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Keywords:

  • Biomarker;
  • chronic allograft nephropathy;
  • fibrosis;
  • kidney;
  • transplantation;
  • TGF-beta

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. The Kidney as a Target of Fibrosis
  5. Are Native Kidneys Like a Transplant?
  6. The Pathway to Kidney Fibrosis—Identifying Potential Targets
  7. The Heart of Interstitial Fibrogenesis—Epithelial Mesenchymal Transformation
  8. Disrupting Matrix Accumulation
  9. Back to the Beginning—Altering Baseline Immunosuppression
  10. Acknowledgments
  11. References

The dramatic improvements in short-term graft survival and acute rejection rates could only have been dreamed of 20 years ago. Late graft loss following kidney transplantation is now the critical issue of this decade. Frequently, graft loss is associated with the development of tubular atrophy and interstitial fibrosis within the kidney (i.e. chronic allograft nephropathy; CAN). Major treatment strategies in this disorder are non-specific and the focus of intervention has been on limiting injurious events. Following graft injury is a fibrogenesis phase featuring both proliferative and infiltrative responses mediated by chemokines, cytokines and growth factors. In particular, TGFβ has been strongly implicated in the pathogenesis of chronic injury and epithelial-mesenchymal transformation (EMT) may be part of this process. The cascade of events results in matrix accumulation, due to either increased production and/or reduced degradation of matrix. Recent investigations into the pathogenesis of tissue fibrosis have suggested a number of new strategies to ameliorate matrix synthesis. While the majority of therapies have focused on TGFβ, this may not be an ideal maneuver in transplant settings and alternative targets identified in other fibrotic diseases will be discussed. Attacking graft fibrosis should be a new focus in organ transplantation.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. The Kidney as a Target of Fibrosis
  5. Are Native Kidneys Like a Transplant?
  6. The Pathway to Kidney Fibrosis—Identifying Potential Targets
  7. The Heart of Interstitial Fibrogenesis—Epithelial Mesenchymal Transformation
  8. Disrupting Matrix Accumulation
  9. Back to the Beginning—Altering Baseline Immunosuppression
  10. Acknowledgments
  11. References

Chronic allograft nephropathy (CAN) is the leading cause of late graft loss following kidney transplantation (1), despite marked improvements in early acute rejection rates (2). Moreover, this injury occurs almost uniformly by 10 years of transplantation, associated with the use of calcineurin-inhibitor therapy (3). This has been a challenging disorder in so far as its multifactorial etiology (4) and lack of specific therapeutic interventions beyond the typical manipulations used in chronic kidney disease.

Complicating our understanding of this disorder is the fact that current pathologic classification includes a number of disorders that are associated with chronic renal failure in a transplant (5). Graft biopsy often can distinguish etiology and may suggest a specific diagnosis such as hypertensive nephrosclerosis, calcineurin inhibitor toxicity, recurrent disease, BK polyomavirus infection and antibody- and immune-mediated injury. These injuries included in the Banff 1997 diagnosis of CAN are manifested by tubular atrophy and interstitial fibrosis (6). Recognizing the identity of graft dysfunction is worthwhile in that it may suggest more specific strategies to ameliorate ongoing injury and CAN.

The events that lead to fibrosis in CAN are summarized in Figure 1 and can be arbitrarily divided into three phases. In the initiation phase, there is tissue injury, which may occur by either antigen dependent or antigen independent insults. Regardless of the etiology of the initiating event, the result is the fibrogenesis phase, consisting of inflammatory and proliferative responses, regulated by cytokines, chemokines and growth factors. This cascade of events results in the matrix accumulation phase, due to either increased production and/or reduced degradation of matrix, ultimately resulting in fibrosis. As has been shown in native kidneys, the magnitude of disease is reflected by the extent of atrophy and fibrosis, as it may directly reflect the loss of nephron mass and thus renal reserve (7). This relationship stays true even with living donor grafts, which may develop interstitial fibrosis and atrophy over time in the absence of other insults, resulting in reduced glomerular filtration rate and graft survival (8).

image

Figure 1. Schema of the development of fibrosis in chronic allograft nephropathy. Following injury, which may be multifactorial and sustained, is a fibrogenic phase, followed by the matrix accumulation phase. For details, see the text.

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Understanding the pathogenesis of fibrosis is critical for two reasons. First, we might identify those at risk for future disease, and then apply more strict post-transplant management and surveillance. Secondly, we may identify new therapeutic targets to directly ameliorate disease. There have been recent excellent reviews regarding events of the initiation phase of allograft nephropathy (1,4). However, the focus of this review will be to highlight the current understanding of fibrogenesis and matrix accumulation phases. A number of potential mediators and inhibitors have now been identified that could be applied to chronic graft injury, many of which have not been thoroughly studied in human transplant settings in a stringent and prospective fashion (Table 1). Moreover, while the focus of this review is in kidney allografts, the fibrogenic cascade outlined may have similar applicability to other transplanted organs.

Table 1. Potential targets and inhibitors of fibrogenesis in transplantation
Fibrogenic pathwayInhibitor
TGFβDecorin
Pirfenidone
Relaxin
Bone Morphogenic Protein-7
TGFβ associatedHepatocyte growth factor
 Ang IIARBs, ACE Inhibitors
 Endothelin-1Bosentan
 CTGFAntisense oligonucleotides, cAMP, TNFα
Epithelial-mesenchymal transdifferentiation
  Rho GTPasesY-27362
Fasudil
Matrix deposition
 Collagen synthesisProlyl-4-hydroxylase inhibitors
 Collagen synthesis & inflammationRetinoids
 MMP 2 and 9BAY 12–9566
 Wnt-4FRP-4

The Kidney as a Target of Fibrosis

  1. Top of page
  2. Abstract
  3. Introduction
  4. The Kidney as a Target of Fibrosis
  5. Are Native Kidneys Like a Transplant?
  6. The Pathway to Kidney Fibrosis—Identifying Potential Targets
  7. The Heart of Interstitial Fibrogenesis—Epithelial Mesenchymal Transformation
  8. Disrupting Matrix Accumulation
  9. Back to the Beginning—Altering Baseline Immunosuppression
  10. Acknowledgments
  11. References

Extracellular matrix deposition closely regulated both temporally and spatially, is critical in organ development and wound healing. However, responses to injury may become aberrant and result in scar formation detrimental to organ function. Within the kidney, tubular epithelial cells, glomerular cells, vascular endothelium and tubulointerstitial fibroblasts may be targeted during an injury or inflammatory response, and also may propagate injury (9). These injuries (Figure 1) may include ischemia/reperfusion injury (10) and acute cellular rejection, manifested by the destruction of normal tubular basement membranes by invading lymphocytes (11). Antibody against donor graft antigens may also result in complement activation, although the target antigens may include both HLA (12) and non-HLA targets (13,14). Both classical and alternative complement activation can occur, with tubular epithelium capable of synthesizing C3 and C4 (15) in the context of exposure to plasma proteins. Moreover, upregulation of adhesion molecules on tubular epithelium and vascular endothelium can facilitate leukocyte adhesion and trafficking, respectively (16).

Injury may also occur in a more distant compartment such as the glomerulus, resulting in a bystander response of interstitial inflammation. For example, transplant glomerulopathy is a distinctive lesion characterized by widening of the subendothelial space with accumulation of flocculent material and duplication of the basement membrane. While some groups have noted an association with antibody mediated injury as detected by C4d staining (17,18), this has not been a consistent finding (19). What is repeatedly recognized, however, is an acceleration in the rate of graft loss with this injury (20,21), which is frequently associated with other features of CAN.

Injury to the glomerulus can be propagated to the tubular epithelium via resultant proteinuria. High grade proteinuria exposes the tubular epithelium to albumin and other bioactive proteins, which may include chemokines, cytokines and complement proteins. For example, glomerular ultrafiltration of high molecular weight bioactive growth factors, such as hepatocyte growth factor (HGF) and transforming growth factor-beta (TGFβ), may activate tubular cells by cognate recognition, and lead to accumulation of extracellular matrix proteins (22). Exposure to abnormal levels of filtered proteins can lead to tubular overproduction of endothelin-1 (23) and monocyte chemoattractant protein-1 (MCP-1) (24), resulting in alterations in renal microcirculation or the propagation of inflammatory injury, respectively. Thus, not only may there be the direct effects of injury within the renal parenchyma but also indirect propagation of injury by tubular epithelial cells. Moreover, in transplantation, the injury may remain and be unabated by standard immunosuppression, providing a constant stimulus of injury.

Are Native Kidneys Like a Transplant?

  1. Top of page
  2. Abstract
  3. Introduction
  4. The Kidney as a Target of Fibrosis
  5. Are Native Kidneys Like a Transplant?
  6. The Pathway to Kidney Fibrosis—Identifying Potential Targets
  7. The Heart of Interstitial Fibrogenesis—Epithelial Mesenchymal Transformation
  8. Disrupting Matrix Accumulation
  9. Back to the Beginning—Altering Baseline Immunosuppression
  10. Acknowledgments
  11. References

Much of our insight into the pathogenesis of renal fibrosis has been obtained by extrapolating results from rodent models of disease, and as such, have limitations in their interpretation to human pathogenesis. Also, the applicability of these findings to rodent models of CAN provides additional limitation. As already discussed, CAN is a multifactorial disease, induced by both immune-dependent and independent factors. The quality of the donor kidney is dependent upon donor age, worsened by ischemic damage, and negatively affected by chronic immunosuppressants that are nephrotoxic. Thus, in transplantation, initial injury and repair may be propagated unintentionally and factors that accelerate disease may be fixed and not be amenable to manipulation as they are in other disease models.

There are a number of unique concerns in applying anti-fibrotic therapy to transplant settings. A practical concern in the use of anti-fibrotic and anti-proliferative agents is the impact on wound healing post-operatively. Indeed, the use of the anti-proliferative immunosuppressant, sirolimus, has been associated with an increased incidence of lymphocele and poor wound healing (25,26). This may be less of an issue if therapy is designed to be initiated at a defined post-operative period. But a more significant concern is the disruption of TGFβ, a significant mediator of matrix deposition and a frequent target in anti-fibrosis studies. However, TGFβ has known immunosuppressive qualities and has critical functions in T-cell regulatory responses and tolerance (reviewed in (27,28). For example, the genetic deletion of TGFβ by homologous recombination in mice is lethal, resulting in progressive organ failure and uncontrolled inflammation (29). In a number of transplant settings, TGFβ may in fact be beneficial. Acute rejection and T-cell immune responses are modified by overexpression of TGFβ in dendritic cells (30) and in humans with acute rejection, overexpression of TGFβ is associated with improved outcome (31). Moreover, TGFβ appears to play an important role in tolerance induction (32) and long-term graft acceptance (33,34) as regulatory T-cell function is TGFβ-linked (35,36). Consequently, TGFβ may be a less than optimal target in transplant settings and a careful assessment of responses in pre-clinical models and ultimately in humans will be needed to determine the appropriateness of these maneuvers. Alternatively, downstream targets might be a more useful approach.

Despite the differences elaborated, the result in both CAN and native kidney disease is kidney scarring and functional loss. Since the outcomes are similar, it may not be unreasonable to apply current models of fibrosis and treatment from native kidneys to a transplanted graft. At the very least, these studies may indicate novel avenues of investigation in pre-clinical and/or clinical transplantation.

The Pathway to Kidney Fibrosis—Identifying Potential Targets

  1. Top of page
  2. Abstract
  3. Introduction
  4. The Kidney as a Target of Fibrosis
  5. Are Native Kidneys Like a Transplant?
  6. The Pathway to Kidney Fibrosis—Identifying Potential Targets
  7. The Heart of Interstitial Fibrogenesis—Epithelial Mesenchymal Transformation
  8. Disrupting Matrix Accumulation
  9. Back to the Beginning—Altering Baseline Immunosuppression
  10. Acknowledgments
  11. References

As shown in Figure 1, the fibrogenesis phase is associated with a series of molecules that can interact with their cognate receptors within the kidney to promote matrix accumulation. Of note, tubular epithelial cells express receptors for growth factors such as TGFβ and insulin-like growth factor-1 (IGF-1) (37) that can interact either with filtered proteins or with proteins expressed within the graft. In particular, TGFβ has been intensely studied in other fibrotic diseases, and has been associated with CAN in a mouse model (38,39) and in human recipients (27,40). It is produced not only by tubular epithelial cells but also infiltrating lymphocytes, and numerous factors can induce its production including angiotensin II, endothelin-1, ischemia and cyclosporine (reviewed in (41)). The biologic effects of TGFβ include matrix production and inhibition of matrix degradation (41), as well as the transformation of epithelial cells into myofibroblasts, a process known as EMT (see below). Thus, it is a seemingly critical target of fibrosis.

A factor relevant to other kidney diseases and one that already has clinically effective inhibitory therapies is angiotensin II (Ang II). This hormone has been identified not only as a regulator of blood pressure and fluid and electrolyte balance, but also in the fibrosis cascade (42). Ang II, acting via aldosterone, increases plasminogen activator inhibitor-1 (PAI-1), and limits plasmin production from plasminogen, resulting in matrix overproduction. Inhibition of this pathway in man using either ACE inhibitors or angiotensin receptor blockers (ARBs) has been associated with reduced proteinuria and intragraft TGFβ expression (43) and improvement in the slope of decline in reciprocal serum creatinine (44). Acceptance of these agents for management of hypertension following transplantation is becoming more common and a portion of the anti-fibrotic effect detected in animal models may be TGFβ-independent. Thus, the theoretical potential for blunting TGFβ responses with detriment to T-cell regulation post-transplant may be in some way be balanced by the effectiveness of hypertensive management and matrix regulation independent of TGFβ.

Pirfenidone (5-methyl-1-phenyl-2-(1H)-pyridone) is an experimental anti-fibrotic agent that suppresses TGFβ expression (45) and matrix deposition (46) in a number of organs and disease models of fibrosis including the kidney (reviewed in (47)). In a rat model of cyclosporine nephrotoxicity, pirfenidone reduced TGFβ mRNA and protein expression and fibrosis by greater than 50% when co-administered with cyclosporine (46) and reduced pro-apoptotic gene expression (48). Moreover, in rat orthotopic lung transplant model, pirfenidone reduced collagen content and peak airway pressure compared to untreated allografts when treatment was initiated immediately post- transplant (49), with suppression of TGFβ and arginase activity. The lack of data in pre-clinical kidney transplant models makes it difficult to predict the practical applicability of this agent in the transplant population although this agent is being used in phase 2 trials in other fibrotic kidney diseases.

Another potential agent that disrupts TGFβ-mediated renal fibrosis is the peptide hormone relaxin, a member of the IGF family. While relaxin causes renal vasodilatation and glomerular hyperfiltration during pregnancy, it has recently been recognized as a potential regulator of fibrosis. For example, in cultured human renal fibroblasts, exposure to relaxin inhibits TGFβ-induced type I collagen and fibronectin synthesis and signaling via Smad 2, and also stimulates MMP-2 and -9 secretion (50). Further, relaxin-deficient mice demonstrate progressive renal fibrosis and loss of function that is reversed with recombinant relaxin (51) and relaxin treatment reduces chemically induced renal fibrosis in rats (52). While there have been no pre-clinical studies in transplantation per se, relaxin has recently been shown to ameliorate ischemia-reperfusion injury in the liver (53) and thus may find its utility in transplantation by limiting organ preservation injury.

Recently, there has been much interest in the downstream effector of TGFβ connective tissue growth factor (CTGF). This 38 Kd cysteine-rich protein of 349 amino acids, a member of the CCN family of growth factors, is produced by a variety of cells, including mesangial cells, fibroblasts and vascular smooth muscle cells (reviewed in (54)). CTGF appears to have a diverse profile of bioactivities in vitro including mitogenic and chemotactic properties, enhanced mRNA expression of type I collagen and fibronectin in fibroblasts and mesangial cells, suggesting a role for CTGF in matrix deposition and remodeling (reviewed in (54)). CTGF has recently been recognized in the pathogenesis of fibrotic renal disease. In rodent models, CTGF is upregulated and appears to mediate the fibrosis in anti-Thy1.1 antibody-mediated glomerulonephritis (55) and in the obstructed kidney (56). In human renal diseases associated with fibrosis, CTGF expression is also enhanced (57,58). Recent studies in our laboratory demonstrate increased CTGF protein systemically in recipients with CAN, and increased mRNA expression in mouse allografts with CAN compared to isografts (59). In the absence of documented immune regulatory properties, CTGF may represent a biomarker of CAN and may be a reasonable therapeutic target that requires further study.

One of the earliest agents used to alter TGFβ-mediated fibrosis is decorin, a small leucine rich proteoglycan that forms complexes with TGFβ leading to inhibition or sequestration within the extracellular matrix. Decorin also interacts with fibril formation and disturbs the stability of type I collagen. While the kidney is normal in decorin deficient mice, these kidneys show a greater degree of tubular damage following obstructive injury than wild type kidneys, not all dependent on TGFβ (60). In experimental models of fibrotic kidney disease including glomerulonephritis and obstruction, decorin treatment reduced proteinuria, as well as collagen deposition and TGFβ expression within the kidney (61,62). Using a mesangial cell vector system, Huijun and colleagues demonstrated that decorin gene transfer reduced expression of TGFβ, fibronectin, and type IV collagen in antithymocyte serum glomerulonephritis, although there was no significant reduction in mesangial proliferation (63). Despite these encouraging results, the clinical utility of this agent has yet to be determined.

Another molecule of substantial clinical interest in other types of organ failure in man is endothelin-1 (ET-1), a vasoactive peptide with potent constrictive properties. ET-1 plays a role in the fibrogenic responses of the kidney in a number of animal models (reviewed in (64)). ET-1 is produced by a variety of cells including tubular epithelium, endothelial cells, macrophages and fibroblasts. In addition to its vasoconstrictive properties, which can result in ischemia damage, ET-1 promotes fibrosis by upregulating TGFβ but also can directly stimulate collagen synthesis and limit collagen degradation. In kidney grafts with arteriosclerosis, ET receptor expression is upregulated in smooth muscle cells of the media (65) and systemic levels of ET-1 are elevated in cyclosporine treated kidney transplant recipients with hypertension (66). Use of a non-selective ET receptor antagonist after ischemic injury in the rat limited the fall in GFR (67). The experience in the blockade of ET-1 receptors in clinical transplantation has been limited despite the availability of bosentan, an antagonist of the ET A and B receptors. Bosentan has had some success in the treatment of pulmonary hypertension trials and in heart failure. A recent study of bosentan in rat tracheal allografts demonstrated an amelioration of obliterative bronchiolitis (68), but whether this strategy will be successful in clinical transplantation is not certain.

The Heart of Interstitial Fibrogenesis—Epithelial Mesenchymal Transformation

  1. Top of page
  2. Abstract
  3. Introduction
  4. The Kidney as a Target of Fibrosis
  5. Are Native Kidneys Like a Transplant?
  6. The Pathway to Kidney Fibrosis—Identifying Potential Targets
  7. The Heart of Interstitial Fibrogenesis—Epithelial Mesenchymal Transformation
  8. Disrupting Matrix Accumulation
  9. Back to the Beginning—Altering Baseline Immunosuppression
  10. Acknowledgments
  11. References

Associated with tubular epithelial cell injury and activation is the development of fibroblasts with myocyte-like properties within the interstitium. These cells may be derived from a number of origins and include circulating mesenchymal cells, resident fibroblasts within the interstitium, as well as tubular epithelial cells that have undergone a phenotypic change through the process of epithelial-mesenchymal transformation (EMT). While EMT is a complex process, it has been described in vitro and in vivo in other chronic kidney diseases associated with fibrosis (69). It has only recently been appreciated that EMT may contribute to kidney graft fibrosis. Grimm et al. have shown that mesenchymal cells of recipient origin are found in the vasculature and interstitium of kidney grafts with chronic graft injury, although histologically, all recipients studied had moderate to severe chronic arteriopathy rather than simply fibrosis and atrophy (70). Even more compelling is recent work by Robertson and colleagues documenting the association of S100A4, a calcium binding protein and a marker of EMT, with the presence of CD8+ T- cells within allografts with CAN (71). This study suggests that infiltrating T-cells, producing TGFβ, may directly induce epithelial cells to transform and migrate into the interstitium. Potential mediators of EMT in human transplantation are the calcineurin inhibitors. Recent studies have shown that cyclosporine A induces EMT in cultured proximal tubular epithelial cells and is associated with a profibrotic transcriptional signature (72,73). The presence of EMT has also been confirmed in human allograft biopsies from recipients with a decline in renal function and the hallmark histology of tubular atrophy and interstitial fibrosis (74), and this process may be linked to oxidative stress (75).

Disruption of clinical EMT may be clinically feasible in the near future. Both bone morphogenic protein-7 (BMP-7) and HGF are inhibitors of EMT in vitro and in vivo (reviewed in (76)). BMP-7 treatment reduced fibrosis and improved renal function in a number of rodent models and attenuated disease via antagonism of TGFβ–mediated EMT (77). BMP-7 signaling is enhanced by kielin/chordin-like protein (KCP) and in its absence, mouse kidneys appear more susceptible to tubular injury and fibrosis (78). Similarly, HGF also appears to block TGFβ–mediated EMT in vitro and attenuated fibrosis in a variety of rodent models (reviewed in (76)). This effect is mediated by HGF modulation of TGFβ expression through transcriptional repressors.

Recently, a Ras-related small guanosine triphosphate, Rho, has been identified in mammals as a regulator of cellular responses including cytokinesis and cell-cycle progression (reviewed in (79)). Activated Rho binds specific effectors, among these, Rho associated coiled-coil forming protein kinase or ROCK. Because of Rho association with actin microfilament assembly, a component of myofibroblast morphology, Rho has been considered a potential regulator of EMT. Treatment of epithelial cells with Y-27632, a specific inhibitor of ROCK, inhibits TGFβ induced CTGF expression (80). In the unilateral ureteral obstruction (UUO) model of interstitial fibrosis in the mouse, Y-27362 suppressed myofibroblast expansion, macrophage infiltration and interstitial fibrosis, with a reduction in gene transcripts for collagen I, αSMA and TGFβ (81). Similarly, fasudil, another ROCK inhibitor, ameliorates fibrosis and inflammation in the UUO model (82). While this agent class has similarly not been tested in transplant models, it is a promising avenue of therapy and is an area that is waiting further testing in man.

Disrupting Matrix Accumulation

  1. Top of page
  2. Abstract
  3. Introduction
  4. The Kidney as a Target of Fibrosis
  5. Are Native Kidneys Like a Transplant?
  6. The Pathway to Kidney Fibrosis—Identifying Potential Targets
  7. The Heart of Interstitial Fibrogenesis—Epithelial Mesenchymal Transformation
  8. Disrupting Matrix Accumulation
  9. Back to the Beginning—Altering Baseline Immunosuppression
  10. Acknowledgments
  11. References

In healthy tissue, matrix synthesis is in balance with matrix degradation. In fibrosing tissue, this state is imbalanced due to either increased synthesis, decreased degradation or a combination of both. Within the kidney, interstitial scar consists of collagens I, III, V and VII, as well as fibronectin, while collagen IV and laminin are in tubular basement membranes (reviewed in (64)). Matrix is further strengthened by cross-linking that involves enzymatic processes including oxidation, transglutamination or glycosylation. Extracellular proteoglycans are another component and may serve as a sink for growth factors within the matrix. Degradation is normally regulated by a series of proteases including serine and lysosomal cysteine proteases. The most critical proteases are the metalloproteinases (MMP), zinc and calcium dependent endopeptidases. Each MMP has substrate specificity and are inhibited by tissue inhibitors of metalloproteinases (TIMPs), all of which are present in normal kidney (reviewed in (64)). Understanding the details of this process may suggest new modes to abrogate matrix accumulation. Blocking matrix formation in and of itself may be quite powerful in clinical transplant settings, as it obviates knowing precisely which insult is the cause. There are difficulties with this approach. They include knowing the correct time to intervene, determining the length and onset of treatment and identifying whether treatment may in some way be detrimental to the overall healing of the graft within its host.

Along these lines, a number of agents have been investigated that are either awaiting additional pre-clinical testing or have been tested in non-transplant situations and are potential therapeutic avenues for our patients. In collagen synthesis, prolyl-4-hydroxylase is the essential enzymatic step, responsible for the post-translation modification of the alpha chains of procollagen (83). Inhibition of prolyl-4-hydroxylase prevents proline hydroxylation of procollagen chains and leads to a terminal unstable folding of the procollagen into a triple helix. The inability to reach its final, stable, conformational state leads to intracellular degradation of the newly formed procollagen thus decreasing the amount of collagen deposition in the interstitium (83,84). Novel phenanthrolinone compounds have been developed that are competitive inhibitors of prolyl-4-hydroxylase both in vivo and in vitro, which do not appear to have systemic toxicity (85). Recent studies in a murine model of chronic allograft nephropathy demonstrated that treatment with one of these agents resulted in not only a reduction of fibrosis and graft inflammation, but also improved graft function compared to vehicle treated controls, with no evidence of toxicity (86). However, this agent was introduced well after the transplant surgery when healing had occurred, and prior to the development of fibrosis. It remains to be seen whether such class of agent may ameliorate established matrix or show clinical utility in transplant surgical settings where wound healing and graft anastomotic healing are a necessity.

As noted above, MMPs are critical regulators of matrix deposition. In rat kidney allografts with fibrosis, these proteins appear to be dysregulated with increased expression of MMP-2 and MMP-9 and a decrease of TIMP-3 (87). A more compelling study by Lutz et al. using BAY 12–9566, an inhibitor of MMP-2, -3, and -9, early post-transplant in this model resulted in improved proteinuria and histology (88). However, institution at a later time-point appeared to aggravate disease.

While HGF ameliorates TGFβ-mediated fibrosis, its effect extends beyond blocking EMT. HGF administration is associated with a reduction of fibrosis, and improved graft survival and renal function of rat kidney allografts (89). Gene transfer of HGF into the kidney limits the extent of acute tubular injury after ischemia, with a reduction in the extent of fibrosis and tubular atrophy long term after transplantation (90). It also ameliorates the fibrosis associated with cyclosporine nephrotoxicity in the rat (91). These effects are mediated not only by reduction in gene transcripts for TGFβ and matrix molecules (89,91), but are associated with a reduction in macrophage infiltration (89), and protection of tubular injury, perhaps by anti-apoptotic effects.

Retinoids have recently been recognized for their anti-inflammatory capacity (reviewed in (92)) and their specific receptors are expressed within the kidney (93), as well as by T-cells and B-cells (94) and macrophages (95). Their effect is mediated by either directly binding to retinoic acid response elements or indirectly, by regulating other transcription factors such as NK-κB or AP-1 (reviewed in (96)). Treatment with isotretinoin (13-cis RA) ameliorated rat anti- Thy 1.1 glomerulonephritis, with a reduction in TGFβ gene expression (97). In rat kidney transplantation, 13-cis RA reduced acute rejection severity (98), with improved graft function and histology. In a chronic rat allograft model, 13-cis RA treatment improved graft function and reduced interstitial fibrosis and inflammatory cell infiltration. (99). These results demonstrate a novel therapeutic approach to CAN, particularly when chronic rejection and immune responses are responsible.

Finally, the Wnt family of secreted glycoproteins is critical in embryogenesis. A number of laboratories are now reporting that abnormal activation of Wnt4 occurs in models of renal fibrosis and may be an important and unrecognized therapeutic target. In a number of models of renal damage with fibrosis, Wnt4 is upregulated, particularly in interstitial fibroblasts (100). Moreover, signaling through β-catenin is upregulated in urinary obstruction and fibrosis may be ameliorated by frizzled-related protein 4, an inhibitor of Wnt signaling (101). Whether this pathway is altered in transplant associated fibrosis has yet to be tested and could represent a novel pathway to be exploited clinically.

Back to the Beginning—Altering Baseline Immunosuppression

  1. Top of page
  2. Abstract
  3. Introduction
  4. The Kidney as a Target of Fibrosis
  5. Are Native Kidneys Like a Transplant?
  6. The Pathway to Kidney Fibrosis—Identifying Potential Targets
  7. The Heart of Interstitial Fibrogenesis—Epithelial Mesenchymal Transformation
  8. Disrupting Matrix Accumulation
  9. Back to the Beginning—Altering Baseline Immunosuppression
  10. Acknowledgments
  11. References

While the focus of this review has been on new agents and potential pathways, a common practice once a diagnosis of CAN is diagnosed is to limit additional calcineurin inhibitor exposure. The data supporting this effort is predominantly from studies documenting the substantial contribution of these agents in CAN development (3). Lower rates of CAN development have been associated with the use of mycophenolate mofetil (102). Such studies suggest that anti-metabolites may uniquely affect fibrogenesis but these agents can also facilitate reduction of calcineurin inhibitors, which may have greater impact. Indeed, recent investigations using calcineurin inhibitor sparing therapies indicate that graft function and/or fibrosis may be improved (103). Until immunosuppressive therapy can be utilized that is non-nephrotoxic but as equally efficacious as standard calcineurin containing regimens, depending on a strategy of immunosuppressive manipulation is not optimal.

In conclusion, while we have learned substantially more about fibrogenesis in the kidney, our understanding of these events and the unique aspects in kidney transplantation continues to grow. EMT should be considered as a critical process that may be initiated by both antigen dependent and independent insults. While a number of promising approaches are being tested in clinical disease and pre-clinical models, many of these involving modulation of TGFβ pathways have theoretical limitations in transplant settings where TGFβ expression may in fact be immunologically beneficial. Thus, exploring alternative pathways and downstream molecules is critical for developing new strategies to ameliorate graft fibrosis and atrophy.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Introduction
  4. The Kidney as a Target of Fibrosis
  5. Are Native Kidneys Like a Transplant?
  6. The Pathway to Kidney Fibrosis—Identifying Potential Targets
  7. The Heart of Interstitial Fibrogenesis—Epithelial Mesenchymal Transformation
  8. Disrupting Matrix Accumulation
  9. Back to the Beginning—Altering Baseline Immunosuppression
  10. Acknowledgments
  11. References

Dr. Roslyn Mannon is supported by the Intramural Research Program of the National Institutes of Health, National Institutes of Diabetes, Digestive, and Kidney Diseases. The author wishes to thank Dr. Jeffrey Kopp and Dr. Peter Mannon for their critical reading of this manuscript.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. The Kidney as a Target of Fibrosis
  5. Are Native Kidneys Like a Transplant?
  6. The Pathway to Kidney Fibrosis—Identifying Potential Targets
  7. The Heart of Interstitial Fibrogenesis—Epithelial Mesenchymal Transformation
  8. Disrupting Matrix Accumulation
  9. Back to the Beginning—Altering Baseline Immunosuppression
  10. Acknowledgments
  11. References