Outline
Overview of T Cells in Kidney Diseases 141
Glomerulonephritis 143
Acute Kidney Injury 144
Ischemic Acute Kidney Injury 144
Sepsis-induced Acute Kidney Injury 146
Nephrotoxic Acute Kidney Injury 146
Kidney Transplantation 146
Conclusion 147
Distinct T-cell subsets play individual roles during kidney injury and repair depending on the type of kidney disease. Understanding of the pathogenic mechanisms of different kidney diseases provide a basis for future studies investigating the contribution of T cells in the regeneration process. Regeneration studies should cover three structural sequelae of injured kidneys: glomerulosclerosis, tubular atrophy and tubulointerstitial fibrosis. Promoting the regeneration process to restore structural and functional integrity in diseased kidneys is as important as reducing damage during the early injury phase, and more frequently required in most kidney diseases since most patients start receiving diagnostic workup and treatment when they already have established disease. Future studies are required to elucidate the precise roles of T-cell subsets during the repair phase of kidney diseases and to harness these findings to develop novel therapies to enhance regeneration in the injured kidney.
Overview of T Cells in Kidney Diseases
T cells are key participants in immune responses occurring in many different kidney diseases. The injured kidney not only is the target of the immune system but also actively participates in aggravating or suppressing intrarenal immune responses. Ischemic acute kidney injury (AKI) is often simulated by animal models of ischemia–reperfusion injury (IRI). Postischemic kidneys per se contribute to immune responses by recruiting inflammatory cells including T cells and other leukocyte subsets, and generating proinflammatory cytokines and chemokines ( Fig. 8.1 ) . Chemokines involved in the recruitment of mononuclear cells, such as CXCR3, stromal cell-derived factor (SDF)-1/CXCL12 and monocyte chemotactic protein-1 (MCP-1)/CCL2, have been directly implicated in the pathogenesis of renal injury after IRI . Postischemic kidneys also recruit leukocytes by upregulating the quantity and avidity of adhesion molecules, and a series of steps occur which in turn increases microvascular permeability. Anti-intercellular adhesion molecule-1 (ICAM-1) antibody was shown to protect normal mice from renal IRI, and many studies have demonstrated similar findings in different experimental models . IRI was shown to increase intrarenal vascular permeability and facilitate extravasation of leukocytes by interrupting the integrity of the renal vascular endothelium . T cells were suggested to contribute directly to the increased renal vascular permeability after IRI, potentially through T-cell cytokine production, since the production of tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ) was increased in CD3 and CD4 T cells from blood and kidney after ischemia and since renal vascular permeability measured with extravasation of Evans blue dye was attenuated in CD3 T-cell-deficient mice after IRI .
Advances in T-cell biology have fostered better understanding of the role of different T-cell subsets in kidney diseases . Among several subpopulations of T cells, regulatory T cells have been suspected to have a potential role in the regenerative processes in diseased kidneys. It is important to review some background about these fascinating cells. Regulatory T cells were first identified about 30 years ago as “suppressor” T cells capable of suppressing antigen (Ag)-specific responses and transferring tolerance in animal models . The current characteristics of regulatory T cells expressing CD4 and CD25 [the α-chain of interleukin-2 (IL-2) receptor] were first elucidated by Sakaguchi et al. . This study demonstrated the lack of CD4 + CD25 + T cells in neonatally thymectomized mice that developed a fatal autoimmune disease. Transfer of CD4 + CD25 + T cells from normal to thymectomized mice prevented the development of autoimmune disease in thymectomized mice. Nuclear transcription factor Foxp3 (forkhead box P3) was reported as the master gene of CD4 + CD25 + T cells regulating the development and function of these cells . Differentiation of most CD4 + CD25 + T cells occurs in thymus after Foxp3 induction in few thymocytes with high affinity to self-Ag–major histocompatibility complex (MHC), and these CD4 + CD25 + T cells are called naturally occurring regulatory T cells (nTregs), while their peripheral counterparts are called inducible Tregs (iTregs) . Transfection of Foxp3 in CD4 + CD25 − T cells was reported to convert them into cells demonstrating suppressor capacity and overexpression of Foxp3 in transgenic mice induced suppressor capacity in CD4 + CD25 − and CD4 − CD8 + T cells . However, the importance of Foxp3 as the key factor of Tregs at present is questionable because expression of Foxp3 is induced after TCR stimulation without Treg development and Foxp3 is restricted to Tregs in mice, but not in humans, in which Foxp3 expression occurs in recently activated naïve T cells as well as in memory T cells . Although no or low expression of CD127 (IL-7) and the presence of CD27 as a surface marker have been reported as characteristics of Tregs, these markers are yet to be validated . There is another subpopulation of CD4 T cells with suppressor capacity called Tr1 cells. Tr1 cells are known to be generated in periphery from naïve CD4 + CD25 − T cells on Ag stimulation with limited costimulation conditions by induction of IL-10 and immature dendritic cells . Tr1 cells inhibit naïve and memory T cell responses by secreting suppressor cytokines including IL-10 and transforming growth factor-β (TGF-β), and migrate to inflammation sites, whereas nTregs basically migrate to lymph nodes. Another subset of iTregs is T H 3 cells, which share several characteristics with Tr1 cells and secrete TGF-β . No specific surface marker has been reported regarding Tr1 and T H 3 cells.
Regulatory T cells expressing CD4 and CD25 (Tregs), the best known subset of regulatory cells, were expected to participate in reducing damage and enhance repair in diseased kidneys based on their regulatory effect found in typical immunological diseases such as autoimmune disease and transplantation . Tregs have been reported to prevent organ-specific autoimmunity and modulate allogeneic immune responses inducing graft tolerance in transplantation, in both experimental and clinical settings . Recently, Tregs have been under active investigation in several kidney diseases.
This chapter will discuss the diverse role of T cells in immune responses that occur in several kidney diseases and focus on the regulatory effect of T-cell subsets contributing to the regenerative processes. Kidney diseases in which T cells are implicated in the pathophysiology can be categorized into three different diseases: glomerulonephritis, acute kidney injury (AKI), and transplantation. The role of T cells will be discussed in more detail for each disease.
Glomerulonephritis
Glomerulonephritis remains a common cause of chronic kidney disease (CKD) and end-stage renal disease (ESRD). Severe acute or persistent chronic inflammation may result in glomerulosclerosis, tubular atrophy and interstitial fibrosis with deterioration of renal function. The reversal of scarring and the restoration of normal glomerular structure and integrity are required for renal repair following glomerulonephritis. It is notable that severe renal inflammation may be completely resolved spontaneously or by immunosuppressive and conservative treatments in most cases of poststreptococcal glomerulonephritis (PSGN) and in some cases of minimal change disease (MCD). However, other types of glomerulonephritis usually leave different forms of sequelae resulting in renal functional and structural deterioration. Glomerulonephritis models can be classified into two different categories according to a traditional concept of pathophysiology: “immune”-mediated and “non-immune”-mediated glomerulonephritis models.
Immune-mediated Glomerulonephritis
Immune-mediated glomerulonephritis models can be divided further into three different entities: Heymann’s nephritis, antiglomerular basement membrane (GBM) disease and lupus nephritis. In Heymann’s nephritis (a rat model of membranous nephropathy), T cells infiltrate in glomerulus and interstitium. Monoclonal antibody treatment against T-cell receptor (TCR) α/β, CD4 and CD8 delayed onset of proteinuria, totally prevented proteinuria and markedly reduced proteinuria, respectively . Permanent CD8 T-cell depletion was reported to prevent proteinuria in active Heymann’s nephritis, suggesting the essential role of CD8 T cells in the development of glomerular injury in Heymann’s nephritis .
Tregs were shown to be protective in an animal model of anti-GBM disease. A recent study performing Treg transfer before induction of anti-GBM disease with rabbit antimouse GBM antibody showed markedly reduced functional and structural renal injury in the Treg transfer group . In this report, green fluorescent protein (GFP)-labeled Tregs demonstrated localization of transferred Tregs in the renal-draining lymph nodes and spleen. However, immune complex formation in the glomeruli was not reduced in Treg-treated mice. These findings suggest that Tregs reduce end-organ damage or enhance repair by limiting kidney-specific immune cell activation within regional lymph nodes. There have been studies reporting the existence of Ag (type IV collagen)-specific Tregs in Goodpasture’s syndrome patients. Collagen-specific T cells were proinflammatory during the active phase of the disease, but were regulatory when disease became convalescent . The subset of T cells with regulatory function was identified as an IL-10-producing population of CD4 T cells .
Studies have shown that Tregs are implicated in the pathogenesis of lupus nephritis . Foxp3-transduced T cells suppressed the production of autoantibodies in autoimmune-prone CD40L transgenic mice . However, adoptive transfer of Tregs did not suppress the development of lupus nephritis despite inhibiting autoantibody production in lupus-prone mice, implying the existence of a specific population of Tregs involved in lupus nephritis . Late-onset treatment with a CCR1 antagonist that inhibits T-cell infiltration prevented progression of lupus nephritis in a murine lupus model, suggesting a role for T cells in the later phase of lupus nephritis .
Non-immune-mediated Glomerulonephritis
Non-immune-mediated glomerulonephritis models include adriamycin nephropathy (AN) and glomerular compromise in mercuric chloride (HgCl 2 )-induced nephrotoxicity.
The important pathogenic mechanisms of AN (a rodent model of focal segmental glomerulosclerosis) were shown to include immune responses, unlike its nomenclature. Several studies have reported that T cells are an important mediator of immune response occurring in AN. In contrast to Heymann’s nephritis, CD4 T cells were shown to have a protective role against the progression of AN. Depletion of CD4 T cells aggravated glomerular and interstitial injury in AN . Another study reported different data with severe combined immunodeficient (SCID) mice developing AN to a similar extent to wild-type mice, suggesting that T cells are not critical in the development of AN . However, a subsequent study showing that Foxp3-transduced CD4 T cells exhibited a protective effect by reducing glomerulosclerosis, tubular damage and interstitial infiltrates suggested a therapeutic potential of Tregs in this model . Another report supported a beneficial role of Tregs in AN in which SCID mice reconstituted with Tregs expressing high levels of Foxp3 showed reduced glomerulosclerosis and tubular injury with significantly less macrophage infiltration . In this study, in vivo blockage of TGF-β using neutralizing antibodies ameliorated the protective effect of Tregs. These findings suggest a TGF-β-dependent Treg–macrophage inhibitory interaction that can explain cognate-independent protection by Treg. Depletion of CD8 T cells protected mice from renal functional and structural deterioration . In contrast, depletion of γδ T cells exacerbated the disease severity in a murine AN model with worse glomerulosclerosis and interstitial inflammation, suggesting a protective role of γδ T cells .
In a rat model of toxic renal injury induced by HgCl 2 , a TGF-β secreting regulatory CD4 T cell line was found to inhibit not only HgCl 2 -induced anti-laminin antibody production, but also increase in serum immunoglobulin E (IgE) concentration and glomerular deposition of immunoglobulin. This implies that TGF-β-producing, autoreactive T cells inhibit both Th1- and Th2-mediated autoimmune diseases . Most studies on T cells in glomerulonephritis have focused on the development or initial injury phase, as reviewed above. Future studies are required to reveal the role of T cells in the regenerative process in glomerulonephritis.
Acute Kidney Injury
AKI models can be classified into three different categories according to the cause of the injury: ischemic, sepsis-induced and nephrotoxic AKI.
Ischemic Acute Kidney Injury
Numerous studies have reported the roles of each immune component in the pathogenesis of renal IRI and most studies examining the immune system have been performed in murine models . Both innate and adaptive immune systems are now well established as being engaged in the immune response occurring in postischemic kidneys after IRI.
T cells were not expected to participate in the initial renal injury of ischemic AKI based on ideas traditionally about the immunological functions of T cells. The pathophysiological role of T cells in the establishment of initial renal injury following IRI has been elucidated in many studies, both directly and indirectly . Previous reports showing that T-cell-targeting medications such as FK506 and mycophenolate mofetil attenuate renal injury after IRI also support the important role of T cells in renal IRI . Blockade of the T-cell CD28-B7 costimulatory pathway with CTLA4Ig (a recombinant fusion protein containing a homolog of CD28 fused to an IgG 1 heavy chain) had a protective effect in the early injury phase of cold renal IRI in rats . A subsequent study revealed that CTLA4Ig treatment both on the day of renal IRI and during the first week after IRI reduced proteinuria in a model characterized by progressive proteinuria in uninephrectomized rats that underwent cold IRI, implying that T cells affect the outcome of renal IRI for longer periods as well as the initial period .
More direct and detailed mechanisms of T-cell involvement in the initial injury phase of renal IRI were demonstrated in a murine renal IRI model using CD4- and CD8-deficient mice . In this study, CD4 and CD8 double knockout mice were significantly protected from early renal injury and T cells showed a two-fold increase in adherence to renal tubular cells in an in vitro hypoxia reoxygenation setting. A subsequent study by the same investigators identified CD4 T cells as a major pathogenic mediator of renal IRI that acts in the early phase similarly to traditional innate immune components . In this study, another T-cell knockout mouse strain, athymic nu/nu mice, was protected from renal injury after IRI, and adoptive T-cell transfer into these mice restored renal injury, demonstrating that T-cell deficiency did indeed confer renal protection in renal IRI. CD4 knockout mice, but not CD8 knockout mice, were significantly protected from renal injury with lower mortality, and adoptive transfer of CD4 T cells into CD4 knockout mice restored renal injury after IRI. CD28 on T cells and T-cell IFN-γ production were reported as key factors of the effects of CD4 T cells on ischemic AKI in this study. A study on CD4 T-cell subsets in a murine renal IRI model demonstrated that CD4 T cells of the Th1 phenotype are pathogenic and that the Th2 phenotype can be protective . This study was performed using mice with targeted deletions in the enzyme signal transducers and activators of transcription (STAT)4 and STAT6 that regulate Th1 (IFN-γ producing) and Th2 (IL-4 producing) differentiation and cytokine production, respectively. Renal injury was aggravated both functionally and structurally in STAT6-deficient mice, but STAT4-deficient mice showed mildly improved renal function after IRI. IL-4 was suggested as a protective mediator of the STAT6 pathway since IL-4-deficient mice showed a similar postischemic phenotype to STAT6-deficient mice. Another report showing that inactivation of IL-16 (a T-cell chemoattractant, strongly expressed in distal and proximal straight tubules of the postischemic kidney) by antibody administration and IL-16 deficiency prevented renal injury with less CD4 T-cell infiltration also supported the importance of CD4 T cells in early renal injury after IRI . The kinetics of early trafficking of T cells into postischemic kidney was described in two recent reports . Flow-cytometric analyses of mononuclear cells directly isolated from the kidney revealed very early T-cell trafficking into postischemic kidneys at 3 h after IRI and sphingosine-1-phosphate receptor was reported to play an important role in T-cell trafficking.
CD8 T cells have not received much attention in renal IRI, although these cells are a major subpopulation of T cells, along with CD4 T cells. A previous study showed that CD8 knockout mice were not protected from renal IRI, suggesting a limited role of CD8 T cells in renal IRI . However, one recent study using germ-free mice reported aggravated renal injury with increased trafficking of CD8 T cells, but not CD4 T cells, into postischemic kidneys in these mice compared with wild-type controls, implying that CD8 T cells may also contribute to early renal injury but that this contribution may be modified by environmental factors including previous exposure to germs. CD8 T cells isolated from postischemic kidneys were recently reported to produce more IFN-γ than did normal and sham-operated kidneys .
The γδ T cells, a minor subset of T cells with T cell receptor (TCR) composed of a γ chain and δ chain, were reported to play a role in early renal injury after IRI since both γδ T-cell-deficient mice and αβ T-cell-deficient mice showed reduced renal injury . Greater proportions of CD3 + CD4 − CD8 − double-negative (DN) T cells were found in normal mouse kidneys compared with spleen and peripheral blood and also in postischemic kidneys following thymoglobulin treatment in a murine renal IRI model . However, the role of DN T cells in ischemic AKI is yet to be determined. Ag–TCR engagement followed by Ag-specific T-cell activation seems to be implicated in the pathogenesis of renal IRI. A recent study using nu/nu mice and transgenic DO11.10 mice that have TCRs specifically recognizing chicken OVA peptide demonstrated that diverse TCR repertoire was important for renal IRI in naïve mice without T-cell activation. However, once T cells were activated in an Ag-specific manner through TCR in DO11.10 mice, the restricted TCR repertoire no longer limited the extent of kidney injury .
With the use of flow-cytometric analyses of kidney-infiltrating mononuclear cells for the elucidation of dynamic kinetics of T-cell trafficking, it has been shown that T-cell infiltration is increased at 3 h but decreased at 24 h after IRI. In a subsequent study, long-term infiltration of activated and effector-memory T cells into postischemic kidneys was found. In addition, T-cell infiltration, which increased with time, into postischemic kidney was followed up until 11 weeks after IRI and the increased T cells expressed an activation and effector-memory phenotype, implying that T cells may play some role in prolonged renal damage or regeneration processes during the repair phase of renal IRI . The same investigators further investigated the phenotype and the role of infiltrated T cells during the repair phase of renal IRI, and found that infiltration of Tregs increased with time in postischemic kidney during the repair phase . In this study, postischemic kidneys had an increased number of TCR-β + CD4 + and TCR-β + CD8 + T cells with enhanced proinflammatory cytokine production, and infiltration of Tregs expressing TCR-β/CD4/CD25/Foxp3 was increased on day 3 and even more on day 10 after IRI. Treg depletion or transfer was performed on day 1 after IRI to avoid affecting initial renal injury and to evaluate the effect of Treg manipulation on the repair process. Treg depletion led to increased proinflammatory cytokine production from kidney-infiltrating T cells and aggravated renal injury. In contrast, Treg transfer reduced proinflammatory cytokine production from T cells in postischemic kidneys and promoted renal regeneration by increasing renal tubular epithelial cell proliferation, measured with Ki-67 (a 360 kDa nuclear protein, expressed by proliferating cells in all phases of the active cell cycle, but absent in resting cells). This study provided the first direct evidence that Tregs are a crucial mediator for renal regeneration in ischemic AKI, even though it was performed in an experimental setting and did not explore the later repair phase when renal fibrosis and tubular atrophy become more apparent. Tregs were also reported to modulate early injury after renal IRI through IL-10-mediated suppression of the innate immune system, and to contribute to the protective effect of ischemic preconditioning in the kidney . Treg transfer was performed 2 weeks before renal IRI and significantly attenuated renal injury, with decreased leukocyte accumulation on day 1 after IRI. Adoptive transfer of wild-type Tregs into RAG-1 knockout mice, which are deficient in mature T cells and B cells, was sufficient to prevent renal IRI, but transfer of IL-10-deficient Tregs did not prevent renal IRI.
Regarding the role of lymphocytes during the repair phase of renal IRI, a recent study identified B cells as another important factor affecting the regeneration process after IRI . Because there are only a few studies that directly deal with regeneration in IRI and since T cells interact with B cells, this report is pertinent to the role of T cells in renal repair. B-cell trafficking kinetics was measured for 4 weeks after renal IRI and the effect of B-cell manipulation was explored. B cells that infiltrated into postischemic kidneys during the repair phase were activated and differentiated into plasma cells that peaked on day 10. Postischemic kidneys of B-cell-deficient mice expressed higher levels of IL-10 and vascular endothelial growth factor, and reduced tubular atrophy with greater tubular proliferation, while adoptive B-cell transfer decreased tubular proliferation and increased tubular atrophy.
Sepsis-induced Acute Kidney Injury
Sepsis-induced AKI (septic AKI) in humans frequently presents as a multiorgan failure accompanied by sepsis. Experimental models of septic AKI are usually induced by administration of lipopolysaccharide (LPS) or cecal ligation and puncture (CLP). There have been few reports regarding the role of T cells in experimental septic AKI models. It would be difficult to investigate this topic because of the difficulty to obtain renal tissue from septic patients with AKI and to simulate a consistent animal model of septic AKI, specifically during the repair phase, given the low likelihood of long-term survival. Therefore, the recent important findings regarding the role of Tregs are reviewed.
Regarding the role of Tregs in sepsis, most studies were performed using blood samples of sepsis patients which revealed an unfavorable role of Tregs, in contrast to experimental data on ischemic AKI. Monneret et al. found a higher percentage of circulating Tregs in the blood samples of septic shock patients . A significant portion of these Tregs expressed CD45RO but not CD69, suggesting that these cells were naturally existing Tregs rather than recently activated by sepsis. Analyses of blood samples obtained between 7 and 10 days after the diagnosis of sepsis showed that the non-survivor group (nine patients) had a higher percentage of Tregs than the survivor group (seven patients). The authors concluded that prolonged existence of Tregs may lead to severe immunoparalysis and result in poor outcome. The same group also demonstrated that the increased proportion of Tregs among CD4 T cells was caused by a selective depletion of CD4 + CD25 − T cells rather than a proliferation of Tregs .
Absence of CD127 was reported as an important feature of Tregs implicated in sepsis in humans and mice . Increased CD4 + CD25 + CD127 − T cells, which inhibit the proliferative response of peripheral blood mononuclear cells (PBMCs) after simulation with mitogens, were found in septic shock patients, and downregulation of Foxp3 expression in Tregs using ex vivo transfection with Foxp3 targeting siRNA restored the PBMCs’ proliferative response to mitogens in a murine CLP model. Another study analyzing Tregs in sepsis patients and healthy controls also reported that sepsis was associated with an increased percentage of Tregs and elevated plasma levels of soluble CD25 . In this study, four out of 13 patients were reassessed several weeks after hospital discharge and their Tregs showed reduced expression of Foxp3, TGF-β and IL-10 compared with their hospitalization period. Tregs limited the capacity of monocytes to induce Ag-specific response and to secrete proinflammatory cytokines in response to LPS, and inhibited LPS-induced monocyte survival through a Fas/Fas ligand-dependent pathway . Coculture study of Tregs and polymorphonuclear neutrophils (PMNs) in a model simulating Gram-negative bacteria infection also demonstrated that the death of PMNs was enhanced by Tregs, and apoptosis of PMNs was more accelerated when Tregs were stimulated with LPS or anti-CD3/CD28 antibodies . Normally, the lifespan of PMNs is prolonged when activated by LPS. The role of human Tregs is well summarized in a recent review paper .
Although there is no direct evidence of Treg involvement in septic AKI, a significant effect of Tregs on septic AKI is predicted based on the accumulated data of circulating Tregs in sepsis patients. To elucidate the role of Tregs on septic AKI, establishment of a more suitable animal model with better animal survival is required.
Nephrotoxic Acute Kidney Injury
Nephrotoxic AKI has been induced with several well-known nephrotoxic drugs including cisplatin, gentamicin and cyclosporine. A study using nu/nu mice and CD4- or CD8-deficient mice in acute cisplatin nephrotoxicity model revealed that T-cell deficiency protected kidneys both functionally and structurally, with decreasing renal myeloperoxidase activity and proinflammatory cytokine production, suggesting T cells as direct mediators of experimental cisplatin nephrotoxicity . Resveratrol was reported to attenuate cisplatin-induced nephrotoxicity in rats by reducing free radicals and inhibiting the infiltration of inflammatory cells such as T cells and macrophages . Tregs were recently identified as a crucial factor in attenuating early renal injury induced by cisplatin both functionally and structurally . However, there are no reports regarding the role of T cells in the repair phase of nephrotoxic AKI.
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