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!

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.

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.

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).

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 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 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.

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).

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.

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).

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.

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.

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).

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

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.

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.

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).

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.

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).

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).

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).