necessary for hyperacute xenograft rejection mediated by non-complement-fixing anti-Gal antibodies (476). Depletion of NK cells with anti-asialo-GM1 prolongs survival of mouse to rat cardiac xenografts, arguing for their participation in acute AMR (477). Antibody-induced chronic allograft vasculopathy in the mouse also depends on NK cells and the Fc portion of the DSA and is independent of complement fixation (374).

Diagnostic Evaluation of Antibody Interaction With the Graft With C4d Stains

At present, C4d deposition in PTC is the most specific indicator in biopsies of the presence of circulating DSA and its interaction with endothelial cells in the graft (142,478,479).
In the older literature, about 85% to 90% of the patients with C4d+ had positive tests for circulating DSA, but these tests were less sensitive than current solid-phase assays. In recent protocol biopsies of presensitized patients, all biopsies with C4d had circulating DSA by solid-phase assay, arguing against the hypothesis that the kidney could absorb enough DSA to render this highly sensitive serologic test negative (480). The deposition of C4d in the absence of detectable DSA to MHC is strong presumptive evidence for non-MHC endothelial target antigens.

FIGURE 29.31 Pathways of C4 activation (389). Activation of C1 (composed of C1q, C1r, and C1s) is initiated by interaction of C1q with IgG or IgM bound to epitopes on the graft endothelium. C4 is cleaved by C1s into C4a and C4b, exposing a sulfhydryl group. The reactive sulfhydryl group of C4b rapidly forms an ester or amide bond with nearby molecules containing hydroxyl or amino groups. C4b combines with the enzymatically active fragment C2a to form C4bC2a, which is known as the classical pathway C3 convertase. C4bC2a cleaves C3 into C3a and C3b (which also has a reactive sulfhydryl group) and with the C3b molecule covalently deposited in the immediate vicinity and forms the C5 convertase C4bC2aC3b. Cleavage of C5 releases a bioactive peptide C5a and C5b. C5b initiates formation of the membrane attack complex (MAC; membrane-bound C5b-9), which causes cell lysis (435). The lectin pathway is stimulated when mannan-binding lectin (MBL), L-ficolin, or H-ficolin binds to the appropriate carbohydrate (typically on pathogens or apoptotic cells) (467). MBL binds to mannose (or glucosamine), and the ficolins bind to N-acetylglucosamine. L-ficolin also binds to elastin and lipoteichoic acid, and H-ficolin also binds to N-acetyl galactosamine. MBL, L-ficolin, and H-ficolin (all homologous to C1q and fibrinogen) activate C4 via their associated serine proteases, MASP-1 and MASP-2 (homologous to C1r and C1s). C4 is also activated via the binding of C-reactive protein (CRP) to the carbohydrate, phosphorylcholine with participation of C1q (1303). Little or no terminal components (C5b-9) are generated, because CRP simultaneously recruits Factor H, and may thus provide an anti-inflammatory effect. (From Rotman S, Collins AB, Colvin RB. C4d deposition in allografts: Current concepts and interpretation. Transplant Rev 2005;19:65.)

A positive C4d stain with the immunofluorescence technique (IF) was defined as “widespread, strong linear circumferential PTC staining in cortex or medulla, excluding scar or necrotic areas,” according to a consensus at the 2003 Banff Conference. For positivity with IHC on paraffin-embedded tissues, strong staining is not required, as tissue pretreatment influences staining intensity. At present, there is debate on the appropriate threshold for positivity in the Banff consensus; 2+ staining intensity (on a scale of 0-4) seems to be a reasonable threshold level for “C4d positivity” by IHC. The 2013 Banff consensus conference proposed that the threshold for C4d positivity should be >10% for IF (C4d2) and >0 for IHC (C4d1) (481). Early biopsies with even one C4d+ cluster of 3 or more PTC with acute rejection were associated with DSA (57%) and grafts failed by 1 year in 38%. The DSA rate was somewhat lower than in patients with biopsies with greater than 50% C4d+ capillaries (86% DSA), but graft failure of the latter was similar (31% at 1 year) (482). Late biopsies with focal C4d (10% to 50%) in paraffin sections had a worse graft survival compared with negative C4d biopsies (483). Among 368 biopsies, focal C4d in frozen sections (10% to 50%) was intermediate in graft survival compared to negative and diffusely positive C4d and intermediate in association with DSA (484). Taken together, it appears that even focal C4d is sufficient to diagnose AMR.

Several pitfalls of C4d staining must be mentioned. Arterial endothelial surfaces and thickened intima in arteriosclerosis and hyaline arteriolar deposits in native kidneys are often C4d-positive in frozen sections or, to a lesser degree, also by IHC. The mechanism is not known. Granular staining of PTC by IHC is of doubtful significance (485). Interpretation of glomerular staining in frozen sections is complicated by the normal presence of C4d in the mesangium, which is therefore not specific for AMR. Additional staining along the GBM occurs in acute AMR, but is difficult to score, and we do not use it as evidence of AMR. In fixed, paraffin-embedded tissues, however, normal glomeruli are entirely negative (486). This difference may be due to fixation blocking access to C4d embedded in the mesangial matrix or GBM (as opposed to cell surfaces). Granular glomerular C4d is typical in immune complex diseases (e.g., membranous glomerulonephritis).

IF in frozen sections remains the technique of choice (487), with the triple layer IF technique probably the most sensitive (488). The sensitivity of immunofluorescence using monoclonal antibody to C4d in frozen sections (IF) is greater than immunoperoxidase stains using polyclonal antibody in paraffin-embedded tissue (IHC) (388,487,488,489). IHC demonstrated a substantially lower prevalence (31% to 87%) and extent (36%)
of C4d deposition in PTC and had a lower reproducibility than IF (kappa 0.3 vs. 0.9, respectively) (487). Furthermore, there is considerable interinstitutional variability in IHC results, as shown by an international quality assurance project involving 73 centers (490). Some variation was attributed to techniques. Heat-induced epitope recovery (pH 6 to 7, 20 to 30 minutes, citrate buffer) with polyclonal antibody incubation (less than 1:80, greater than 40 minutes) appeared to be the best practice (490). Not uncommonly, the plasma in the capillaries is fixed by the formalin processing and also stains for C4d by IHC, which interferes with interpretation. Extravasation of C4d into the connective tissue is also common and should not be mistaken for capillary wall deposition. If extensive, these samples are not interpretable.

The sensitivity of C4d deposition for AMR is probably around 80%, as judged by patients who have other evidence of AMR, such as DSA with capillaritis or increased levels of endothelial gene expression (491,492). Capillaritis with DSA and little or no C4d is more common in “smoldering” and chronic AMR than acute AMR, although the frequency in the last category has not been reported.


Introduction and Clinical Presentation

Hyperacute rejection refers to immediate rejection of the kidney upon perfusion with recipient blood (typically within 60 minutes). Hyperacute rejection is a variant of acute AMR, in which donor-specific antibody titers are sufficient at the time of transplantation to cause immediate rejection. The graft rapidly becomes cyanotic and flaccid, despite good pulses at the hilum and swells poorly on venous compression (493). In the first 10 minutes, the graft sequesters platelets, neutrophils, complement, fibrinogen, and coagulation factors, and the level of circulating DSA decreases (494). The clinical signs are anuria, high fever, and no perfusion on renal scan. Microangiopathic hemolytic anemia with thrombocytopenia and increased circulating fibrin split products can develop and reverses on removal of the graft (495). Fortunately, hyperacute rejection is now rare, due to effective crossmatch screening, and is encountered in less than 0.1% of transplants (496).

Pathologic Changes


The kidney becomes livid, mottled, and cyanotic soon after reperfusion in the operating room (33,497,498,499). The kidney is initially flabby and soft, but subsequently swells and develops widespread hemorrhagic cortical necrosis, with medullary congestion (Fig. 29.32). The large vessels are sometimes thrombosed.

Light Microscopy

The pathologic features are the same as severe acute AMR (Fig. 29.33). Neutrophil and platelet margination occurs in the first hour along damaged endothelium of glomerular and PTC, and the capillaries fill with sludged, compacted red cells and fibrin (493). Neutrophils form “chain-like” figures within the PTC without obvious thrombi (493). The endothelium is stripped off the underlying basal lamina, and the interstitium becomes edematous and hemorrhagic. Intravascular coagulation occurs, and cortical necrosis ensues over 12 to 24 hours. The medulla is relatively spared, but is ultimately affected as the whole kidney becomes necrotic (33). Widespread microthrombi are usually found in the arterioles and glomeruli and can be detected even in totally necrotic samples. The larger arteries may be spared, but small arteries often also show neutrophilic infiltration or fibrinoid necrosis. Mononuclear cell infiltrates are typically sparse. One case showed CD3+ cells in the adventitia of small arteries and in the surrounding interstitium, probably indicating a component of TCMR (497).

FIGURE 29.32 Hyperacute rejection, nephrectomy. The cut surface of the markedly swollen kidney is grossly hemorrhagic and glistening with edema fluid (hence the reflections).

Immunofluorescence Microscopy and Immunohistochemistry

Fibrin, IgM, and C3 are occasionally quite prominent in the vascular and glomerular lesions (493,497). The nature of the antigen influences the distribution of the staining and the isotype of the antibody. ABO antibodies are primarily IgM and deposit in all vascular endothelia. Antibodies to HLA class I or II cause little or no IgG or IgM deposition in the microvasculature (500,501).

C4d is deposited in the PTC and glomeruli, as in acute AMR (see Fig. 29.33) and is more useful diagnostically than immunoglobulin deposition. Occasional cases biopsied at the time of operation may be negative for C4d (502), perhaps related to focally decreased perfusion, necrosis, or insufficient time to generate substantial amounts of C4d. Careful search for viable tissue, in particular in the medulla, can sometimes reveal areas of
C4d positivity along PTC. Lack of C4d deposition in one case of hyperacute rejection was attributed to non-complement-fixing donor-specific antiendothelial antibodies (502).

FIGURE 29.33 Hyperacute rejection due to preexisting antidonor class II HLA antibodies (509). A: Interstitial edema and hemorrhage are conspicuous as are neutrophils in PTC. Glomeruli are congested and have lost endothelial nuclei. (H&E, original magnification 200×.) B: C4d on paraffin section shows widespread, circumferential deposition along the PTC (stain done on an unstained paraffin section stored for 25 years). (C4d IHC, original magnification 400×.)

Electron Microscopy

Neutrophils are abundant in the glomerular and PTC (i.e., in the microcirculation), where they seem to attach to injured endothelial cells (493) (Fig. 29.34). Electron-dense deposits are rare or absent (493). The endothelium is swollen, separated from the GBM by a lucent space. Capillary loops and PTC are often bare of endothelium. Platelet, fibrin thrombi, and trapped erythrocytes occlude capillaries.

Etiology and Pathogenesis


ABO blood group antigens were the first identified target of hyperacute rejection (493,499). Eluates from the rejected kidney contain anti-ABO IgM or IgG antibodies (503). HLA class I (33,493,498,499,504) and class II (417,501) antigens can also be targets of hyperacute rejection, and DSA have been eluted from hyperacutely rejected kidneys (505). The rapid graft destruction in humans by anti-MHC antibodies contrasts with that in rodents and may be explained by the fact that normal murine endothelium has less class I and class II antigen expression and a less efficient complement system.

Occasional cases of hyperacute rejection still arise despite a negative lymphocyte crossmatch. These have been attributed to non-HLA antigens on endothelium (205,502,506). These recipients are typically multiparous females or recipients of prior transplants (497), and some have received HLA-identical kidneys (429,430). The nature of the antigen(s) has not been determined in most cases, even whether they are allospecific. In one instance, selective reactivity to donor endothelial cells in contrast to donor lymphocytes was demonstrated (502). Antibodies have been eluted from hyperacutely rejected kidneys that stain the endothelium of PTC and arterioles (507,508). Some of these cross-react with monocytes (508), and others do not (502,507). Testing of the pretransplant serum on the donor kidney sometimes shows binding of immunoglobulin to
the PTC in patients with antiendothelial antibodies (205,507). Preexisting donor kidney reactive antibodies were detected by immunoperoxidase techniques in 19% of 70 patients who had a negative T-cell cytotoxic crossmatch, and 50% reacted with endothelium (some also with epithelium) (509).

FIGURE 29.34 Hyperacute rejection due to anti-HLA-DR antibodies, biopsied at 24 hours after transplantation. Electron micrograph of glomerular capillaries shows fibrin platelet thrombi (thick arrows), a degranulated neutrophil (arrow), and compacted red cells; the endothelium is absent from most of the GBM. (×5000.)

Other antibodies include cold-reactive IgM agglutinins reactive with recipient red cells in kidneys that are not rewarmed before blood flow is reestablished (510,511). These cause immediate graft dysfunction due to intravascular aggregation of recipient erythrocytes and thrombosis, as described in five cases (512). Exogenous antibodies can also cause hyperacute rejection. Perfusion of the donor kidney with third-party human plasma-containing donor-reactive cytotoxic antibodies is a rare cause of hyperacute rejection (513,514), which provides proof that antibody alone is sufficient to initiate this injury, even a single exposure. ATG has been implicated in rare instances, in one of which the batch of rabbit ATG (Thymoglobulin) reacted with activated endothelial cells; no C4d was detected in PTC in that case (515). Equine ATG (Atgam) was also implicated in early acute AMR in two cases; these had C4d deposition in PTC and no DSA (515).

Effector Mechanisms

Hyperacute rejection is caused by binding of circulating antibodies to the surface of endothelial cells, complement fixation, platelet activation, lysis of the endothelium, and activation of the clotting system with thrombosis. The sequence of events is similar, if not identical, to that in acute AMR, only developing more rapidly and vigorously, in a setting with no opportunity for the endothelium to develop resistance (accommodation).

Renal allografts in monkeys presensitized to donor antigens develop a marked reduction in renal blood flow due to vasoconstriction, as the earliest and most abnormal finding (516). At 5 minutes, endothelial immunoglobulin and faint C3 deposits were detectable but never became prominent; fibrin formation was sparse at all times. Glomeruli were the most sensitive, and arterial injury became more prominent at higher antibody titers. Early red cell sequestration and stasis were marked, followed by progressive aggregation of platelets and infiltration of neutrophils. Renal venous studies revealed marked consumption of C3 but no evidence of intrarenal activation of the coagulation, fibrinolytic, or kinin-forming systems. Platelet aggregates in glomeruli and arteries and IgM deposition on the surface of glomerular endothelial cells were beautifully demonstrated by electron microscopy in hyperacute rejection in rabbits (517). Increased expression by endothelial cells of leukocyte adhesion molecules CD31 (PECAM-1) and CD62E (E-selectin); increased production of tissue factor, PAI, and platelet-activating factor; and decreased thrombomodulin also occur (517,518). A similar sequence occurs in discordant xenografts (i.e., those in which the recipient has preformed “natural” IgM antibodies) (519).

In general, only the complement-fixing antibodies mediate hyperacute rejection. IgG3 (4% of circulating IgG) and IgM are better complement-fixing antibodies than IgG1 (65% of circulating IgG) or IgG2 (25% of IgG); IgG4 (5% of IgG) does not fix complement. Sera that are positive in microcytotoxicity assays (complement fixation required) contain predominantly IgG3 with or without other IgG isotypes; sera negative by microcytotoxicity but positive by flow cytometry (only antigen binding required) contain predominately IgG2 and IgG4. About 80% of patients with high titers of antidonor cytotoxic antibodies in pretransplant crossmatch tests reject their kidney hyperacutely (520). IgM antibodies to HLA antigens curiously do not always trigger hyperacute rejection; only about half of those with IgM anti-class I antibodies have hyperacute rejection. One reason may be low affinity, as some only react in the cold or dissociate after multiple washes. IgA antibodies have not been associated with hyperacute rejection.

Hyperacute rejection does rarely occur in the absence of demonstrable antidonor antibody, presumably due to primed cytotoxic T cells present in the circulation at the time of transplantation. Such a phenomenon has been described in presensitized pigs, which reject renal allografts hyperacutely, but have no detectable humoral antibody (521). The first visible lesion within 30 minutes consists of lymphocytes attached to the arterial endothelium; after a few hours, the graft develops florid mononuclear infiltrate and necrosis. T-cell-mediated hyperacute rejection of mouse heart allografts has also been described in the absence of preexisting donor-reactive antibodies (522). Hyperacute rejection was occasionally reversed in humans by anti-T-cell antibody (OKT3), arguing for a T-cell-mediated component in rare cases (497).

Clinical Course, Prognosis, Therapy, and Clinicopathologic Correlations

Removal of the necrotic graft is often necessary to prevent the development of systemic toxicity. Recovery is extraordinarily rare, but has been reported (497,524). In one case, a follow-up biopsy at 30 days showed resolution of the glomerular thrombi (497). In another case, transplant glomerulopathy was evident at 39 days posttransplant (524).

Preventive desensitization protocols are now being tried that involve various combinations of plasmapheresis, IVIG, rituximab, and immunosuppressive drugs (525,526). New drugs that block complement activation, such as eculizumab, are under evaluation (527). Splenectomy is also added in some protocols and immunoabsorption with antigen (ABO) or protein A columns (528,529). If the titer of antibodies diminishes to low or undetectable, transplantation has been safely undertaken, even though antibodies were previously present (525,530). In some patients, the antibodies return with either
an episode of acute rejection or no immediate effect on graft function (accommodation). The long-term outcome of these recipients is unknown but of great interest.



Acute antibody-mediated rejection (acute AMR or acute humoral rejection) became a well-defined diagnostic category in 2003 (82). Acute AMR occurs in patients who either develop a threshold level of antidonor antibodies after transplantation or were presensitized and transplanted after desensitization. The histologic features of acute AMR are not absolutely specific and quite variable and thus not sufficient alone for definitive diagnosis (141,150,219,531). Detection of C4d in graft endothelium and the new solid-phase methods for detecting antidonor antibody have led to better diagnosis of this condition.

Halloran and colleagues described a short-term worse outcome in patients who developed acute rejection in the presence of circulating antibodies to donor HLA class I antigens (416,500). Certain morphologic features (neutrophils in PTC, thrombi, fibrinoid necrosis) were more common in patients with anti-HLA antibodies, but no histologic feature was a specific or sensitive indicator of circulating DSA (169). Furthermore, deposition of immunoglobulin or C3 was not conspicuous in these cases. The first diagnostically useful immunologic marker of AMR was identified by Feucht in the early 1990s, who reported that deposition of complement split fragments C4d and C3d in PTC could be detected in the majority of transplanted kidneys with “cell-mediated rejection” (532,533). C4d deposition was associated with “high immunologic risk” (i.e., previous transplants or high levels of PRA) and with a poor prognosis. They suggested that humoral rejection should be considered, despite negative crossmatch before transplantation and paucity of immunoglobulin deposition. The C4d deposition in PTC, circulating DSA, and neutrophils in capillaries were later documented as the diagnostic triad for acute AMR (387,418,534). However, peritubular capillaritis rich in mononuclear cell elements (macrophages, lymphocytes) rather than neutrophils is common in acute AMR, and the lack of neutrophils or even PT capillaritis does not argue against a diagnosis of acute AMR. On the other hand PT capillaritis, in particular in early graft biopsies, can occur in the absence of acute AMR (478). These observations have been confirmed in many centers, and the criteria are now widely accepted Banff consensus (82,461). As outlined above, the reader should also be aware that AMR often concurs with TCMR with overlapping histologic changes.

Prevalence, Clinical Presentation, and Risk Factors

The clinical presentation of acute AMR is generally that of severe rejection, with more frequent oliguria (35% vs. 10% without antibodies) and need of dialysis (40% vs. 10%), compared with rejection in the absence of HLA class I antibodies (416). However, there is no clinical feature that permits distinction from pure T-cell-mediated acute rejection. Acute AMR is most common 1 to 3 weeks after transplantation in particular in desensitized patients, but can develop suddenly at any time (150). In one series, the mean day of onset was 15 ± 11 days (earliest 3 days), not different from that of acute cellular rejection (14 ± 10; earliest 6 days) (387). The latest case reported was 30 years after transplant (535). Late onset is often associated with iatrogenic or patient-initiated decreased immunosuppression (536,537).

TABLE 29.5 Conditions caused by antibody-induced graft injurya

Major Forms

Hyperacute rejection

Acute antibody-mediated rejection

Chronic antibody-mediated rejection


Smoldering/indolent antibody-mediated rejectionb


aMost of these have C4d deposition in PTC and circulating DSA to HLA antigens. Variants with DSA but little or no C4d (≤C4d 1 by IF and C4d 0 by IHC) have also been described, particularly for the chronic AMR and smoldering AMR. These are indicated as “C4d-negative” added to the diagnostic category, for example, C4d-negative chronic AMR.
Antibody induced graft injury, especially acute, smoldering and chronic antibody-mediated rejection, can show concurrent T-cell mediated rejection (acute and/or chronic).

b “Smoldering” refers to biopsies with mononuclear cell capillaritis and/or glomerulitis without chronic or acute changes.

c “Accommodation” is defined here as C4d deposition in PTC or DSA without evidence of active rejection.

The primary risk factor for acute AMR is presensitization (blood transfusion, pregnancy, prior transplant) as judged by a historical positive crossmatch or high levels of PRA (538). About 24% of biopsies for acute rejection meet the criteria for acute AMR (either solely mediated by antibodies or with a concurrent T-cell component) (Table 29.5). The overall frequency of acute AMR in transplant recipients is about 6%. However, among presensitized patients with DSA, the frequency increases to 28% (range 8% to 43% among 11 centers) (539). Some crossmatch-negative patients in the past with acute AMR would be reclassified as presensitized with the current more sensitive DSA assays (540). The risk of acute AMR is increased if the pretransplant sera DSA fix complement in vitro as judged by C1q or C4d deposition on Luminex beads (541) or if the levels of DSA are higher as judged by the mean fluorescence channel in solid-phase assays (540,542). Acute AMR occurs with all traditional drug regimens, even in protocols that cause profound T-cell depletion (538,543,544). Experience with the newer agents, such as belatacept, is still limited, but there is a suggestion that alemtuzumab (anti-CD52) induction is associated with a higher frequency of acute AMR (543,545).

Pathologic Findings

Gross Pathology

The kidneys are swollen and congested; in severe cases, widespread hemorrhage and patchy infarction are present (Fig. 29.35).

FIGURE 29.35 Gross appearance of an allograft with severe acute AMR 16 days after transplantation that failed after initially functioning for several days; ABOi graft (A2 into O).

Light Microscopy

Acute AMR has been divided into three types based on light microscopy (82,387): type I, acute tubular injury; type II, microcirculation inflammation with neutrophils and mononuclear cells in capillaries; and type III, fibrinoid necrosis of arteries (82). Acute AMR commonly is accompanied by features of TCMR, that is, these cases are classified as mixed acute TCMR and AMR, but AMR can occur in isolation. Acute AMR in late biopsies is typically superimposed on chronic AMR or TCMR (546).


Glomerular capillaries have neutrophils in 10% to 55% of cases (169,387,388,416) and mononuclear glomerulitis in 19% to 90% (150,169,387,388) (Fig. 29.36). Intraglomerular mononuclear cells are mostly monocytes/macrophages (CD68+) (389,547), in contrast to T-cell predominance in acute TCMR (168). Fibrin thrombi are present in about 20% of cases (169,387,416).


Evidence of acute tubular injury is common (loss of brush borders, thinning of cytoplasm, paucity of nuclei); in one series, these were found in 75% of cases (387). Indeed, acute tubular injury may be the only manifestation of acute AMR (Fig. 29.37) (387). Focal coagulative necrosis of tubules can be found in a minority of cases (Fig. 29.38) (387). Neutrophilic tubulitis is found in rare cases of acute AMR (Fig. 29.39) (387). Mononuclear tubulitis is seen in 30% to 80% of cases and is considered evidence of a concurrent T-cell-mediated component (150,169,387,388), as is increased expression of HLA-DR (142,531).


In cases with pure acute AMR, edema with a scant mononuclear infiltrate may be present in the interstitium, insufficient for the diagnosis of (concurrent) acute TCMR following current Banff criteria. In one series, the majority fell within the Banff “suspicious/borderline” range (Banff category 3) (388). Whether this represents a component of TCMR or is caused by the antibody/complement interaction with the tissue is not known. Interstitial hemorrhage and edema can be prominent, but is not necessarily indicative of an antibody component (387,409). Frank cortical infarction is present in a minority of cases (5%) (387). B cells can be present in aggregates, and plasma cells can be detected (Figs. 29.40 and 29.41); these latter features are not diagnostic for acute AMR and can also be seen in TCMR.


Trpkov et al. (169) pointed out the association of neutrophils in PTC with class I DSA, a feature long recognized in hyperacute rejection (Fig. 29.42). In three series totaling 78 cases, 54% had neutrophils in PTC (169,387,548). However, neutrophils were rarely (less than 3%) found in a series from Vienna (388). Mononuclear cells, especially monocytes/macrophages, are also present in PTC. The PTC are often markedly dilated (169). Capillaritis can be present with little or no C4d deposition, which may follow a few days later (549).

In about 25% of cases, the arterial media shows myocyte necrosis, fragmentation of elastica, and accumulation of brightly eosinophilic material called “fibrinoid” with little mononuclear infiltrate in the intima or adventitia (Fig. 29.43). This lesion is not dissimilar to microscopic polyangiitis. Among the patients with anti-class I antibody, 25% had fibrinoid necrosis (vs. 5% of those without such antibodies) (169). Another study noted 53% of 17 patients had either fibrinoid necrosis (24%) or transmural arterial inflammation (18%), or both (12%) (550). Arterial thrombosis is uncommon. However, acute AMR may also manifest as TMA, with mucoid intima thickening and trapped red cells. These cases have a higher risk of graft loss than those without TMA (551). Epidemiologic evidence argues that endarteritis with mononuclear cells in the arterial intima may in some cases be mediated by antibody (153), a feature also noted in experimental studies with adoptive transfer of DSA (374). Approximately 40% to 50% of acute rejection episodes with transplant endarteritis (many also with tubulo-interstitial cellular rejection) are C4d positive and have a component of concurrent AMR (142,150,151,153,532). Whether the cellular component of transplant endarteritis in AMR is different from that due to TCMR is not apparent.

Immunofluorescence Microscopy and Immunohistochemistry


Glomerular staining for C4d is considered nondiagnostic in frozen sections (Fig. 29.44). In fixed tissue stained by IHC, about 30% of cases have glomerular C4d (388) (Fig. 29.45A), while normal glomeruli do not stain (Fig 29.45B). No distinctive patterns are found by immunofluorescence for immunoglobulins
(387). Mesangial IgM and IgG may be more prominent than in non-AMR, but the difference (43% vs. 17%) is not diagnostically useful (169).

FIGURE 29.36 Acute AMR with glomerular inflammation (compare to Figure 29.7). A: Transplant glomerulitis with mononuclear cells, neutrophils, and reactive endothelial cells. An endothelial mitosis is shown (arrow, insert) C4d stain along PTC was positive. (PAS stain, original magnification 400×.) B: Transplant glomerulitis with neutrophils and thrombi. (H&E original magnification 20×.) C: Transplant glomerulitis in acute AMR with a few CD3+ cells (T cells). (Immunoperoxidase stain.) D: Same graft showing many CD68 cells (macrophages). The graft had prominent C4d deposition along PTC. (Immunoperoxidase stain.) (C, D courtesy of Alex Magil, Vancouver.)


C3 and C5b-9 (MAC) deposits have been reported primarily in tubular basement membranes, rather than PTC. The reason for this is not clear, but may relate to the ability of tubules to activate the alternative complement pathway (552). C5b-9 and C3 also are deposited in TCMR along the TBM. C4d is occasionally present segmentally along the TBM, in both acute AMR and TCMR, often in cases with tubular atrophy and TBM thickening.


Immunoglobulin is usually not demonstrable in PTC (150); however, in a small minority of anti-class I antibody cases, IgM and IgG may be detectable (169,387). IgM is usually present in ABO-incompatible (ABOi) grafts with acute AMR (553).

Intense immunofluorescence staining for C4d is usually detected in a widespread, uniform distribution in the PTC of the cortex and medulla (142,150,531,532,534) (Fig. 29.46). Focal deposition may also be found. In classic cases, at low power, the smaller oval and elongated ring-like fluorescent profiles of dilated PTC are readily evident between the larger, negative tubular cross sections although dilated PTC may mimic tubular cross sections by IF. The capillary staining is crisp, linear, and continuous, but also may have a finely granular pattern at high power, which extends into the lumen from the more linear deposits. Medullary vessels are typically positive and can be the only place of C4d positivity in some cases with marked edema and cortical injury (141,531). In IHC, C4d has a similar pattern, diffuse, linear, and circumferential in the PTC wall, although the intensity typically is less and variable (see Fig. 29.45). Intraluminal and interstitial C4d may also be seen, but is an artifact of fixation. C4d-negative cases of acute AMR have been described, although
the frequency is uncertain. In a series with serial biopsies of presensitized patients, C4d became positive later after the capillaritis had been present for several days (549).

FIGURE 29.37 Acute AMR present on day 9 of DGF. A: Pattern of acute tubular injury, without evidence of inflammation. (H&E stain, original magnification 400×.) B: Immunofluorescence C4d stain on cryostat section shows widespread staining of PTC. (Original magnification, 400×.)

FIGURE 29.38 Acute AMR with glomerular and tubular necrosis. (H&E original magnification 200×.)

C4d is present in most PTC, even those that lack endothelial cell markers, and colocalizes with anti-type IV collagen and endothelial cells in frozen tissue (531,534). This location fits with the known ability of C4b to crosslink to nearby proteins at the site of complement activation. The covalent linkage of C4d to structural proteins may explain why C4d remains for several days after alloantibody disappears, since antibody binds to cell surface antigens that can be lost by modulation, shedding, or cell death. C4d can be detected on the surface of the endothelial cells and in intracytoplasmic vesicles by immunoelectron microscopy (554). Reduced CD34 expression has been described as well as platelet fragments that stain for CD61 (also found in acute TCMR) (555).

Protocol biopsies have shown that C4d deposition can precede histologic evidence of acute AMR. Haas et al. (118) found focal or diffuse C4d staining in two of eighty-two 1-hour biopsies taken at the time of transplantation after reperfusion. Both patients later developed an acute AMR(days 5 and 34) (118). The recipients had been treated with plasmapheresis before transplantation because of a positive crossmatch and had a weakly positive

flow crossmatch at the time of transplantation (118). In 1-week protocol kidney biopsies, Sund showed PTC endothelial C4d deposition in 30% of cases; 33% of these did not meet histologic criteria of acute rejection, but 82% developed rejection during further follow-up. Koo reported C4d in 13% of 48 one-week protocol biopsies (556). C4d was present in 33% of samples with rejection and 3% of samples without rejection; all 5 with C4d and rejection had DSA (556). Outcome at 1 year was not affected by C4d status at 7 days, despite the lack of specific treatment.

FIGURE 29.39 Acute AMR (A) Widespread interstitial hemorrhage is present. B: Neutrophils are in tubules, resembling acute pyelonephritis. (H&E, original magnification 400× (A) and 200× (B).)

FIGURE 29.40 Plasma cell-rich late acute TCMR, C4d-positive (compare to Figure 29.13). The combination of plasma cell infiltrates, that is, acute plasma cell-rich TCMR and acute AMR, is a very rare occurrence that seems to carry a poor prognosis. (H&E original magnification 400×.)

FIGURE 29.41 Acute AMR with a nodule of B cells, a finding that has been reported by some to carry an adverse prognostic significance. At present, there is no linkage between B cells in the interstitial infiltrate and the presence of antidonor antibodies. (Immunohistochemical stain for CD20, original magnification 100×.)

FIGURE 29.42 Acute AMR with characteristic intraluminal PTC leukocytes. Primarily neutrophils (A) and primarily mononuclear (B) cells. (H&E (A) and PAS (B). Original magnification 400× (A) and 600× (B).)

FIGURE 29.43 Acute AMR (A) Fibrinoid arterial necrosis (Banff type III). Neutrophils and fibrin are seen in the wall of the arcuate sized artery. Nephrectomy specimen. (C4d stain-positive. H&E original magnification 200×.) B: Mucoid intimal thickening resembling TMA. Patient had anti-class I antibodies (134). Red cell fragment in intima (arrow). (H&E, original magnification unknown.) (Courtesy of Kim Solez.)

FIGURE 29.44 Normal kidneys stained for C4d using a monoclonal antibody cryostatin cryostat sections. A: Negative PTC and faint staining along the TBM. B: Prominent mesangial deposits are present in normal glomeruli. (Original magnifications 400×.)

The precise time course of C4d clearance and relationship to DSA levels has not been documented in humans. C4d deposition disappears a few days after treatment, provided the antibody disappears, as judged by sporadic repeat biopsies and experimental studies. Loss of C4d has been observed in as early
as 7 to 8 days after a positive biopsy (150) and in rat heart transplants in 5 days (557).

FIGURE 29.45 Paraffin sections stained using a polyclonal anti-C4d (347). A: Acute AMR showing widespread, circumferential deposits in characteristically dilated PTC containing leukocytes. The glomerular capillary walls also stain prominently. B: Normal kidney stained with the same technique demonstrates the absence of C4d in the glomeruli in paraffin sections (compare with Fig. 28-45B). (Original magnification 200×.)

C3d, produced by the classical pathway after C4b, has been suggested as an indicator of more complete complement activation. However, the normally high background of C3 deposition in the tubular basement membranes makes C3d much harder to interpret than C4d (558). Two series found C3d in 44% to 60% cases of C4d+ acute AMR (534); a minority had C3d without C4d. In other studies, C3d in PTC was not associated with neutrophils in PTC (548,558) or DSA (548).


Fibrinoid necrosis in arteries usually stain for IgG and/or IgM, C3, C4d, and fibrin (217,534). Nondiagnostic C4d deposition occurs along endothelial surfaces of arteries and arterioles, in the thickened intima of arteries and in arteriolar hyaline, whether or not acute AMR is present; this staining pattern is seen also in native kidneys.

FIGURE 29.46 Acute AMR, C4d stain using monoclonal antibody to C4d in cryostat sections. Bright widespread, circumferential staining is present in (A) and (B). A: Has a slightly granular appearance, and (B) is purely linear. Both patients had antibodies to donor HLA antigens. (Original magnification 400×.)

Electron Microscopy


The appearance may resemble TMA, with platelets, fibrin, and neutrophils in glomerular capillaries (Fig. 29.47). The glomerular endothelium is reactive with loss of fenestrations. Endothelial cell swelling (88%), separated from the GBM by a widened lucent space (100%) and early GBM duplication (76%), was present in C4d+ biopsies within the first 3 months in a series mostly of presensitized patients (559). Similar ultrastructural changes were seen with little or no C4d deposition in patients with DSA and glomerulitis/capillaritis. These changes are more evident in allografts that later develop transplant glomerulopathy (220,560).

FIGURE 29.47 Acute AMR. A: Neutrophils are in PTC whose endothelium shows the subtle changes of injury (loss of fenestrations). B: A glomerulus has one capillary plugged with fibrin (arrows) and another filled with compacted red cells surrounded by a reactive endothelial cell (C ); a third loop has a few platelets (arrowheads). (Electron micrographs. (A) ×3,700; (B) ×3,300.)


Neutrophils and monocytes are found in PTC with platelets, and fibrin (560,561). Interstitial edema and red cell extravasation can be found. Intact platelets are few, but microvesicles presumably derived from platelets are common (555). Endothelial cells show swelling, detachment, and expansion of the subendothelial space with electron-lucent “fluffy” material and sometimes trapped red cells (561). Lysis, apoptosis, and fragmentation of endothelial cells are evident (560). These changes are more severe and extensive than the endothelial swelling and apoptosis that occurs in ischemic renal injury (560). Apparent new capillary sprouts have been illustrated (560). After 2 to 4 weeks, the endothelial cells show cytoplasmic processes extending into the lumen and early multilayering of the basement membrane (560,561). Liapis et al. (224) showed in approximately 30% of late biopsies with acute AMR or with mixed acute TCMR and AMR evidence of severe PTC multilamination, that is, ultrastructural evidence of chronic rejection (as judged by ≥7 circumferential layer in one PTC and greater than five layers in two additional PTC). These observations illustrate that electron microscopy can uncover signs of chronic rejection before they become more prominent and detectable by standard light microscopy. In severe cases, after 2 to 3 months, some capillaries are completely destroyed, with disappearance of the endothelial lining and remnants of the basement membrane; those that remain have a thickened, multilayered basement membrane (561).


The small arteries with fibrinoid necrosis show marked endothelial injury and loss, smooth muscle necrosis and deposition of fibrin tactoids.


Most cases of acute AMR have detectable DSA to donor class I and/or II antigens (543,550). DSA react with MHC expressed on donor endothelium, particularly in peritubular and glomerular capillaries, and sometimes target arteries and arterioles. Whether other donor antigen-expressing cells in the allograft can be targeted (such as epithelial cells, smooth muscle) is unknown. A small minority (less than 5%) have evidence of reactivity to non-MHC or other endothelial antigens (567), for example, the rarely reported acute AMR in HLA-identical sibling grafts (42,43). The nature of these antigens is unknown, with the exception of autoantibodies to the angiotensin 1 receptor (567). These autoantibodies may act by amplifying the damage initiated by MHC antibodies, as shown for AT1R antibodies experimentally (433).

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Jun 21, 2016 | Posted by in UROLOGY | Comments Off on Renal Transplant Pathology

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