Takeshi F. Andoh

Synergistic effects of cyclosporine and rapamycin in a chronic nephrotoxicity model

Rapamycin (RAPA) acts synergistically with cyclosporine (CsA) to achieve powerful immunosuppression in several animal models of organ transplantation and autoimmune disease. If these drugs are to be used together, they should not enhance toxicity. Thus, we examined the effects of combining CsA and RAPA on renal structure and function in a rat model of chronic CSA nephropathy. Rats were given placebo, CSA (2, 4, and 8 mg/kg), RAPA (0.01 and 0.1 mg/kg), or CsA+RAPA for 28 days while on a low-salt diet. RAPA at a subtherapeutic dose of 0.1 mg/kg worsened glucose metabolism and potentiated chronic nephrotoxicity induced by CsA at 8 mg/kg in terms of both renal function and structural injury. Since hyperglycemia is known to accelerate fibrotic processes, the impairment of glucose metabolism may play a role in tubulointerstitial fibrosis (plasma glucose vs. tubulointerstitial fibrosis, r=0.72, n=18, P<0.001). RAPA had to be given at a dose 10-fold lower (0.01 mg/kg) and CsA at a dose 4-fold lower (2 mg/kg) than the dose required for complete immunosuppression to minimize nephrotoxicity. Although the CsA+RAPA combination acts synergistically on immunosuppression, the combination at the subtherapeutic dose of each drug may be synergistically nephrotoxic, perhaps due to hyperglycemia. Clinical combinations of CsA and RAPA must be tested carefully for chronic nephrotoxicity.

CHRONIC CYCLOSPORINE NEPHROTOXICITY

Cyclosporine has improved patient and graft survival rates in solid organ transplantation, and has been increasingly applied with considerable clinical benefit in the treatment of autoimmune diseases. However, the therapeutic benefits of immunosuppressive therapy for transplant and autoimmune indications have frequently been limited by the occurrence of chronic nephrotoxicity, Cyclosporine nephrotoxicity therefore remains an important clinical challenge. The clinical aspects and pathophysiology of chronic Cyclosporine nephrotoxicity, which is characterized by a decrease in glomerular filtration rate, afferent arteriolopathy, and striped tubulointerstitial fibrosis, are reviewed. Insights gained from experimental models of chronic nephrotoxicity associated with tubulointerstitial fibrosis are presented to elucidate the pathophysiology.

Mechanism of fibrosis in experimental tacrolimus nephrotoxicity

The clinical use of tacrolimus (FK506) is limited by nephrotoxicity. The pathogenesis of fibrosis in chronic FK506 nephrotoxicity remains unknown. Because transforming growth factor (TGF)-beta plays a key role in the fibrogenesis of many diseases, including cyclosporine nephrotoxicity, we studied a salt-depleted rat model of chronic FK506 nephropathy in which clinically relevant FK506 blood levels are obtained and which shows similarities to the lesions described in patients receiving FK506. Pair-fed rats were treated with either FK506 (1 mg/kg/day s.c.) or an equivalent dose of vehicle and were killed at 7 or 28 days. Characteristic histologic changes of tubular injury, interstitial fibrosis, and arteriolopathy developed in FK506-treated rats at 28 days and were accompanied by worsening kidney function, decreased concentrating ability, and enzymuria. FK506-treated kidneys had a progressive increase in the expression of TGF-beta1 and matrix proteins (biglycan, tenascin, fibronectin, and type I collagen). This effect seems to be specific because the expression of type IV collagen, a basement membrane collagen, was not affected. Matrix deposition was present mostly in the tubulointerstitium and vessels in accordance with the FK506 chronic lesion. The expression of plasminogen activator inhibitor-1, a protease inhibitor influenced by TGF-beta, followed TGF-beta1 and matrix proteins, suggesting that the fibrosis of chronic FK506 nephropathy likely involves the dual action of TGF-beta1 on matrix deposition and degradation. Since both peripheral and tissue renin expression were elevated with FK506, the renin-angiotensin system may play a role in the pathogenesis of this condition.

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‐β (TGF‐β). Salt‐depleted rats were administered CsA, CsA + PFD, vehicle (VH) or VH + PFD and sacrificed at 28 days. Physiologic and histologic changes were studied in addition to TGF‐β1, plasminogen activator inhibitor‐1 (PAI‐1) and biglycan mRNA expressions by Northern blot. TGF‐β1 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‐β1, 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‐β1 protein expression by 80%. These results demonstrate that PFD can attenuate renal fibrosis in this model. PFD was associated with a decrease in TGF‐β1 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.

Protective effects of dietary L-arginine supplementation on chronic cyclosporine nephrotoxicity.

BACKGROUND L-Arginine (L-Arg), the substrate for nitric oxide (NO) synthase producing NO, and the NO synthase inhibitor, N-nitro-L-arginine-methyl ester (L-NAME), have both been shown to modify acute cyclosporine (CsA)-induced intrarenal vasoconstriction. However, the mechanism of chronic CsA nephrotoxicity characterized by progressive tubulointerstitial fibrosis (TIF) remains unclear. Thus, we examined the pathogenetic role of NO in a rat model of chronic CsA nephropathy. METHODS Rats were given vehicle, CsA (7.5 mg/kg), CsA + L-Arg (1.7 g/kg), CsA + D-arginine (1.7 g/kg), and CsA + L-NAME (3.5 mg/kg) for 28 days on a low-salt diet. NO production, glomerular filtration rate (GFR), blood and urine chemistry, and histology were assessed. RESULTS L-Arg treatment significantly enhanced NO biosynthesis and protected animals from impaired GFR and development of TIF induced by CsA, whereas D-arginine did not. In contrast, L-NAME strikingly reduced urinary NO and worsened both GFR and TIF compared to the CsA alone group, whereas L-NAME did not change renal function and histology in the vehicle group. CONCLUSIONS Chronic CsA nephrotoxicity can be aggravated by NO blockade and ameliorated by NO enhancement, suggesting that NO has an important role in the mechanism of chronic CsA nephropathy.

Expression of apoptosis-related genes in chronic cyclosporine nephrotoxicity in mice

To define the mechanism of cyclosporine (CsA)‐induced apoptosis, we investigated the expression of apoptosis‐related genes in experimental chronic CsA nephrotoxicity. Mice on a low‐salt (0.01%) diet were given vehicle (VH, olive oil, 1 mg/kg/day), or CsA (30 mg/kg/day), and sacrificed at 1 and 4 weeks. Apoptosis was detected with deoxynucleotidyl transferase‐mediated dUTP nick end‐labeling (TUNEL) stain, and the expressions of apoptosis‐related genes were evaluated by reverse transcription‐polymerase chain reaction, immunoblot or immunohistochemistry. The activity of caspase 1 and 3 was also evaluated. The CsA group showed increases in apoptotic cells compared with the VH group (54 ± 41 vs. 3 ± 3, p < 0.05), and the number of apoptotic cells correlated well with interstitial fibrosis scores (r = 0.83, p < 0.01). The CsA group showed a significant increase in Fas‐ligand mRNA (0.20 vs. 0.02 amol/μg total RNA, p < 0.05) and Fas protein expression (146% vs. 95%, p < 0.05), compared with the VH group. The CsA group showed significant increases in ICE mRNA (0.21 vs. 0.03 amol/μg total RNA at 4 weeks, p < 0.05) and CPP32 mRNA (0.18 vs. 0.03 amol/μg total RNA at 4 weeks, p < 0.05), compared with the VH group. The enzymatic activity of ICE (16.6 vs. 7.9 ρmol/μg/ h, p < 0.05) and CPP32 protease (15.6 vs. 2.7 ρmol/μg/ h, p < 0.05) proteases were increased in the CsA group, compared with the VH group. The ratio between bax and bcl‐2 protein increased significantly in the CsA group (5.3‐fold), compared with the VH group. Levels of p53 protein also increased in the CsA group. Immunohistochemical detection of Fas, Fas‐ligand, ICE and CPP32 revealed strong immunoreactivity in renal tubular cells in areas of structural injury. These findings suggest that local activation of the apoptosis‐related genes is associated with CsA‐induced apoptotic cell death.

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‐β (TGF‐β) was shown to play a central role.

Enhancement of FK506 nephrotoxicity by sodium depletion in an experimental rat model

FK506 can show efficacy in transplant rejection even after other immunosuppressive drugs have been ineffective. However, the lack of a suitable animal model has hindered the study of FK nephrotoxicity, which has been noted as a common adverse effect in human trials. In this paper, we report a model of chronic FK nephrotoxicity in which renal structure and function are worsened by sodium depletion. Pair-fed male Sprague-Dawley rats were given FK (6 mg/kg p.o.) or vehicle for 21 days on a low-salt or normal diet. There was no significant difference in body weight between FK and vehicle groups. The FK whole-blood trough levels (3-10 ng/ml) in rats are similar to those in FK treated transplant patients. In sodium-depleted rats, FK clearly decreased GFR (0.09 +/- 0.03 ml/min/100 g vs. 0.94 +/- 0.06 ml/min/100 g in the vehicle group, P < 0.01), urinary osmolarity (UOsm, P < 0.01) and plasma magnesium (P < 0.01) and increased plasma creatinine (Pcr, P < 0.01), fractional excretion of magnesium (P < 0.01), urine volume (P < 0.01), plasma renin activity (PRA, P < 0.05), and alanine aminopeptidase (AAP, P < 0.05) as compared with those in the vehicle group. Salt depletion significantly potentiated these functional changes as compared with those in the normal salt group (GFR, UOsm, Pcr, PRA, and AAP of the low salt group vs. those of the normal salt group, P < 0.05 by ANOVA). In the sodium-depleted rats, the main lesion in the rat kidneys was focal collapse and vacuolization in proximal tubules, but there was also significant interstitial fibrosis. In contrast, no injury was observed in the sodium-replete rat kidneys. In conclusion, an experimental model of FK nephrotoxicity in sodium-depleted rats has been developed that is characterized by reduced GFR and structural damage to the proximal tubule accompanied by interstitial fibrosis. Sodium depletion appears to potentiate these changes at blood levels similar to those achieved in patients receiving FK.

Expression of vascular endothelial growth factor and its receptors Flt-1 and KDR/Flk-1 in chronic cyclosporine nephrotoxicity.

BACKGROUND Vascular endothelial growth factor (VEGF) is an endothelial cell mitogen involved in angiogenesis, wound healing, and inflammation. METHODS Rats placed on low salt diet (LSD) or normal salt diet (NSD) were treated with cyclosporine (CsA) or vehicle (VH) and killed at 7 or 28 days. We studied the expression of VEGF and its receptors Flt-1 and KDR/Flk-1 mRNA by Northern and that of VEGF protein by Western blot. RESULTS CsA induced VEGF mRNA and protein expressions at 7 and 28 days in LSD rats. At 7 days, CsA up-regulated the expression of Flt-1 and KDR/Flk-1 receptors; however, at 28 days, Flt-1 remained unchanged whereas KDR/Flk-1 expression declined. In NSD rats, in which the lesion did not develop, the expression of VEGF and its receptors remained similar to control. CONCLUSIONS What causes VEGF to be up-regulated remains unclear. Further studies are needed to study the role of hypoxia and other cytokines in relation to VEGF in this model.

Effect of pirfenidone on apoptosis-regulatory genes in chronic cyclosporine nephrotoxicity

Background. Apoptosis was shown to play a role in the progression of fibrosis in a chronic cyclosporine A (CsA) nephrotoxicity animal model. In addition, the antifibrotic molecule pirfenidone (PFD) was shown to ameliorate fibrosis in this model. We evaluated the role of PFD on the expression of apoptosis-regulatory genes in the kidneys of CsA-treated rats. Methods. Rats were administered CsA 7.5 mg/kg per day, CsA+PFD (250 mg/kg/day), vehicle (VH), or VH+PFD, and sacrificed at 28 days. Physiologic and histologic changes were studied, and apoptosis was detected by terminal deoxynucleotide transferase-mediated dUTP nick-end labeling stain. The mRNA expression of pro-apoptotic genes p53 and Fas-ligand was evaluated by quantitative polymerase chain reaction, and that of Bcl-xL, an anti-apoptotic gene, was evaluated by Northern blot analysis. In addition to mRNA expression, immunohistochemical studies of caspase 3 were performed. Result. PFD administration to CsA-treated rats significantly ameliorated nephrotoxicity. Apoptosis-positive cells were increased by CsA but significantly reduced by PFD treatment (68±19 vs. 3±1, P<0.01). In addition, PFD down-regulated the mRNA expression of CsA-induced p53 and Fas-ligand (P<0.01) and increased that of Bcl-xL, previously reduced by CsA (P<0.01). Finally, PFD significantly down-regulated caspase 3 expression, present mostly on renal tubular epithelial cells. None of these changes were observed in VH-treated rats. Conclusion. Whereas CsA favored the expression of pro-apoptotic genes, that effect was ameliorated by PFD. Because apoptosis can partly explain the loss of cells associated with fibrosis, the influence of PFD on apoptosis-regulatory genes in a manner that reduces apoptosis may explain some of its antifibrotic properties.