Cyclosporine A sensitizes the kidney to tubulointerstitial fibrosis induced by renal warm ischemia

BACKGROUND: Renal warm ischemic injury and immunosuppression with cyclosporin A (CsA) may contribute to chronic allograft nephropathy after cadaveric transplantation. This study establishes whether CsA can sensitize the kidney to injury and fibrosis induced by renal warm ischemia (RWI). METHODS: The left kidney of Sprague-Dawley rats was subjected to 30 min of warm ischemia and/or intraperitoneal CsA (15 mg/kg/d) for 30 days (n=6 per group). Renal injury and fibrosis were assessed histologically together with immunohistochemistry for collagen III, transforming growth factor (TGF)-beta1, ED1 (macrophage marker), and alpha-smooth muscle actin. Renal mRNAs for collagen III, TGF-beta 1, matrix metalloproteinase (MMP)-2, and tissue inhibitor of metalloproteinase-1 together with MMP enzyme activity were also determined. RESULTS: RWI or CsA alone produced only minor effects on renal injury and fibrosis. However, in CsA-treated rats, RWI produced a marked increase in tubulointerstitial fibrosis, as shown by the potentiation of collagen III and TGF-beta1 determined by immunochemistry and mRNA analysis. The up-regulation of tissue inhibitor of metalloproteinase-1 mRNA was associated with a decrease in MMP enzyme activity. In CsA-treated rats, RWI was also associated with an increase in inflammatory infiltrates, elevated immunostain for ED1 (indicating extensive macrophage influx), and elevated immunostain for alpha-smooth muscle actin (indicating myofibroblast activation). CONCLUSIONS: In the rat, CsA can sensitize the kidney to fibrosis induced by renal warm ischemia. In renal transplantation, when cadaveric donor kidneys have been subjected to a period of warm ischemia, CsA may be an inappropriate choice for immunosuppressive therapy.     

Cyclosporine-induced renal injury induces toll-like receptor and maturation of dendritic cells

BACKGROUND: The toll-like receptor (TLR) is stimulated by not only pathogen-associated molecular patterns but also endogenous TLR ligands provided by injured cells. The influence of cyclosporine A (CsA)-induced renal injury on TLR expression and subsequent signaling pathway was evaluated. METHODS: Induction of chronic CsA nephropathy was made by administering CsA (15 mg/kg/day) for 28 days in rats. The TLR2 and TLR4 mRNA and protein expression, TLR-signaling pathway (MYD88, NF-kappaB and AP-1), putative TLR ligand (heat shock protein 70 [HSP70]), and maturation of dendritic cells were evaluated in CsA-treated rat kidneys. RESULTS: Long-term CsA treatment upregulated TLR2 and TLR4 mRNA and protein expression on renal tubular cells, and these were accompanied by increased MYD88, NF-kappaB and AP-1 expression. Putative TLR ligand (HSP70) was also significantly increased in CsA-treated rat kidney compared with vehicle-treated rat kidney. CsA-treatment increased expression of TNF-alpha mRNA, the number of dendritic cells, and expression of MHC class II antigen. Double-labeling of markers of dendritic cells and MHC class II antigen revealed that matured dendritic cells increased in CsA-treated rat kidney. CONCLUSIONS: CsA-induced renal injury stimulates components of innate immunity, and this finding suggests close association between CsA-induced renal injury and activation of innate immunity.     

Colchicine suppresses osteopontin expression and inflammatory cell infiltration in chronic cyclosporine nephrotoxicity

BACKGROUND: Colchicine (Col) is beneficial to renal injury because of its anti-inflammatory effect, but its mechanism has yet to be elucidated. The present study was designed to evaluate the inhibitory effects of colchicine on osteopontin (OPN) expression and the macrophage accumulation in chronic cyclosporine (CsA) nephrotoxicity in rats. METHODS: Male adult Sprague-Dawley rats on a low salt diet (LSD, 0.05% sodium) were treated daily with Col (30 microg/kg), CsA (15 mg/kg), and both CsA and colchicine or vehicle (olive oil 1 ml/kg) for 4 weeks. The effects of colchicine on chronic CsA nephrotoxicity were evaluated by examining renal function, histopathology, and ED-1 positive cells. The expressions of OPN mRNA and protein were estimated respectively by Northern blot and immunohistochemistry. RESULTS: Compared with vehicle-treated rats, CsA-treated rats showed an increase in serum creatinine, a decline in creatinine clearance rate, and tubulointerstitial fibrosis (all p < 0.01). Concomitant administration of colchicine reversed all of the above parameters (all p < 0.01). Of note, the upregulated expression of osteopontin mRNA and protein seen in CsA-treated rats was significantly decreased after colchicine treatment. Furthermore, the expression of osteopontin mRNA was strongly correlated with the number of ED-1 positive cells (r = 0.712, p < 0.001) and the tubulointerstitial fibrosis score (r = 0.586, p = 0.007). CONCLUSION: Colchicine is capable of abrogating the upregulation of chemotactic OPN expression and macrophage influx, and this is associated with improved renal tubulointerstitial fibrosis in chronic CsA nephrotoxicity.     

TGF-beta stimulates collagen (I) in vascular smooth muscle cells via a short element in the proximal collagen promoter

BACKGROUND: Accumulation of extracellular matrix contributes to the development of intimal hyperplasia. Transforming growth factor beta (TGF-beta) stimulates the production of several matrix proteins in vascular smooth muscle cells (SMC) including type I collagen, but the underlying mechanisms of TGF-beta's effects are not well understood. MATERIALS AND METHODS: The effect of TGF-beta on type I collagen biosynthesis was determined by a [3H]proline incorporation assay and Northern blotting. The promoter of human alpha2(I) procollagen (COL1A2) gene was analyzed by transient transfection analysis and gel mobility shift assay. RESULTS: Treatment of human vascular SMC with TGF-beta stimulated collagen synthesis and increased the level of alpha2(I) collagen mRNA. A collagen-luciferase reporter gene, constructed by linking the human COL1A2 promoter with the firefly luciferase gene, was transiently expressed in human SMC. Treatment with TGF-beta significantly stimulated the activity of this collagen-luciferase reporter. Using deletion analysis, we identified a 150 bp DNA fragment (-334 to -184) in the human COL1A2 promoter as the site through which TGF-beta mediates collagen gene expression in human SMC. Gel mobility shift assays demonstrated that this 150 bp DNA fragment formed conjugates with multiple nuclear factors derived from SMC, a process that was further enhanced by TGF-beta. CONCLUSIONS: TGF-beta stimulates the human type I collagen gene via a DNA element located in the proximal region of its promoter. Interventions that disrupt interaction between this DNA element and nuclear factors may block the production of collagen in response to TGF-beta and consequently may have a significant effect on the development of intimal hyperplasia.     

Dual response of animals with chronic cyclosporine nephrotoxicity to an acute ischemic injury

Normal kidneys regenerate after acute injury with little development of chronic fibrosis. However, the long-term effects of an acute injury in kidneys with established chronic toxicity induced by cyclosporine (CsA) are not entirely clear. To study the consequences of an ischemia and reperfusion (IR) injury in long-term CsA-treated rats, male Wistar rats (250-300 g) were treated daily with CsA (10 mg/kg) or vehicle (olive oil 1 mL/kg) for 28 days. On day 21, ischemia was performed by clamping the renal vessel for 1 hour. Blood samples were collected on days 0 and 21 (before IR) as well as days 22 and 28. On day 28, the kidneys were collected to examine the mRNA expression of MCP-1 by real-time PCR. For renal function, serum creatinine levels were measured. Twenty-four hours after reperfusion, long-term CsA-treated animals showed better renal function compared with the control group, as demonstrated by serum creatinine levels: 2.2 +/- 0.13 mg/dL vs 2.9 +/- 0.18 mg/dL, respectively (P < .05). However, 1 week after IR, the renal function was worse among the long-term CsA-treated group than the controls: 1.16 +/- 0.08 mg/dL vs 0.8 +/- 0.09 mg/dL, respectively (P < .05). Interestingly, CsA treatment was associated with lower MCP-1 mRNA expression than that in the control group: mean MCP-1 mRNA expression 0.58 +/- 0.13 vs 1.02 +/- 0.12, respectively (P < .05). In conclusion, animals with chronic CsA nephrotoxicity were protected from an acute renal injury, possibly through decreased chemokine production, although at later time points, renal function was clearly impaired, probably by the acceleration of vasculopathy caused by nephrotoxicity.     

Pentosan polysulfate treatment reduces cyclosporine-induced nephropathy in salt-depleted rats

BACKGROUND: Long-term cyclosporine (CsA) treatment leads to a decreased glomerular filtration rate, hyalinosis of afferent arterioles, and striped cortical tubulo-interstitial fibrosis. We showed previously that pentosan polysulfate (SP54) prevented the development of microvascular and interstitial lesions in mouse models of progressive glomerulosclerosis. In this study, we examined the effect of pentosan polysulfate on the development of CsA nephropathy. METHODS: Pair-fed Sprague-Dawley rats were fed a low-sodium (0.03%) diet and received CsA (15 mg/kg, subcutaneously, in olive oil)/5% glucose, pentosan polysulfate (10 mg/kg, subcutaneously in 5% glucose) plus CsA, olive oil/pentosan polysulfate, or olive oil/5% glucose for 30 days. Creatinine clearance (CrCl) was determined at three time points. Afferent arteriolar lesions, glomerular volume, and tubulo-interstitial lesions were quantitated. RNA was extracted from cortex. RESULTS: Severe lesions were found in the CsA group. A reduction in the number of affected arterioles (32%) and the degree of chronic tubulo-interstitial lesions (44%) was found in pentosan polysulfate/CsA-treated rats. A 20% decrease in glomerular volume was found in CsA rats, but not in pentosan polysulfate/CsA-treated rats. Pentosan polysulfate treatment did not prevent the CsA-induced decrease in CrCl (approximately 30%) at 4 weeks. CsA did not affect cortical endothelial or neuronal nitric-oxide synthase or mRNA levels, but there was small increase in neuronal nitric-oxide synthase mRNA levels in the pentosan polysulfate/CsA-treated group. CONCLUSIONS: Pentosan polysulfate reduced structural renal lesions in CsA-treated, salt-depleted Sprague-Dawley rats.     

In vivo effect of the natural antioxidant hydroxytyrosol on cyclosporine nephrotoxicity in rats.

BACKGROUND: Cyclosporine A (CsA) is the first-line immunosuppressant used in transplant patients and in auto- immune diseases. Nephrotoxicity is the major limitation of CsA use. Although the mechanisms of nephrotoxicity have not been completely defined, some evidence suggests that reactive oxygen species (ROS) play a causal role. The present study was designed to investigate in vivo effects of hydroxytyrosol (DOPET), a natural olive oil antioxidant, on oxidative stress, renal histology and haemodynamic alterations induced in rats by CsA treatment. METHODS: Adult Sprague-Dawley rats were treated i.p. with CsA (15 mg/kg) alone or in combination with DOPET (20 mg/kg) for 3 weeks. At the end of the treatment, superoxide concentration within the cells of the abdominal aorta and renal artery was quantified from the oxidation of dihydroethidium (DHE) using fluorescence microscopic imaging analysis. In kidney tissues, lipid peroxidation was measured by thiobarbituric acid-reacting substances (TBARS) assay, glutathione level was assessed enzymatically and the expression of haem oxygenase-1 (HO-1) gene was evaluated by semiquantitative RT-PCR. Renal morphology was studied by classical histological techniques, while the glomerular filtration rate (GFR) was estimated by inulin clearance. Systemic blood pressure was monitored by the tail method and through the catheterization of the carotid artery. RESULTS: CsA administration increased superoxide concentration both in the aorta and in the renal artery, while DOPET completely prevented this effect. Higher levels of TBARS, a significant decrease in GSH and an upregulation of HO-1 mRNA were observed in the kidneys of CsA-treated rats. DOPET treatment reversed quantitatively these effects. However, CsA-dependent changes in renal histology were only partially reversed by DOPET. Finally, CsA induced a severe reduction in GFR and a significant increase in both systolic and diastolic blood pressure; the DOPET treatment had no significant effect on these haemodynamic alterations. CONCLUSION: The reported data indicate that effective DOPET protection from CsA-induced oxidative stress is associated with a mild effect on histological damages and does not affect the altered glomerular function and the hypertension, thus indicating that kidney injury by CsA is only in part dependent on oxidative stress.     

Effect of FTY720 on chronic cyclosporine nephropathy in rats

BACKGROUND: Long-term treatment with cyclosporine A (CsA) causes tubulointerstitial inflammation and fibrosis in the kidney. To define the role of lymphocytes in this process, the novel lymphocyte-specific inhibitor FTY720 was administered to rats with experimental model of chronic CsA nephropathy. METHODS: Sprague-Dawley rats were treated daily for 4 weeks with CsA (7.5 mg/kg), or both CsA and FTY720 (0.125 mg/kg). The effects of FTY720 on CsA-induced renal injury were evaluated using renal function tests and histopathology, and the expression of mediators of CsA-induced renal injury (osteopontin, transforming growth factor-beta1 [TGF-beta1], betaig-h3, and angiotensin II). RESULTS: FTY720 treatment significantly decreased T-lymphocyte accumulation in kidneys compared with CsA treatment alone. FTY720 treatment improved not only CsA-induced renal dysfunction but also renal histopathology, demonstrated by decreased macrophage infiltration and interstitial fibrosis. Increased osteopontin, TGF-beta1, betaig-h3, and angiotensin II expression in CsA-treated rat kidneys were decreased with FTY720 treatment. CONCLUSIONS: FTY720 treatment prevents CsA-induced renal injury.     

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.     

Cyclosporine induces myocardial connective tissue growth factor in spontaneously hypertensive rats on high-sodium diet

BACKGROUND: The introduction of cyclosporine (CsA) has led to an improvement in the prognosis of solid organ transplantation. However, drug-induced hypertension and nephrotoxicity, associated with the development of atherosclerosis and coronary heart disease, still worsen the long-term outcome of CsA-treated patients. Whether the CsA-induced myocardial changes are associated with the induction of connective tissue growth factor (CTGF), a recently found polypeptide implicated in extracellular matrix synthesis, is not known. METHODS: Spontaneously hypertensive rats (8-9 weeks old) were treated with CsA (5 mg x kg(-1) x d(-1) subcutaneously) for 6 weeks. The influence of angiotensin-converting enzyme inhibition (enalapril 30 mg x kg(-1) x d(-1) orally) and angiotensin-1 receptor blockade (valsartan 3 and 30 mg x kg(-1) x d(-1) orally) on CsA toxicity was also investigated. Myocardial morphology was examined, and vascular lesions were scored. Localization and the quantitative expression of CTGF, as well as collagen I and collagen III, mRNA were evaluated by in situ hybridization and Northern blot. RESULTS: CsA-induced hypertension and nephrotoxicity were associated with myocardial infarcts and vasculopathy of the coronary arteries. CsA increased myocardial CTGF, collagen I, and collagen III mRNA expressions by 91%, 198%, and 151%, respectively. CTGF mRNA expression colocalized with the myocardial lesions. Blockade of the renin-angiotensin system prevented vascular damage and the CsA-induced CTGF, collagen I, and collagen III mRNA overexpressions in the heart. CONCLUSIONS: CsA increases CTGF, collagen I, and collagen III mRNA expressions in the heart. The induction of CTGF gene is mediated, at least in part, by angiotensin II.     

转化生长因子β1在实验性慢性环孢素A肾病中的作用

目的　观察转化生长因子 β1(TGFβ1)在环孢素A(CsA)所致大鼠肾小管间质纤维化中的作用 ,探讨阻断肾素血管紧张素系统对TGFβ1表达的影响。 方法　低盐饮食 (钠质量分数 0 .0 5 % )饲养 7d后随机分成⑴对照组 ;⑵CsA处理组 ;⑶CsA 盐酸维拉帕米处理组 ;⑷CsA 依那普利处理组 ;⑸CsA 氯沙坦处理组。大鼠皮下注射CsA(15mg·kg-1·d-1) 4周 ,制成慢性CsA肾病模型 ,分别用Northernblot检测TGFβ1mRNA、免疫组织化学方法检测TGFβ1、二聚糖蛋白表达。 结果　CsA增加大鼠肾脏TGFβ1mRNA及蛋白水平表达 ,增加二聚糖蛋白水平。依那普利、氯沙坦可显著减少TGFβ1、二聚糖蛋白表达 (P <0 .0 5 ) ,并能明显减轻肾小管间质纤维化。结论　TGFβ1可能介导了CsA引起的肾小管间质纤维化 ;阻断肾素血管紧张素系统 ,可明显减轻肾小管间质纤维化程度     

Expression of H60 on mice heart graft and influence of cyclosporine

OBJECTIVES: It has been reported that the MHC class I chain-related antigen (MIC), a ligand of NKG2D, an activating receptor of natural killer cells, is expressed on rejected renal allografts. This study investigated whether heart transplantation induced expression of a homologue of MIC (H60) in outbred Kun Ming (KM) mice, widely used in China, and whether CsA had an influence on the process. METHODS: Forty-eight KM female mice were divided into untreated and CsA-treated groups, after cervical heart allotransplantation. Grafts were harvested 1, 3, 5, and 7 days postoperatively. H60 mRNA was analyzed by RT-PCR, and the protein detected by immunohistochemistry. RESULTS: Compared with no mRNA expression in the normal heart, H60 mRNA was observed at day 5 and upregulated on day 7 in the untreated group. It was detected on day 3, peaked on day 5, but was lower than untreated group, and decreased on day 7 in the CsA-treated group. H60 protein was detected in cardiocytes only on day 7 in the untreated group. CONCLUSION: Our study suggested that expression of the NKG2D ligand, H60, may activate natural killer cells playing a significant role in innate immunity associated with transplantation. The early expression of H60 mRNA on day 3 in the CsA-treated group might relate to the toxicity of CsA. The expression peaked on day 5 and decreased on day 7, possibly induced by CsA. The results suggested that H60 might be a new target for prevention of rejection.