Amlodipine Prevents Kidney Fibrosis By Regulating Smad Expression

PART ONEAmlodipine prevents gentamicin-induced rat tubulo interstitial fibrosis by regulating Smad2, Smad7expressionObjective The model of gentamicin-induced rat tubulo interstitial fabrosis was established to observe effect of amlodipine on gentamicin-induced rat renal tubular injury by regulating Smad2, Smad7expression.Methods In this investigation,28healthy male Sprague-Dawley rats were used. The animals were divided into four groups of seven animals each. Amlodipine (2.5mg/kg/day,5mg/kg/day) was administered through an intragastric gavage (i.g.) daily for8consecutive days. The next day in the administration of amlodipine, rats in all groups except the control group then received100mg/kg/day gentamicin intraperitoneal injection (i.p.) daily for7consecutive days, rats received simultaneously distilled water for the control and gentamicin groups. The treatment protocols were repeated daily for8consecutive days before the animals were sacrificed and samples collected for biochemical measurements. Twenty-four-hour urines were collected at the8th day after administration of amlodipine to measure the content of urinary protein, the activity of y-Glutamyl-transpeptidase (GGT), N-acetyl-β-D-glucosaminidase (NAG), and alkaline phosphatase (ALP). After that, the rats were anaesthetized with45mg/kg sodium pentobarbital and approximately3mL of blood was collected via a femoral artery, sera were centrifuged at200×g for5min at+4℃to measure blood urea nitrogen (BUN); and serum creatinine (SCr). The content of malondial dehyde (MDA), anti-oxidase activity of superoxide dismutase (SOD) in renal were detected. The content of nitric oxide (NO) and the activity of nitric oxide synthase (NOS) in renal cortex were measured. Rat kidneys were soaked in10%formaldehyde solution and embedded in paraffin. The tissue sections were stained with hematoxylin and eosin and lesions of glomerulus and tubules were observed under optical microscope. Apoptosis was evaluated by terminal dexoynucleotidl transferase mediated d-UTP nick and labeling (TUNEL); immunohistochemistry assays were used to detect the levels of Smad2, Smad7expression in tubules.Results Results showed that treatment with gentamicin alone caused significant changes in the levels of urinary protein/24h, NAG, GGT, ALP, BUN, and SCr. Amlodipine effectively reversed the effect of gentamicin on these parameters. The histopathological changes of kidney suggest that the serious congestion of swelling of kidney tubules and the casts including erythrocyte casts and protein casts in gentamicin groups, amlodipine effectively reversed gentamicin-induced rat renal tubular injury. TUNEL Analysis found more apoptotic cells in gentamicin group and the cells were focused on kidey tubules. Amlodipine can significantly decreased the number of apoptotic cells. Immunohistochemistry assays indicated that amlodipine significantly reduced Smad2expression, and increased Smad7expression in gentamicin-induced tubules.Conclusion Amlodipine protected against gentamicin-induced nephrotoxicity by reduced Smad2expression, and increased? expression. PART TWOAmlodipine prevents adriamycin-induced toxicity in cultured rat mesangial cells by up-regulation of Smad6, Smad7expressionObjective The aim of this study was to investigate effects of amlodipine on adriamycin-induced expression of Smad6, Smad7in rat mesangial cells.Methods Mesangial cell line from Sprague-Dawley rats was cultured for experiment. Cytotoxicity was measured using LDH release assay. Real-time PCR and Western blot analyzed that effect of amlodipine on Smad6, Smad7mRNA and protein expression in rat mesangial cells. Immunohistochemical analyzed that effect of amlodipine on Smad7expression in the cells.Results Results showed that amlodipine (10-8to10-5mol/L) significantly decreased LDH activity in rat mesangial cells when given in combination with TGF-β1(P<0.01). Amlodipine (10-7,10-6mol/L) significantly increased Smad6,7mRNA and protein expression in cells treated with adriamycin and TGF-β1(P<0.01). Immunocytochemistry also showed that amlodipine significantly up-regulated adriamycin-induced Smad7expression in mesangial cells.Conclusion amlodipine protects against adriamycin-induced toxicity in rat mesangial cells by up-regulation of Smad6,7expressions.