The Protective Effects Of Fasudil In Lipopolysaccharide Induced Acute Kidney Injury Rats And The Related Mechanisms

Object:Sepsis-induced acute kidney injury (Sepsis-AKI) remains a leading cause of acute renal failure in hospital. This study aimed to investigate the protective effects of fasudil in Sepsis-AKI rats and the related mechanisms.Method:1.24 Male Wistar rats (250-300g) were kept on a 12-h-light/dark cycle and had free access to water and standard rat diet. The rats were randomly allocated into the followi ng groups:(1) Control group (Control), treated with normal lml saline by intravenous injection (n=8); (2) Lipopolysaccharide group (LPS), administered an i.v. injection of 6mg/kg Lipopolysaccharide diluted with saline to lml (Schering AG, Germany) at a rate of 0.5ml/min (n=8); (3) Fasudil group (FAS), identical to the LPS group, with the addi-tional pretreatment with fasudil (30 mg/kg FAS), diluted with saline to 1ml, given single intravenous (i.v.) one hour before the LPS injection (n=8);2. Biochemical parameters and inflammatory cytokines:Starting at 24 hours after the injection of lipopolysaccharide or saline, the urine was collected for 24 hours (i.e., 1440 mins) for creatinine measurement. Blood samples were obtained at 48 hours after the injection of lipopolysaccharide. Blood urea nitrogen (BUN), serum creatinine (Scr), urine creatinine and urine albumin were measured using automatic biochemistry analy-zer at the Clinical Laboratory, in Zhongnan Hospital of Wuhan University. The crea-tinine clearance (CrCl) was calculated according to the formula:CrCl=UV/P, where U represents the urinary creatinine concentration (?mol/L); V is the total urine volume collected in milliliters divided by the duration of the collection in minutes (ml/min); P is serum creatinine concentration (?mol/L). The CrCl was then corrected for a body weight of 100 g and was thereby expressed as ml/min/100g. Detect TNF-? and IL-10 content in renal and plasma by ELISA kit.3. Histological observation:Forty-eight hours after administration of lipopoly-saccharide or saline, the rats were sacrificed, kidney removed and then fixed in 10% formalin for 24h for routine dehydration and paraffin embedding. Sections were cut at 4 ?m and stained with hematoxylin and eosin (H&E). Histological assessment of medullary damage, including tubular vacuolization and degeneration, necrosis, renal tubular casts, and inflammatory infiltration.4. Terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay:To detect apoptotic DNA fragmentation, TUNEL assay was performed on paraffin-embedded kidney tissue with an In Situ Cell Death Detection Kit (Roche, Mannheim, Germany), according to the manufacturer's instructions. After fluorescein staining, the apoptotic cells (i.e., Tunel positive cells) appeared brightly green in color under a fluorescent microscope.5. Immunoblotting:The expression of regulatory proteins(GRP78, CHOP, caspase-12 and ROCK-1) were determined Immunoblotting.The immunoblots were then visualized using an enhanced chemiluminescence system, and photographs were assessed by densitometry by using Image-Pro Plus-6.0 software in a blinded manner. Results were expressed as the ratio of the density of the protein of interest to that of ?-actin.Result1. Biochemical parameter and inflammatory cytokines:The biochemical parameters were presented in Table 1.Compared with the rats in Normal control group, LPS significantly reduced CrCl in AKI rats (0.84vs1.68 ml/min/100 g,P<0.01=, while it significantly increased serum creatinine, blood urea nitrogen and urinary (P<0.01=. In contrast, CrCl was much higher in FAS group (1.03 ml/min/100 g, respectively) than the LPS group. Serum Cr and BUN were lower in FAS group than in the LPS group, (tabel 1);For TNF-? and IL-6, it showed higher level both in renal and plasma after LPS administration than Control group,and Fasudil reduced the expression of TNF-a and IL-6.(tabel 2)2. Histological changes:In the renal medullary and cortex region, there were tubular vacuolization and degeneration, necrosis, and hyaline or cellular casts associated with infiltration of mononuclear cells in the Sepsis-AKI rats. Notably, there were much fewer pathological changes in FAS groups. (Fig.1)3. TUNEL assay:Renal tubular cells in Sepsis-AKI rats exhibited more TUNEL-positive cells than in Control rats, indicating that LPS induced significant apoptosis. TUNEL-positive cells in FAS group were significantly fewer than those of the LPS group. (Fig.2)4. Expression of ROCK-1:The expression of ROCK-1 increased in the LPS group vs. the Control group, additionally, the expression of ROCK-1 in FAS group was lower than LPS group. (Fig.3)5. Expression of caspase-12:Compared with 23% in Normal controls, the caspase-12 activity significantly increased to 62% in Sepsis-AKI rats. Its activity gradually decreased to 51% in Fasudil group. (Fig.4)6. Expression of GRP78 and CHOP:For GRP 78 and CHOP, it showed higher expression after LPS administration than Control group. The up-regulation of GRP 78 and CHOP induced by LPS was significantly and progressively attenuated in FAS group. (Fig.5 and 6)Conclusuon:The present results suggest that early application of fasudil could prevent the development of LPS induced renal tubular cells apoptosis by suppression of ER-stress pathway and inflammatory.