Sirolimus increases transforming growth factor-beta1 expression and potentiates chronic cyclosporine nephrotoxicity

BACKGROUND: Sirolimus (SRL) is increasingly being used to decrease cyclosporine (CsA) exposure. SRL is not known to be nephrotoxic and has a mechanism of action distinct from CsA. We investigated the effect of combining CsA and SRL on renal structure and function and on transforming growth factor-beta1 (TGF-beta1) and extracellular matrix (ECM) proteins in a model of chronic CsA nephrotoxicity. METHODS: Rats treated with vehicle, SRL 0.3 mg/kg/day, CsA 5 or 10 mg/kg/day, or CsA5+SRL were sacrificed at 7 or 28 days. Physiologic and histologic changes were studied in addition to TGF-beta1 mRNA and protein expressions, and mRNA expression of plasminogen activator inhibitor-1 (PAI-1) and ECM proteins biglycan and types I and IV collagen. RESULTS: While SRL alone did not alter renal function and structure, it potentiated the nephrotoxic actions of CsA when used in combination with low-dose CsA5 and resulted in significant changes similar to high-dose CsA10. In addition, SRL alone increased TGF-beta1 by 44% to 49% (P < 0.05 vs. VH). When used in combination with low-dose CsA5, SRL potentiated TGF-beta1 mRNA and protein by 121% and 176%, respectively (P < 0.05 vs. VH and CsA5), to levels achieved with high-dose CsA10. The expression of the ECM proteins followed that of TGF-beta1; a similar effect was observed with PAI-1, suggesting a decrease in ECM degradation. CONCLUSION: Because SRL augments nephrotoxicity, caution should be exercised when it is used in combination with CsA. More studies are needed to determine the long-term clinical impact of SRL on nephrotoxicity and allograft function.     

Mycophenolate Mofetil Ameliorates Arteriolopathy and Decreases Transforming Growth Factor-β1 in Chronic Cyclosporine Nephrotoxicity

Afferent arteriolopathy is the most characteristic lesion of chronic cyclosporine (CsA) nephrotoxicity. We investigated the effect of therapeutic doses of mycophenolate mofetil (MMF) in a model of chronic CsA nephrotoxicity where transforming growth factor-beta (TGF-beta) was shown to play a central role. Rats treated with vehicle, MMF 10 mg/kg/day, CsA 10 mg/kg/day or CsA + MMF were sacrificed at 7 or 28 days. Physiologic and histologic changes were studied in addition to TGF-beta1 mRNA and protein expressions, and mRNA expression of plasminogen activator inhibitor-1 (PAI-1) and the extracellular matrix (ECM) proteins biglycan and types I and IV collagen. While MMF markedly ameliorated afferent arteriolopathy, it had no significant effect on interstitial fibrosis and tubular atrophy. In addition, MMF treatment reduced both TGF-beta1 mRNA and protein levels by 39% and 32%, respectively (p < 0.05 vs. CsA only). The expression of the ECM proteins followed that of TGF-beta1 and was significantly decreased with MMF; a similar effect was observed with PAI-1, suggesting an increase in ECM degradation. These results suggest that MMF exerts a beneficial effect on CsA arteriolopathy and that it decreases TGF-beta1. While this drug combination may be useful clinically, long-term studies are needed to determine if MMF has a lasting benefit.     

Effect of nitric oxide modulation on TGF-beta1 and matrix proteins in chronic cyclosporine nephrotoxicity

BACKGROUND: Chronic cyclosporine (CsA) nephrotoxicity is characterized by interstitial fibrosis and afferent arteriolar hyalinosis. L-arginine (L-Arg), the substrate for nitric oxide (NO) synthase and N-nitro-L-arginine-methyl ester (L-NAME), the NO synthase inhibitor, were shown to modulate acute CsA nephrotoxicity. However, the mechanism of fibrosis in chronic CsA nephrotoxicity remains unclear. Thus, we examined the effect of NO modulation on fibrosis and the expression of transforming growth factor-beta1 (TGF-beta1) and matrix proteins in chronic CsA nephrotoxicity. METHODS: Rats were administered CsA (7.5 mg/kg), CsA + L-Arg (1.7 g/kg), CsA + L-NAME (3.5 mg/kg), vehicle (VH), VH + L-Arg, and VH + L-NAME, and were sacrificed at 7 or 28 days. NO production, physiologic parameters, and histology were studied in addition to the mRNA expression of TGF-beta1, plasminogen activator inhibitor-1 (PAI-1) and the matrix proteins biglycan and collagens type I and IV by Northern and the protein expression of PAI-1 and fibronectin by enzyme-linked immunosorbent assay. RESULTS: While L-NAME strikingly reduced NO biosynthesis and worsened the glomerular filtration rate and CsA-induced fibrosis, L-Arg had the opposite beneficial effect. In addition, the CsA-induced up-regulated expression of TGF-beta1, PAI-1, and the matrix proteins biglycan, fibronectin, and collagen I was significantly increased with L-NAME and strikingly improved with L-Arg. Collagen IV expression was not affected. Also, NO modulation did not affect VH-treated rats. CONCLUSIONS: Chronic CsA nephrotoxicity can be aggravated by NO blockade and ameliorated by NO enhancement, suggesting that NO maintains a protective function. NO modulation was associated with a change in TGF-beta1 expression, which, in turn, was associated with alterations in matrix deposition and matrix degradation through its effect on PAI-1.     

Aldosterone and PAI-1: implications for renal injury

Aldosterone modulates cardiovascular and renal injury and fibrosis in animal models. This review explores the hypothesis that aldosterone causes injury and fibrosis, in part, through effects on plasminogen activator inhibitor-1, the major physiological inhibitor of plasminogen activators in vivo. Aldosterone interacts with angiotensin II to increase plasminogen activator inhibitor-1 expression in vitro and in vivo. Plasminogen activator inhibitor-1, by inhibiting the production of plasmin from plasminogen, tips the balance in favor of extracellular matrix accumulation and promotes fibrosis. Aldosterone receptor antagonism decreases both PAI-1 expression and fibrosis in animal models. These findings have implications for the clinical management of cardiovascular and renal disease.     

Effect of cyclosporine and sirolimus on the expression of connective tissue growth factor in rat experimental chronic nephrotoxicity

BACKGROUND/AIMS: Connective tissue growth factor (CTGF) is a pro-fibrotic growth factor that acts downstream of transforming growth factor (TGF)-beta. However, CTGF regulation remains unknown. We tried to determine the effect of two commonly used immunosuppressants, cyclosporine (CsA) and sirolimus (SRL), on CTGF expression in a model of chronic nephrotoxicity. METHODS: Adult Sprague-Dawley rats kept on a low-salt diet were treated daily for 4 weeks with vehicle (VH), SRL (0.3 mg/kg), CsA5 (5 mg/kg), CsA10 (10 mg/kg) or both CsA5 and SRL. CTGF and TGF-beta1 expressions were evaluated by Northern blot. Functional and histologic parameters in addition to number of apoptotic cells were determined. RESULTS: At 28 days, both CsA doses were capable of inhibiting CTGF mRNA expression to levels similar to control. On the other hand, SRL increased CTGF expression by 3.5-fold. However, addition of CsA to SRL completely reversed that trend and returned levels to control. The results were different for TGF-beta1, which was increased by both CsA and SRL and to a greater extent by the drug combination. CONCLUSION: Unlike TGF-beta, CTGF does not seem to play an important role in CsA-induced chronic nephrotoxicity. In addition, calcineurin-dependent pathways are likely involved in CTGF regulation.     

Combined effects of moderately elevated blood glucose and locally produced TGF-beta1 on glomerular morphology and renal collagen production.

BACKGROUND: There is a correlation between renal graft rejection and blood glucose (BG) levels. Furthermore, diabetic patients may develop non-diabetic renal diseases, which in some circumstances progress rapidly. Since transforming growth factor-beta1 (TGF-beta) levels are elevated in many renal diseases, the accelerated progression may be due to interactions between glucose and locally produced TGF-beta1. Therefore, we investigated the effect of mild hyperglycaemia on glomerular morphology and collagen production in TGF-beta1 transgenic mice. METHODS: To achieve BG concentrations of approximately 15 mmol/l in TGF-beta1 transgenic and non-transgenic mice, we used multiple streptozotocin (STZ) injections, and after 8 weeks, we measured the changes in glomerular morphology and total collagen content. We also analysed extracellular matrix (ECM) and protease mRNA levels using real-time polymerase chain reaction (PCR) and phosphorylated extracellular signal-regulated kinase 1/2 (pERK) expression by immunohistochemistry. RESULTS: Mild hyperglycaemia alone had no effect on glomerular structure or ECM deposition. Over-expression of TGF-beta1 increased basement membrane thickness (BMT) and the mesangial volume fraction. Furthermore, it augmented ECM, Matrix metalloproteinase-2 (MMP), MMP-9, plasminogen activator inhibitor-1 (PAI) and tissue inhibitor of metalloproteinase-1 (TIMP) gene expression and pERK1/2 immunostaining. Elevated BG in combination with TGF-beta1 resulted in enlargement of glomerular volume, total mesangial volume and renal collagen content. Moreover, high BG exaggerated TGF-beta1-induced changes in the BMT, MMP-2 and TIMP-1 expression and pERK1/2 staining. CONCLUSION: Even moderate elevations in BG accelerate the progression of those kidney diseases in which TGF-beta1 is involved. This emphasizes the importance of strict BG control in renal transplant patients and diabetic patients with renal malfunctions unrelated to diabetes.     

Hepatocyte growth factor modulates matrix metalloproteinases and plasminogen activator/plasmin proteolytic pathways in progressive renal interstitial fibrosis

Evidence suggests that hepatocyte growth factor (HGF) ameliorates renal fibrosis in animal models of chronic renal disease by promoting extracellular matrix catabolism. This study examined the molecular mechanisms of HGF-induced alterations in matrix degradation both in vitro and in vivo. In vitro, HGF increased the collagen catabolizing activity of human proximal tubular epithelial cells (HKC) that were treated with TGF-beta1. Increased collagen catabolism was associated with enhanced activity of both matrix metalloproteinases (MMP) and plasminogen activators (PA)/plasmin proteolytic pathways. HGF abrogated TGF-beta1-induced production of the profibrotic tissue inhibitor of metalloproteinase-2 (TIMP-2) and plasminogen activator inhibitor-1 (PAI-1). In addition, HGF induced the production of MMP-9. In vivo, continuous infusion of HGF in the rat remnant kidney model ameliorated renal fibrosis and tubulointerstitial collagen deposition. This was associated with increased tubular expression of MMP-9, enhanced in situ gelatinolytic activity, partially restored plasmin activity and decreased expression of TIMP-2 and PAI-1 in tubular cells, and upregulation of renal TIMP-3 expression. Conversely, blocking of endogenous HGF by an anti-HGF neutralizing antibody increased renal fibrosis and interstitial collagen. This was accompanied by decreased tubular expression of MMP-9, less in situ proteolytic activity, and elevated expression of TIMP-2 and PAI-1 in tubular cells. Collectively, these findings demonstrate that HGF ameliorates renal fibrosis by enhancing extracellular matrix catabolism via both MMP and the PA/plasmin proteolytic pathways.     

Temporal expression of urokinase plasminogen activator, plasminogen activator inhibitor and gelatinase-B in chronic wound fluid switches from a chronic to acute wound profile with progression to healing

The plasminogen activator/plasmin system is known to initiate a proteolytic cascade resulting in the activation of matrix metalloproteinases in vitro leading to the degradation of extracellular matrix. To investigate whether or not this cascade is present during delayed wound healing and contributes to the pathophysiological basis of impaired healing we examined the temporal expression of urokinase plasminogen activator, plasminogen activator inhibitor-1 and gelatinase-B in fluid collected from chronic venous leg ulcers compared to acute surgical mastectomy wounds. Using a chromogenic substrate assay, levels of active urokinase plasminogen activator in chronic wounds were found to be about five fold higher compared to sera and two fold higher compared to mastectomy wounds. Levels of active plasminogen activator inhibitor-1 in chronic wounds were four times higher than those found in sera and two times higher than those found in mastectomy wound fluid. Using a fibrin overlay system and reverse zymography, we found that when the wound was not healing, the expression of urokinase plasminogen activator in chronic wound fluid was initially detected in the active forms (50 and 33 kDa), but that as the wound healed and decreased in size, was detected as an inhibitor- bound urokinase plasminogen activator-plasminogen activator inhibitor-1 complex ( congruent with 80-116 kDa). When the expression of active urokinase plasminogen activator was highest, no plasminogen activator inhibitor-1 was detectable. In contrast, urokinase plasminogen activator was always detected in the inhibitor bound form as a urokinase plasminogen activator-plasminogen activator inhibitor-1 complex in blood- and plasma-derived serum and mastectomy wound fluid. Plasminogen activator inhibitor-1 was detected in blood-derived serum and mastectomy wound fluid but not in plasma derived serum. Expression of matrix metalloproteinase-9 in chronic wound fluids, analyzed by gelatin zymography, showed that when urokinase plasminogen activator was detected in the active forms, matrix metalloproteinase-9 was overexpressed by approximately twice that found in mastectomy wounds and approximately 30 times that detected in blood-derived sera. When urokinase plasminogen activator appeared almost entirely as an enzyme- inhibitor complex, the level of expression of matrix metalloproteinase-9 was similar to that seen in mastectomy wound fluid. We conclude that the switch in urokinase plasminogen activator expression from an active to inhibitor bound form correlates with the decrease seen in matrix metalloproteinase-9 expression suggesting the presence of a proteolytic cascade initiated by the plasminogen activator/plasmin system during wound healing leading to the activation of matrix metalloproteinase-9. In addition, expression of urokinase plasminogen activator and matrix metalloproteinase-9 appear to be useful biomarkers to determine clinical wound healing status.     

Plasmin is not protective in experimental renal interstitial fibrosis

BACKGROUND: The plasminogen-plasmin system has potential beneficial or deleterious effects in the context of renal fibrosis. Recent studies have implicated plasminogen activators or their inhibitors in this process. METHODS: The development of renal interstitial fibrosis was studied in mice genetically deficient in plasminogen (plg-/- mice) and littermate controls (plg+/+ mice) by inducing unilateral ureteric obstruction (UUO) by ligating the left ureter. RESULTS: Collagen accumulation in the kidney was decreased in plg-/- mice at 21 days compared with plg+/+ mice by hydroxyproline assay (plg+/+ 19.0 +/- 1.2 microg collagen/mg tissue, plg-/- 15.6 +/- 0.5 microg collagen/mg tissue, P= 0.04). Macrophage accumulation in plg-/- mice was reduced at 21 days, consistent with a role for plasmin in macrophage recruitment in this model. Myofibroblast accumulation, assessed by the expression of alpha-smooth muscle actin (alpha-SMA), was similar in both groups at both time points. Endogenous plasmin played a role in the activation of transforming growth factor-beta (TGF-beta), as plg-/- mice had lower ratios of betaig-h3:TGF-beta1 mRNA than plg+/+ mice. Matrix metalloproteinase (MMP)-9 activity was unchanged in the absence of plasmin, but MMP-2 activity was decreased. CONCLUSION: Plasminogen, the key proenzyme in the plasminogen-plasmin system, does not protect mice from experimental interstitial fibrosis and may have significant pathogenetic effects. These findings, together with other recently published studies in the biology of renal fibrosis, imply that effects of proteins such as plasminogen activator inhibitor-1 (PAI-1), tissue-type plasminogen activator (tPA), and urokinase-type plasminogen activator receptor (uPAR) on renal fibrosis occur independently from the generation of plasmin.     

Pirfenidone Treatment Decreases Transforming Growth Factor-β1 and Matrix Proteins and Ameliorates Fibrosis in Chronic Cyclosporine Nephrotoxicity

Chronic cyclosporine (CsA) nephrotoxicity is characterized by tubulointerstitial fibrosis. Pirfenidone (PFD) is a novel antifibrotic compound that was shown to prevent and even reverse fibrosis. The mechanism of action of PFD is unclear but involves inhibition of transforming growth factor-beta (TGF-beta). Salt-depleted rats were administered CsA, CsA + PFD, vehicle (VH) or VH + PFD and sacrificed at 28days. Physiologic and histologic changes were studied in addition to TGF-beta1, plasminogen activator inhibitor-1 (PAI-1) and biglycan mRNA expressions by Northern blot. TGF-beta1 immunohistochemistry was also performed. Treatment with PFD ameliorated CsA-induced fibrosis by about 50% (p < 0.05). CsA-induced decrease in creatinine clearance improved with PFD but the difference was not significant. TGF-beta1, PAI-1 and biglycan mRNA expressions increased with CsA (p < 0.05 vs. VH) but strikingly improved with PFD treatment (p < 0.05 vs. CsA), which brought the levels down to VH levels. PFD treatment also decreased TGF-beta1 protein expression by 80%. These results demonstrate that PFD can attenuate renal fibrosis in this model. PFD was associated with a decrease in TGF-beta1 expression, which, in turn, was associated with a decrease in matrix deposition. These experiments suggest that PFD can be clinically useful for preventing chronic CsA nephrotoxicity and may prove to be helpful in other progressive renal diseases.     

Spironolactone prevents early renal injury in streptozotocin-induced diabetic rats

BACKGROUND: Glomerular and tubulointerstitial injury leads to chronic impairment of renal function, and thus, reversal of the injury may improve renal function and survival. The present study investigated whether and how mineralocorticoid receptor antagonist spironolactone ameliorates early renal injury in streptozotocin-induced diabetic rats. METHODS: Streptozotocin (65 mg/kg, single intraperitoneal injection)- or vehicle-administered rats were used as diabetic or control rats, respectively. The streptozotocin-administered rats were treated with spironolactone (50 mg/kg/day sc) for 3 weeks. Among the 3 groups of rats, we compared renal fibrosis and renal hypertrophy, using picro-sirius red staining and immunohistochemistry of ED-1 macrophage marker, plasminogen activator inhibitor-1 (PAI-1), and transforming growth factor (TGF)-beta1. RESULTS: Three weeks after administration of streptozotocin, rats exhibited increased collagen deposition in glomerular, tubulointerstitial, and perivascular areas in the kidney, which was completely attenuated by spironolactone treatment. In rats given streptozotocin alone, there were increases in ED-1-positive cell, PAI-1 expression, and TGF-beta1 expression in glomeruli and tubulointerstitiums, which were also suppressed by spironolactone treatment. Maximal glomerular and proximal tubular areas were not significantly different among the 3 groups. Rats given streptozotocin alone revealed an increase in proximal tubule wall-to-lumen ratio that was not influenced by treatment with spironolactone. CONCLUSION: Streptozotocin-induced renal fibrosis, PAI-1 expression, TGF-beta1 expression, and macrophage infiltration occur via mineralocorticoid receptor, and spironolactone ameliorates renal fibrosis presumably via the inhibition of macrophage infiltration, PAI-1 expression, and TGF-beta1 expression in streptozotocin-induced early diabetic injury.     

Synergistic effects of mycophenolate mofetil and losartan in a model of chronic cyclosporine nephropathy

BACKGROUND: Combined treatments of mycophenolate mofetil (MMF) and losartan (LSRT) have synergistic effects on various renal diseases through their hemodynamic and anti-inflammatory effects. This study investigated whether MMF treatment is effective in inhibiting inflammatory processes in chronic cyclosporine A (CsA) nephrotoxicity, and whether combined treatment using MMF and LSRT affords superior protection compared with the respective monotherapies. METHODS: Rats on a low-salt diet were given vehicle (VH group, olive oil, 1 mg/kg per day), CsA (15 mg/kg per day), CsA and LSRT (CsA+LSRT group, 100 mg/L per day), CsA and MMF (CsA+MMF group; 40 mg/kg per day), or CsA, LSRT and MMF (CsA+LSRT MMF group). Control groups received each drug without CsA treatment. Renal function, histologic parameters (arteriolopathy, tubulointerstitial fibrosis, and inflammatory cell infiltration), and mediators of CsA-induced nephrotoxicity (angiotensin-II, osteopontin, and transforming growth factor [TGF]-beta1) were studied. RESULTS: The CsA-treated rats showed decreased renal function and increased histologic parameters compared with the VH-treated rats. The CsA+MMF treatment significantly improved renal function and histopathologic parameters compared with the CsA group, and combined treatment with MMF and LSRT further improved those parameters compared with the CsA+LSRT and CsA+MMF groups. At a molecular level, increased expression of angiotensin II protein, osteopontin, and TGF-beta1 mRNAs in the CsA group were significantly decreased with MMF, and further decrease was observed with the combined treatment using MMF and LSRT. CONCLUSIONS: MMF treatment decreases CsA-induced nephrotoxicity, and combined treatment with LSRT has a synergistic effect in preventing chronic CsA nephrotoxicity.     

The effects of different immunosuppressants on chronic allograft nephropathy by affecting the transforming growth factor-beta and Smads signal pathways

OBJECTIVE: This study investigated the effects of various immunosuppressants on chronic allograft nephropathy (CAN) by affecting transforming growth factor-beta (TGF-beta) and Smads signal pathway. METHODS: Vascular smooth muscle cells (VMSC) from rat aorta were incubated for 6 or 12 hours with various immunosuppressants. Cyclosporine (CsA) (3 microg/mL), FK506 (1 microg/mL), mycophenolate mofetil (MMF) (0.3 microg/mL), rapamycine (Rapa) (10 microg/mL), CsA (1 microg/mL/MMF 0.3 microg/mL). We used the Sprague-Dawley Wistar rat accelerated kidney sclerosis model. Before transplantation, the kidney was preserved 1 hour in 0 degrees C to 4 degrees C heparin sodium chloride solution to reinforce the cold ischemia injury. The rats were divided into eight groups (each group n = 8): group A, pseudo-OP; group B, isotransplantation; group C, CsA 6 mg/kg . d; group D, FK506 0.15 mg/kg . d; group E, MMF 20 mg/kg . d; group F, Rapa 0.8 mg/kg. d; group G, CsA 3 mg/kg . d + MMF 20 mg/kg . d. The serum creatinine levels and pathological changes, according to the Banff scheme, were observed at 2, 4, 6, 8 and 12 weeks posttransplantation. Immunohistochemistry and quantitative fluorescence polymerase chain reactions were used to end localize and quantitate the expression of TGF-beta1 and Smad 2, 3, 7 in VMSC and in the transplanted kidney. RESULTS: CsA and FK506 stimulated gene expression and protein production of TGF-beta1, smad2, and smad3, but inhibited expression of smad7 both in VSMC and in the transplanted kidney. In contrast, MMF and Rapa down-regulated gene expression and protein production of TGF-beta1, smad2, 3 while up-regulating expression of smad7. There was no significant difference between the CsA group and the FK506 group, as well as the MMF group and the Rapa group. The group treated with CsA + MMF was similar to the MMF and the Rapa groups. CONCLUSION: Our study suggested that various immunosuppressants affected differentially TGF-beta1 and Smads signal pathways in rat VSMC and kidney grafts. CsA and FK506 can cause CAN, owing to up-regulated expression of smad2 and smad3, and down-regulation of smad7 expression. MMF and Rapa can prevent the CAN progression, because of down-regulation of the expression of smad2 and smad3, with increased smad7 production.     

Transforming growth factor-beta is a potent inhibitor of extracellular matrix degradation by cultured human mesangial cells

Accumulation of the glomerular extracellular matrix (ECM) is a pivotal event in the progression from acute glomerular injury to end-stage renal disease. Although enhanced ECM synthesis has been demonstrated to contribute to ECM accumulation, the role of decreased ECM degradation is largely unknown. It was previously shown that glomerular ECM degradation is mediated by a plasminogen activator (PA)/plasmin/matrix metalloproteinase 2 (MMP-2) cascade. However, little information is available regarding the factors that regulate the activity of this degradative cascade in normal or pathologic states. Transforming growth factor-beta1 (TGF-beta1) is shown here to be a potent inhibitor of ECM degradation by cultured human mesangial cells. Using human mesangial cells grown on thin films of 125I-labeled Matrigel, dose-dependent inhibition of ECM degradation in the presence of TGF-beta1 was observed, reaching >90% inhibition with 0.4 ng/ml TGF-beta1. Addition of anti-TGF-beta antibodies (4 microg/ml) in the absence of exogenous TGF-beta increased ECM degradation (1.8+/-0.2-fold versus controls, P<0.05). In contrast, platelet-derived growth factor, at concentrations up to 10 ng/ml, had no effect on ECM degradation. TGF-beta completely blocked the conversion of plasminogen to plasmin and markedly reduced the conversion of latent MMP-2 to active MMP-2. TGF-beta did not significantly alter the levels of tissue PA, total MMP-2, or tissue inhibitor of metalloproteinase-1, but did increase the levels of PA inhibitor- (1.8-fold, P<0.05), the major physiologic inhibitor of PA. These data document that TGF-beta is a potent inhibitor of ECM degradation by cultured human mesangial cells, and they suggest that decreased mesangial matrix degradation, caused by TGF-beta-mediated decreases in the activity of the PA/plasmin/MMP-2 cascade, may contribute to the glomerular matrix accumulation that occurs in progressive renal disease.     

Glomerular extracellular matrix accumulation in experimental anti-GBM Ab glomerulonephritis

Thickening of the glomerular basement membrane (GBM) results from excessive accumulation of extracellular matrix (ECM) proteins following glomerular injury. We studied the temporal relationship between the expression of growth factors, ECM accumulation, ECM degrading proteinases, and their inhibitors in a rat model of anti-GBM antibody (Ab) glomerulonephritis (GN) by the RNase protection assay and immunohistochemistry. There were two- or fourfold increases in the expression of transforming growth factor-beta(1) (TGF-beta(1)) and platelet-derived growth factor (PDGF) A and B chain mRNAs 4 days after anti-GBM Ab administration. These changes were temporally associated with increased accumulation of alpha1(III) and alpha2(IV) collagens, fibronectin, and heparan sulfate proteoglycan along the GBM. The increase in matrix accumulation was associated with little or no increases in the proteinases, urokinase plasminogen activator (u-PA) and transin, respectively. There was a 1.6x increase in the u-PA/28s mRNA ratio on day 4 in rats with anti-GBM Ab GN, but this was not associated with an increase in u-PA biologic activity. By comparison, the mRNAs of the proteinase inhibitors, plasminogen activator inhibitor-1 (PAI-1) and tissue inhibitor of metalloproteinase (TIMP) were 5x greater than that of control rats on day 4. PAI-1 mRNA correlate with increased biologic activity. These data demonstrate a temporal association between TGF-beta(1) and PDGF expression and matrix accumulation within the GBM in anti-GBM Ab GN. In addition, it suggest that this matrix accumulation results from an imbalance between matrix synthesis and degradation.     

The renin-angiotensin-aldosterone system and fibrinolysis in progressive renal disease

Renal glomerular and interstitial fibrosis is widely viewed as the final common pathway to renal failure, regardless of the initiating injury. Similarly, the renin-angiotensin-aldosterone system (RAAS) plays an important role in the progression of renal disease. This review explores the hypothesis that the RAAS causes injury and fibrosis, in part, through effects on plasminogen activator inhibitor-1 (PAI-1), the major physiologic inhibitor of plasminogen activators in vivo. PAI-1, by inhibiting the production of plasmin from plasminogen, tips the balance in favor of extracellular matrix accumulation and promotes fibrosis. Interruption of the RAAS decreases both PAI-1 expression and fibrosis in animal models. These findings have implications for the clinical management of renal disease.     

Selective lymphocyte inhibition by FTY720 slows the progressive course of chronic anti-thy 1 glomerulosclerosis

BACKGROUND: Progression is a hallmark of chronic renal disease and histologically characterized by fibrosis and inflammation of the tubulointerstitial compartment. To define the role of lymphocytes in this process, the novel lymphocyte-specific inhibitor FTY720 was administered to rats with anti-thy 1-induced chronic progressive glomerulosclerosis. In this model, the initial and short-term inflammatory glomerular injury progresses self-perpetuatedly toward tubulointerstitial fibrosis by not primarily immune-mediated, intrarenal mechanisms. METHODS: Chronic progressive glomerulosclerosis was induced by murine anti-thy 1 antibody injection into uninephrectomized rats. Treatment with FTY720 (0.3 mg/kg body weight) was started 7 days after disease induction. Proteinuria was measured every 4 weeks. In week 20, the following parameters were determined: blood lymphocyte number, kidney function, both glomerular and tubulointerstitial histologic matrix accumulation, protein expression of transforming growth factor-beta1 (TGF-beta1), fibronectin, and plasminogen activator inhibitor-1 (PAI-1) as well as infiltration with macrophages and lymphocytes. RESULTS: Treatment with FTY720 lowered blood lymphocyte count and renal lymphocyte infiltration highly significantly. In comparison to the untreated chronic progressive glomerulosclerosis animals, the lymphocyte depletion achieved significantly limited the progression of the disease, as shown by lowered proteinuria, tubulointerstitial matrix expansion, and TGF-beta1, fibronectin, and PAI-1 expression, as well as improved renal function. Glomerular matrix protein expression and accumulation was moderately lowered by FTY720. Glomerular macrophage infiltration was not, tubulointerstitial macrophage infiltration was moderately, but not significantly, decreased by FTY720 treatment. CONCLUSION: Lymphocyte depletion by FTY720 limits the progression of anti-thy 1-induced glomerulosclerosis toward chronic tubulointerstitial fibrosis and renal insufficiency. The data suggest that lymphocytes actively participate in the progression of chronic experimental kidney disease, and that FTY720 may be a novel approach to slow the progressive course of human chronic renal diseases.     

Glomerular matrix accumulation is linked to inhibition of the plasmin protease system.

TGF-beta plays a pivotal role in the pathological accumulation of extracellular matrix in experimental glomerulonephritis. Increased TGF-beta expression leads to increased synthesis and deposition of extracellular matrix components while administration of anti-serum to TGF-beta suppresses the major manifestations of the disease. We hypothesized that TGF-beta might also enhance matrix accumulation by decreasing matrix turnover via effects on protease/protease inhibitor balance. Plasmin is a potent protease capable of degrading a variety of matrix molecules. Plasmin generation from plasminogen is regulated by plasminogen activator(s) (PA) and plasminogen activator inhibitor(s) (PAI). In this study PA activity was markedly reduced and PAI-1 synthesis dramatically increased when TGF-beta was added to normal glomeruli. Diseased glomeruli also showed decreased PA activity, increased PAI-1 synthesis and increased PAI-1 deposition into matrix. Administration of anti-TGF-beta serum to glomerulonephritic rats blocked the expected increase in glomerular PAI-1 deposition. Thus changes in the PA/PAI balance favoring accumulation of matrix are induced by TGF-beta in normal glomeruli and are present in nephritic glomeruli when endogenous TGF-beta production is high. Our findings implicate the plasmin protease system in tissue repair following acute glomerular injury and suggest another mechanism by which TGF-beta enhances the matrix accumulation characteristic of many glomerular diseases.     

Effects of retinoids on the TGF-beta system and extracellular matrix in experimental glomerulonephritis.

Transforming growth factor-beta1 (TGF-beta 1) overexpression plays a key role in the glomerular accumulation of extracellular matrix proteins in renal disease. Retinoids have previously been shown to significantly limit glomerular damage in rat experimental glomerulonephritis. Therefore, the effects of all-trans retinoic acid and isotretinoin on the components of the TGF-beta system and extracellular matrix proteins in anti-Thy1.1-nephritis (Thy-GN) were investigated. Vehicle-injected control rats were compared with rats treated with daily subcutaneous injections of 10 mg/kg body wt all-trans retinoic acid or 40 mg/kg body wt isotretinoin (n = 9 per group) either with a pretreatment (day -2 through 8) or posttreatment protocol (day +3 through 8), i.e., starting before or after induction of Thy-GN, respectively. Urinary TGF-beta 1 excretion was 60% lower in all-trans retinoic acid-treated animals with Thy-GN (P < 0.025). The increase of cortical TGF-beta 1 gene expression in Thy-GN rats was significantly attenuated with all-trans retinoic acid and even more with isotretinoin treatment as compared with untreated animals (P < 0.025). Cortical expression of TGF receptor II, but not receptor I gene expression, was significantly lower in animals treated with all-trans retinoic acid or isotretinoin (P < 0.05). In all-trans retinoic acid-treated animals with Thy-GN, the increase of glomerular TGF-beta 1 protein (P < 0.008) and TGF-beta 1 (P < 0.025) and TGF receptor II mRNA (P < 0.015) was significantly less. Immunohistochemistry revealed less glomerular staining for TGF-beta 1 and TGF receptor II in the presence of all-trans retinoic acid. TGF-beta 1 immunostaining was not restricted to monocytes and macrophages, as indicated by double-staining. Glomerular staining for collagen IV and collagen III was less in animals treated with isotretinoin (P < 0.02 for both) in contrast to all-trans retinoic acid, whereas fibronectin remained unchanged. It was concluded that the beneficial effects of retinoids on glomerular damage are presumably due to a marked reduction in renal TGF-beta 1 and TGF receptor II expression.