Studies On The Mechanisms Of Cyclosporin-induced Chronic Nephrotoxicity

Cyclosporine A (CsA) is a potent immunosuppressive agent currently most widely employed in the management of solid organ transplantation and autoimmune diseases. CsA has significantly reduced both incidence and severity of acute rejection, and brought excellent graft survival rates. Especially,the survival rate of kidney graft reached 90%. Despite its efficacy, the clinical utility of CsA is limited by the severe side effects, mainly nephrotoxicity and arterial hypertension.It was regarded in the past that the pathogenesis of CsA-induced nephrotoxicity was the activation of the renin angiotensin system.In our study ,we successfully established experimental chronic CsA-induced nephropathy models in rat and in human kidney tubular epithelial cellsl(HK-2 cell line).Based on the models,We have investigated the roles of the reactive oxygen metabolites(ROM), NF-kB, iNOs and TGF- B1 and the changes of apoptosis and mitochondrial membrane potential in the CsA-induced nephropathy models.The aim is to supplement the basic theory about the mechanisms of CsA-induced chronic nephrotoxicity ,and provide the experimental and theoretical basis of nephrotoxicity for the development of more effective and less toxic immunosuppressive agents in the future.In the experiments,96 wistar male rat were randomly divided into six groups of sixteen rats as follows. Vehicle:rats received a daily subcutaneous injection of olive oil; LNAME: rats received a daily subcutaneous injection of olive oil and were allowed free access to LNAME water (LNAME was dissolved in distilled water at a concentration of 5mg/100mL); PDTC: rats received a daily subcutaneous injection of olive oil and PDTC 100mg.kg-1.d-1 concurrently;CsA: rats received a daily subcutaneous injection of CsA (CsA was dissolved in olive oil) 15 mg.kg-1.d-1;CsA plus LNAME: rats received a daily subcutaneous injection of CsA 15 mg.kg-1.d-1 and were allowed free access to LNAME water at a concentration of 5mg/100mL;CsA plus PDTC: rats received a daily subcutaneous injection of CsA 15 mg.kg-1.d-1 and PDTC lOOmg.kg-1.d-1 concurrently.After 28 days,twenty-four-hour collections of urine and blood were performed for the determination of blood urea nitrogen (BUN), serum creatinine (SCr) , N-acetyl-beta-d-glucosaminidase (NAG) and urineroutines.After the rats were anesthetized ,the abdomen was opened and the kidneys were removed for the measurements of MDA and GSH-Px ,other kidneys were processed for light and electronic microscopy, immunocytochemistry and TENUL.HK-2 cell were treated with CsA at concentrations of 0.1,1 ,10mmol . L-1 for 24 hours. As inhibitors of NO, 1mmol .L -1 N-nitro-L-arginine-methyl ester (LNAME ) and inhibitors of NF-KB, 25umol . L~1 pyrrolidine dithiocarbamate (PDTC) were added respectively. Apoptosis was detected by Hoechest 33342 staining assay. Mitochondria membrane potential was detected by flow cytometry. The activation of NF-kB was studied by the assessment of NF-KB P65 measured by laser scanning confocal microscope and immunocytochemistry. TGF-B1 was aslo assayed by immunocytochemistry. The expression of iNOs was assessed by western blot.The results are as follows:We established successfully the experimental chronic CsA-induced nephropathy models. It can be observed from light microscopy that CsA resulted in renal cortex swelling ,some renal glomerular capsule disappeared, the structures of renal tubules near the glomerulus were destroyed, CsA also caused vacuolation, nucleus pycnosis and infiltration of neutrophilic granulocyte and macrophage in the proximal tubule.lt can be observed that tubules in the juncture between cortex and medulla markedly diminished and some of the structures destroyed. Electron microscopy studies revealed glomerulus were approximately normal but some parts of basement membranes were unclear, podocyte apophysis turned into flat, mesangial cell apophysis shrinked, mitochondria swelled and nucleolus shrinked into isotrope. CsA enhanced basement membrane thickness and dilatation of the endoplasmic reticulum, giant mitochondria disr...