| Objective: Diabetic nephropathy (DN) is frequent complication of diabetesand one of the major death causes in patients with diabetes. The study exploredthe effects of Rho/Rho kinase (ROCK) signaling pathway on hepatocyte growthfactor (HGF) and transforming growth factor-β1(TGF-β1) in the kidney tissue ina rat model of type2diabetes and observed the effects of Fasudil on DN,withthe objective of providing a novel strategy for the treatment of DN.Methods: The experimental rat model of type2diabetes was establishedby high-fat diet combined with low-dose streptozotocin (STZ) intraperitonealinjection, and the control group (NC group) was established. At week10,Glucose infusion rate (GIR)was measured by hyperinsulinemic-euglycemicclamp. The fasting blood glucose (FBG), fasting insulin (FINS), triglycerides(TG) and cholesterol (TC) were measured. Then, the diabetic rats wererandomly divided into two groups: untreated diabetic rats (DM group) thatreceived vehicle and treated diabetic rats (DF group) that received ROCKinhibitor fasudil hydrochloride hydrate (Fasudil,10mg/kg/d,i.p.). Systolicarterial blood pressure (SABP), body weigh (BW) and blood glucose wasmeasured weekly. At week24, Fasting plasma was collected for furthermeasurement of FINS, FBG, TG, TC, creatinine (Cr), urea nitrogen (BUN) andglycosylated hemoglobin (HbA1c). The homeostasis model assessment forinsulin resistance (HOMA-IR) was calculated. The kidneys were harvested andweights (KW) were recorded.24h urinary albumin excretion rate (UAER)andhydroxyproline were measured；The renal histological changes were observedby hematoxylin-eosin (HE), and the volume of collagen was evaluated bymasson staining. The mRNA expression of HGF and TGF-β1were examined byreverse transcriptase polymerase chain reaction (RT-PCR).The phosphorylationof MYPT1was examined by Western blot. Results:1Changes of body weight：At week10, the BW of the high-fat diet rats was significantly higher thanthat of the control rats(P＜0.05).At week24, the BW of the rats in DM groupand the DF group were lower than that in NC group(P＜0.05). There was nosignificant differences in BW between DM group and DF group(P＞0.05).2The changes of biochemical indexAt week10, FBG,FINS,TG and TC of rats in DM group were significantlyhigher than those in NC group(P＜0.01). At week24, FBG,FINS,TG,TC andHbA1c of the rats in DM group and DF group were significantly higher thanthose in NC group(P＜0.01). There was no significant differences in these indexbetween DM group and DF group(P＞0.05).3Insulin sensitivityAt week10, GIR of the diabetic rats were significantly lower than those inNC group(P＜0.01). At week24, the HOMA-IR was calculated as FBG×FINS/22.5, HOMA-IR of the rats in DM group and DF group were significantlyhigher than those in NC group(P＜0.05). There was no significant differences inHOME-IR between DM group and DF group(P＞0.05).4SABPAt week24, There was no significant differences in SABP among threegroups(P＞0.05).5RW, RW/BWAt week24, RW and RW/BW of rats in DM group were significantly higherthan those in NC group(P＜0.01),RW and RW/BW of rats in DF group weresignificantly lower than those in DM group(P＜0.01). There was no significantdifferences in RW and RW/BW between DF group and NC group(P＞0.05).6Renal function and UAERAt week24, the BUN of rats in DM group and DF group were significantlyhigher than those in NC group (P＜0.01). But there were no significantdifferences in BUN between DM group and DF group(P＞0.05).There was nosignificant differences in Cr among three groups (P＞0.05). The UAER of rats in DM group were significantly higher than those in NC group(P＜0.01). TheUAER of rats in DF group were significantly lower than those in DM group(P＜0.01).7Concentrations of HYP in renal tissuesAt week24, the concentrations of HYP in renal tissues in DM group weresignificantly higher than those in NC group (P＜0.01).The concentrations ofHYP in renal tissues in DF group were significantly lower than those in DMgroup(P＜0.01). There was no significant differences in HYP between NCgroup and DF group(P＞0.05).8HE stainingIn the renal tissues of rats of NC group, the structure of kidney glomeruluswas normal and the kidney tubules epithelial cells which were rich in cytoplasmlined up in order. However, the kidney glomerulus hypertrophy, mesangialregion extension, increased matrix and the kidney tubules epithelial cells whichwas structure-disordering cytoplasm-lost and foam-like all could be observed inthe renal tissues of rats in DM group. The alterations mentioned above were allobviously ameliorated in DF group.9Masson stainingThere was a small quantity of collagen fiber in renal interstitium andaround vascular in renal tissues of rats of NC group. Compared with NC group,collagen fiber in renal interstitium and around vascular in renal tissues of ratssignificantly increased in DM group(P＜0.01). Collagen fiber in renalinterstitium and around vascular in renal tissues of rats was markedly reduced inDF group compared with DM group(P＜0.01). There were no significantdifferences in collagen fiber in renal interstitium and around vascular betweenNC group and DF group (P>0.05).10mRNA expressions of HGF and TGF-β1in renal tissuesThe mRNA expression of HGF in renal tissues in DM group weresignificantly lower than NC group (P＜0.01). The mRNA expression of HGF inrenal tissues in DF group were significantly higher than those in DM (P＜0.01).There was no significant differences in HGF between NC group and DF group(P ＞0.05). The mRNA expression of TGF-β1in renal tissues in DM group weresignificantly higher than those in NC group (P＜0.01), There was no significantdifferences in TGF-β1between NC group and DF group(P＞0.05).11Protein expressions in renal tissuesThe phosphorylation MYPT1(p-MYPT1) in renal tissues in DM groupwere significantly higher than those in NC group (P＜0.01). The p-MYPT1inrenal tissues in DF group were significantly lower than those in DM (P＜0.01).There was no significant differences in p-MYPT1between NC group and DFgroup(P＞0.05).Conclusions:1The rats made by high-fat diet combined with low-dose STZintraperitoneal injection exhibit hyperglycemia, hyperlipidemia and insulinresistance. These suggested a rat model of type2diabetes is successfullyestablished.2The results show that hyperglycemia activates the Rho/ROCK pathway inkidney, and the Rho/ROCK pathway results in extracellular matrix expansion inthe mesangial region via the regulation of HGF and TGF-β1in kidney, whichplay an important role in the pathological process of DN.3Fasudil, ROCK inhibitor, can suppress renal fibrosis and proteinuria indiabetic rats in part through the upregulation of HGF and the balance betweenHGF and TGF-β1. ROCK may be a novel therapeutic target for DN. |
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