THE ROLE OF TRANSFORMING GROWTH FACTOR BETA IN CHRONIC RENAL ALLOGRAFT NEPHROPATHY (2000)

Chronic allograft nephropathy (CAN) remains a major problem in renal transplantation. Although treatment of acute rejection episodes has advanced and is now successful in the majority of cases, there is no effective therapy for this long-term deterioration. The term chronic rejection is often used for this condition but is not strictly correct because as well as ongoing immune attack, a transplanted kidney bears other nonimmunological insults including ischemia reperfusion (IR) injury and immunosuppressant drug toxicity (Table 1; reviewed in 1). Histologically, failing transplants are characterized by marked fibrotic changes throughout the kidney, similar to those that occur in many chronic renal diseases. The glomeruli show sclerosis, shrinkage, and collapse of the glomerular tuft. In the interstitial compartment there is a light infiltrate of mononuclear cells associated with extracellular matrix (ECM) deposition, and variable degrees of tubular atrophy or loss. Arteries exhibit concentric intimal hyperplasia resulting from infiltration of smooth muscle cells, and this occurs even in very small vessels. Capillaries may show thickening and multi-layering of the basement membrane. Once the features of CAN are present, they are usually progressive and eventual graft loss is inevitable. The molecular events behind these changes are being intensively investigated, and there is much evidence to support an important role for the cytokine transforming growth factor (TGF)-β. The aim of this review is to critically appraise this evidence. Table 1: Risk factors for chronic allograft nephropathyTRANSFORMING GROWTH FACTOR-β TGF-β is a cytokine that exists structurally as a homodimer, with each monomer having a molecular mass of 12.5 kD. There are three isoforms of TGF-β in humans. TGF-β1 is the one for which most experimental data are available and will be the main focus of this article. TGF-β is released as an inactive precursor, in a complex bound with a latency-associated peptide (LAP) (2). Various factors can release TGF-β from this complex including alterations in pH, several proteolytic enzymes, and the protein thrombospondin (3). This is one level at which its activity is controlled. Active TGF-β can also be inhibited by local factors such as the proteoglycan decorin to which it binds in a reversible manner (4). There are three known classes of TGF-β receptors (TGFR), but only two of these, TGFR I and TGFR II, are involved in signaling pathways. The third class, TGFR III, comprises two related proteins betaglycan and endoglin, which bind TGF-β on the cell surface and present it to the other receptors (5). The intracellular processes that occur after activation of TGF-β receptors have now been elucidated in great detail. They involve a class of proteins called Smads, which are responsible for transferring information from activated receptors to the nucleus where they act directly on DNA promoter sequences (6). TGF-β can both stimulate and inhibit cell growth and proliferation. It has been seen as a regulatory molecule, acting to restore balance after deviations from normal. It prevents the adhesion of neutrophils to endothelium (7) and their subsequent transmigration (8). It also inhibits the proliferation of monocytes and lymphocytes (9, 10) and can induce lymphocyte apoptosis (11). Indeed TGF-β seems to trigger the reduction of inflammation, in preparation for healing, and this ties in with its role in ECM turnover and fibrosis. Fibroblasts are stimulated by TGF-β to produce matrix proteins such as collagen and fibronectin (12). TGF-β is also important in preventing matrix degradation, both by decreasing synthesis of proteolytic enzymes and also increasing the local levels of their specific inhibitors (13, 14). Inappropriate fibrosis has been linked with TGF-β overexpression in several pathological conditions (15). Measurement of TGF-β Levels The accurate assessment of TGF-β levels is necessary to assess its role in biological processes. Unfortunately this is not altogether straightforward. In particular, though many studies measure absolute levels of TGF-β either at the protein or messenger RNA (mRNA) level, this may not actually reflect biological activity because of the complexities of TGF-β release and activation. As described above, TGF-β is released as an inactive complex associated with LAP, and most antibodies that have been used in immunohistochemical studies detect this complex as well as the active form. Once activated, TGF-β can also be inhibited by local factors. Hence although measuring gene transcription in mRNA analysis may indicate that increased levels of TGF-β are being produced, it will not allow for the fact that there may also be increased inhibition or destruction of the active form. Some studies have used plasma levels of TGF-β in an attempt to reflect tissue activity, but this approach is questionable because of the high TGF-β content of platelets (16). At present no "gold standard" method of assessing TGF-β activity exists. Some groups have used differential antibody staining of active and latent TGF-β (17), however, at present there are few studies to confirm the accuracy of this method. Another approach is the use of ligand binding assays using TGF-β receptors that in theory should only bind to the active form of TGF-β (18). Unfortunately these approaches are suitable only for unprocessed specimens because of concerns that tissue fixation may cause the dissociation of TGF-β from LAP. TGF-β in Renal Fibrosis There is a substantial body of evidence supporting the role of TGF-β in renal fibrosis, and only a brief summary can be provided here. In early in vitro experiments, TGF-β administration to mesangial cells was shown to cause proliferation (19) and also synthesis of matrix proteins (20). Cultured glomerular epithelial cells also produce ECM proteins such as fibronectin and collagen in response to TGF-β (21, 22). In an elegant in vivo model, Isaka and colleagues (23) transfected the TGF-β gene into rat kidneys, using a viral vector introduced via the renal vein. The overexpression of TGF-β was confirmed by immunohistochemistry, and there was a clear increase in mesangial matrix production, evident as early as 3 days after transfection. Recently, the same group has managed to introduce antisense oligonucleotides, against TGF-β, into rats with glomerulonephritis (24). The oligonucleotides neutralized TGF-β's action, with a consequent significant reduction in fibrosis. Yamamoto et al. (25) investigated the mechanism by which TGF-β might cause fibrosis using an experimental model of renal fibrosis produced by the administration of antithymocyte serum to rats. A single injection caused mesangial cell injury resulting in a peak of TGF-β mRNA and protein levels approximately 7 days after injection. This was associated with ECM synthesis, but subsequently because TGF-β levels fell there was resolution with return of the glomeruli to normal. If, however, before resolution a second injection of antithymocyte serum was given, TGF-β levels remained elevated and this was accompanied by severe glomerulosclerosis and interstitial fibrosis. The authors described this as the "two-shot" model of fibrosis and proposed that in response to continuing injury a defect in TGF-β regulation occurred, perhaps due to the development of an autocrine loop. Other experimental models of renal injury associated with increased TGF-β expression include streptozotocin-induced diabetes (26), obstructive uropathy (27), and fibrosis induced by adriamycin administration (28). RISK FACTORS FOR CAN AND THEIR EFFECTS ON TGF-β There are a number of proven risk factors that are associated with the development of CAN, and all have been shown to cause graft damage. Indeed there is a widely held view that CAN represents a stereotypic response to injury of a renal allograft, irrespective of the initiating agent or agents (1, 29). The study of individual risk factors in isolation either in vitro or using animal models has demonstrated that all can influence the expression of TGF-β, supporting the proposition that it has a role in the development of CAN. Acute Rejection Acute rejection episodes, particularly those that are severe or recurrent, are a major risk factor for CAN (30). They cause injury to the graft by a variety of mechanisms including direct cellular cytotoxicity and complement-mediated damage. Nagano et al. (31) studied acute rejection in a rat model using a strain combination in which acute rejection developed almost immediately after transplantation. They demonstrated that TGF-β mRNA levels in the kidney increased significantly by 3 days after the onset of acute rejection, a feature not seen in isograft controls. Chronic Immunological Injury There are numerous rodent models of renal transplantation, and the posttransplantation outcomes depend upon the two strains involved. This has allowed studies in situations where there is continuing immune activation but not acute rejection. For example transplantation from Lewis (Lew)-to-Fisher 344 (F344) rats leads to an initial inflammatory infiltrate followed by resolution by 1 month, whereas when F344 kidneys are transplanted into Lew recipients, inflammation continues and eventually the kidneys develop histological changes similar to those found in humans with CAN. Paul's group (32) examined the expression of TGF-β mRNA in these models using Northern blotting and showed that in both it was increased at 2 weeks compared with normal kidneys or isograft controls. By 1 month, levels in the F344-to-Lew kidneys that developed CAN continued to rise, but in the other group they were no longer different from those in controls. Another group also used Northern blotting to measure TGF-β mRNA in the F344-to-Lew model and showed that at time points between 4 and 52 weeks after transplantation levels were significantly elevated compared with isograft controls (33). This was associated with marked fibrosis and led the authors to suggest that TGF-β has a causative role in the matrix expansion that occurs in CAN. In a mouse model of renal transplantation that develops changes similar to CAN, TGF-β expression was increased by both immunohistochemistry and Northern blotting (34). Interestingly MHC-deficient mice that did not express high levels of TGF-β still developed changes of CAN after allotransplantation. This suggests that in these animals other cytokines and growth factors are assuming a more prominent role. Ischemia Reperfusion Injury There is an inevitable ischemic time associated with renal transplantation. Injury is caused not only as a consequence of ischemia, but also during reperfusion, and this involves activation of endothelial cells and the release of oxygen free radicals and various inflammatory mediators (35). TGF-β is induced and activated by oxygen free radicals (36, 37), and it is likely that TGF-β has a physiological role in protecting against IR injury (38). Indeed in an experimental model in which rats undergo transient clamping of the renal pedicle to mimic IR injury, TGF-β mRNA and protein levels are up-regulated concurrently with the period of tubular regeneration, suggesting a role in recovery from damage (39). In the long term, rats with renal IR injury develop marked fibrosis similar to that found in CAN (40), and this is associated with persistent TGF-β up-regulation. Drug Toxicity It is increasingly recognized that modern immunosuppressive drugs can have adverse effects on renal allografts (41). In experimental models using rodents, cyclosporine administration produces similar histological appearances to those found in transplant recipients with CAN and is associated with increased TGF-β expression (42, 43). Administering anti-TGF-β antibody to these animals abrogates these effects, supporting the proposition that cyclosporine toxicity occurs via a TGF-β-dependent mechanism (44). A more recently introduced immunosuppressant tacrolimus has also been shown to be associated with fibrosis and TGF-β overexpression (45). Hyperlipidemia Because the vascular lesions in CAN show similarities with atherosclerosis, hyperlipidemia after transplantation has been considered a risk factor (46). Interestingly administering a high cholesterol diet to rats induces renal interstitial fibrosis within 12 weeks, and this is associated with raised levels of TGF-β (47). Human glomerular cells in vitro are stimulated to produce TGF-β by administration of oxidized low-density lipoproteins (48). Hypertension A recent study of over 29,000 patients confirmed that hypertension is associated with chronic renal transplant dysfunction (49). Recently Tilney's group (50) induced hypertension in the F344-to-Lew model and showed that it led to more severe histological changes and was associated with further up-regulation of TGF-β measured by immunohistochemistry. Investigation of the role of hypertension in renal fibrosis has revealed evidence of a complex interplay between the renin-angiotensin system and TGF-β that will be discussed in detail later. CLINICAL EVIDENCE FOR THE ROLE OF TGF-β IN CAN Clinical studies of TGF-β expression after renal transplantation are complicated by the fact that it is difficult to dissociate the various risk factors involved. In general, most human studies have shown up-regulation of TGF-β after renal transplantation (Table 2) (51–57). Table 2: Studies of TGF-β in human renal transplantationOnly three studies have contained more than 50 patients. The first of these measured plasma levels of TGF-β (51), and so the results are of doubtful validity. The other two measured tissue levels of TGF-β in renal biopsy specimens using reverse transcriptase-polymerase chain reaction and immunohistochemistry, respectively. Sharma et al. (53) found a clear increase in TGF-β mRNA expression in the group of patients with CAN (72% vs. 47%, P =0.01). Pankewycz et al. (56) did not specifically mention CAN in their patient group but did find a higher level of TGF-β protein expression in biopsy specimens with histological features of cyclosporine nephrotoxicity. As previously mentioned, often these two diagnoses are difficult to distinguish. Both of these studies suffer from the previously mentioned criticisms of not distinguishing latent and active TGF-β, but the results are still of interest. The study by Horvath et al. (54) at first glance seems to contradict the above findings by showing a fall in TGF-β mRNA in kidneys with CAN. However the patients studied had all had nephrectomies and many had stopped immunosuppressive therapy before kidney explantation, making it an atypical group. The remaining three studies in Table 2 all had small numbers of patients and failed to detect any difference in TGF-β expression between different patient groups. Four of the studies used immunohistochemistry and were able to study the differential distribution of TGF-β within the kidney after renal transplantation (52, 54–56). Previous studies on rats had shown that renal tubules were the only compartment that showed strong immunopositivity in normal kidney (58), and this was confirmed in two of the human studies that contained normal controls. Overall staining was much stronger after transplantation compared with normal kidneys. The interstitial compartment was the most strongly positive, reflecting the inflammatory infiltrate, and TGF-β was also seen more prominently in glomeruli and blood vessels. Decreased levels were found in the tubules after transplantation especially during rejection. There were some differences in expression between TGF-β isoforms, with for example TGF-β2 staining particularly strongly in blood vessels (55). None of the studies showed a clearly different staining pattern for TGF-β in CAN. THE INTERPLAY OF TGF-β WITH OTHER FACTORS DURING CAN At the site of chronic injury there are a number of inflammatory cells producing cytokines and growth factors, chiefly macrophages and T cells. Phenotypic changes in resident cell populations also occur, and both mesangial and tubular epithelial cells contribute to the ongoing fibrotic process. Resident fibroblasts are characterized by expression of α-smooth muscle actin, and they seem to take on characteristics of smooth muscle cells leading to their description as myofibroblasts (59). In an experimental study of renal scarring, the cytoplasm of myofibroblasts showed immunopositivity for TGF-β, suggesting an important role in the development of fibrosis although this remains to be clearly defined (60). In a small immunohistochemical study on human biopsy specimens, myofibroblasts as identified by staining for α-smooth muscle actin did seem to be associated with CAN (61). In the setting of ongoing renal damage there are numerous growth factors and cytokines, and many interact with TGF-β; one of the most important of these is angiotensin II. Although mainly known for its role in blood pressure and fluid balance control, there is no doubt that this peptide has an important role in chronic renal disease. Most of the evidence has come from experimental models of renal fibrosis in which blockade of angiotensin II with antagonists or angiotensin-converting enzyme inhibitors has been shown to reduce the pathological changes and substantially negate the up-regulation of TGF-β (62–64). Although some of this effect could be due to reduction in blood pressure, a known risk factor for fibrosis, in vitro work showing that angiotensin II administration to mesangial cells increases both ECM and TGF-β production supports a direct effect also (65, 66). This complex subject has recently been well reviewed (67). The endothelins are another group of vasoactive mediators that are implicated in the pathogenesis of fibrosis. Although first described as products of endothelial cells, they are now known to have numerous sources (68) and act in vitro to stimulate the production of ECM proteins (69). Endothelin mRNA levels are raised in the F344-to-Lew model of CAN, and the administration of an antagonist is effective in reducing the histological changes (70). TGF-β stimulates endothelin release from endothelial (71) and tubular epithelial (72) cells in culture. There is also a complex relationship with angiotensin II, with both molecules acting at least in part by stimulating synthesis of the other (73). Platelet-derived growth factor (PDGF) is important in renal fibrosis, being a potent mitogen that stimulates proliferation of myofibroblasts (74) and mesangial cells (75). The action of TGF-β on mesangial cell growth is at least partly mediated via the stimulation of PDGF (19). Gene transfection studies have shown that TGF-β and PDGF act in tandem to cause glomerulosclerosis, stimulating ECM synthesis and cell proliferation, respectively (23). There are several growth factors and cytokines whose interactions with TGF-β in fibrosis remain to be fully elucidated. The fibroblast growth factors are a family of molecules produced by macrophages, endothelial cells, and smooth muscle cells and thought to be involved in the response to injury. They stimulate fibroblast proliferation and production of ECM proteins (76). mRNA levels are raised in the F344-to-Lew model of CAN (32). Vascular endothelial growth factor, a promoter of angiogenesis, is found at increased levels in the interstitium of kidney biopsy specimens from patients with CAN (77) and is produced by cultured mesangial cells in response to angiotensin II (78). In vitro interleukin 1 causes fibroblast proliferation (79) and platelet-activating factor stimulates ECM deposition from mesangial cells, probably via a TGF-β-dependent mechanism (80). THE POTENTIAL USE OF TGF-β MEASUREMENT IN CLINICAL PRACTICE Because of the reservations about measurement expressed earlier, TGF-β measurement has limited usefulness in clinical practice at present. It shows promise as a surrogate marker of CAN with preliminary evidence suggesting that it correlates with protein levels of collagen III at 6 months after transplantation (57), a putative predictor of subsequent graft dysfunction (81). It is possible then that the effect of drugs on TGF-β may indicate potential success in treating CAN. Recently Hutchinson's group (82) has shown that polymorphisms in the TGF-β gene promoter influence expression of active cytokine, and therefore patients of different TGF-β genotypes have differing baseline levels of TGF-β and different propensities for developing fibrosis. With advances in immunosuppression, this may mean that regimens are increasingly tailored to individual patient characteristics. There is as yet little information on the effects of drugs on TGF-β expression. Some early in vitro work suggests tacrolimus may be less stimulatory to TGF-β than cyclosporine (83), and clinical studies are in progress to see if this translates into decreased fibrosis. Mycophenolate mofetil is an immunosuppressant that in the F344-to-Lew rat model of renal transplantation has been shown to prevent both the rise in TGF-β levels and the morphological changes that usually occur (84). Large trials of its use have shown a promising reduction in acute rejection rates (85), but it is too early to comment on late graft function. Initial trials introducing MMF in patients with established CAN have shown some improvement in renal function (86, 87), and in one a fall in plasma TGF-β levels was demonstrated (88). Angiotensin-converting enzyme inhibitors might be expected to have an effect in reducing CAN. Although extensively studied in many chronic renal diseases, there is a surprising lack of data about their effects in renal transplantation, with only two small uncontrolled trials reported (89, 90). A recent study used the angiotensin II receptor antagonist losartan in 14 patients with CAN and found a reduction in plasma TGF-β levels (91), but tissue levels were not measured and there was no correlation with functional outcome. DIRECT MANIPULATION OF TGF-β SIGNALING: POSSIBLE EXPERIMENTAL THERAPIES In the last few years the mechanisms of TGF-β activation and signaling to the cell have been defined in great detail. This has led to a raft of potential new anti-TGF-β therapies being proposed, which interrupt this pathway at some stage. Initial studies have looked at antagonising its action with antibodies (92, 93) or with soluble LAP (94). While effective in preventing fibrosis, the problem with these strategies is that TGF-β is a multifunctional molecule and effects of its inhibition in other tissues are not fully understood. Because it has a role in many physiological processes, a more targeted approach is probably required. For example it has been suggested that the two TGF-β receptor groups involved in signal transduction may have different functions and that the type I receptor is the important one in ECM deposition (95). This may make selective antagonists a possible future therapeutic development. Recently tissue growth factor has been described as a of TGF-β's in fibrosis It is that it only on tissue cells and not on epithelial or immune cells. this were confirmed in further the development of agents that inhibit production or action be an new for Another possible for therapeutic are local matrix of these, thrombospondin has been shown to be increased in human kidney transplants chronic rejection It seems to be important in the release of TGF-β from LAP. In a study on fibrosis in thrombospondin 1 expression was associated with increased free TGF-β and decreased TGF-β as well as increased fibrosis (32). In cultured mesangial cells, thrombospondin increases both TGF-β and fibronectin levels As mentioned earlier, another is a of TGF-β activity in and it has been shown to prevent renal fibrosis in a rat model (55). There is no doubt that TGF-β has an important part to in the development of CAN. as with so many putative remain a long from a of its and this is due to the fact that it is by a number of factors and has such a of As its role in fibrosis is defined more with perhaps the of differences or mediators of its TGF-β may as a marker and perhaps a potential