| Objective: Diabetic nephropathy (DN) is one of the most common andsevere microvascular complications of diabetes. At present, there are noeffective measures that can prevent the development and progression of DN.This study investigated the roles of Rho/Rhokinase (ROCK) and transforminggrowth factor beta1(TGF-β1) signaling pathways in the pathogenesis ofdiabetic nephropathy in rats with type2diabetes and the effect of fasudil，aspecific inhibitor of ROCK，on DN, with the objective of providing a novelstrategy for the prevention or treatment of DN.Methods: The model of type2diabetes in rats was established by highfat diet combined with one-time intraperitoneal injection of low dosestreptozotocin (STZ,30mg/kg).Meanwhile, the control group was established.At week10, insulin resistance in the diabetic rats was confirmed byhyperinsulinemic–euglycemic clamp. The fasting blood glucose (FBG),fasting insulin (FINS), triglycerides (TG) and cholesterol (TC) were measured.Then, diabetic rats were randomly divided into untreated diabetic rats(diabetes group) and treated diabetic rats (fasudil group) that received fasudil(10mg/kg/day, i.p.). The body weight (BW) and systolic arterial bloodpressure (SABP) of rats were recorded weekly. At week24,urine samples over24h were collected and the urinary albumin excretion rate(UAER) wasmeasured; FBG, FINS, TG, TC, creatinine(Cr), urea nitrogen(BUN) andglycosylated hemoglobin (HbA1c) were measured. Made the left kidneysweighed (KW) and the ratio of KW to BW (KW/BW) calculated.Themorphology of renal tissue was observed by hematoxylin and eosin (HE)staining.The volume of collagen was evaluated by masson staining and thehydroxyproline (HYP) determination. The mRNA expressions of TGF-β1andconnective tissue growth factor (CTGF) in renal cortex were assessed by reverse transcription polymerase chainreaction (RT-PCR).The phosphorylationof myosin phosphatase target subunit1(MYPT1), representing ROCK activity,was measured by Western blot analysis.Results:1General stateEating, drinking, movement and color of fur of the rats in control groupwere normal. The diabetic rats appeared polyphagia, polydipsia and polyuria.The movement was significantly reduced and the color of fur was dark indiabetic rats.2Insulin sensitivityAt week10, compared with control group, the glucose infusion rate (GIR)was significantly lower in diabetic rats (5.45±0.84mg/kg/min vs11.34±0.76mg/kg/min, P<0.01). At week24, homeostasis model assessment for insulinresistance (HOMA-IR=FBG×FINS÷22.5) was calculated. The HOMA-IR wassignificantly higher in diabetes group and fasudil group compared with that incontrol group (110.86±5.27and109.17±4.88vs12.23±1.99, respectively, P<0.01). However, there were no significant differences in HOMA-IR betweendiabetes group and fasudil group (110.86±5.27vs109.17±4.88, P>0.05).3BW, KW and KW/BWAt week10, compared with control group, the BW was significantlyincreased in diabetic rats (P<0.05). At week24, compared with control group,the BW was markedly decreased in diabetes group and fasudil group (P<0.05).There were no significant differences in BW between diabetes groupand fasudil group (P>0.05).The KW and KW/BW were significantly increasedin diabetes group compared with control group (P<0.01). Compared withdiabetes group, the KW and KW/BW were markedly decreased in fasudilgroup (P<0.01). There were no significant differences in KW and KW/BWbetween control group and fasudil group (P>0.05).4Blood biochemical parametersAt week10, compared with control group, FBG, FINS, TG weresignificantly increased in diabetic rats (P<0.01) and TC was significantly increased in diabetic rats too (P<0.05). At week24, compared with controlgroup, FBG, FINS, TG, TC, HbA1c were markedly increased in diabetes groupand fasudil group (P<0.01). There were no significant differences in FBG,FINS, TG, TC, HbA1c between diabetes group and fasudil group (P>0.05).5Parameters of kidney and SABPAt week24, compared with control group, the UAER was significantlyincreased in diabetes group (0.35±0.04μg/min vs0.05±0.01μg/min, P<0.01).The UAER was markedly reduced in fasudil group compared with diabetesgroup (0.08±0.04μg/min vs0.35±0.04μg/min, P<0.01). Compared withcontrol group, the UAER was significantly increased in fasudil group(0.08±0.04μg/min vs0.05±0.01μg/min, P<0.05). There were no significantdifferences in serum Cr among three groups (P>0.05). Compared with controlgroup, the BUN was markedly increased in diabetes group and fasudil group(13.56±2.19mmol/L and13.36±1.81mmol/L vs8.01±1.11mmol/L,respectively,P<0.01). There were no significant differences in BUN between diabetesgroup and fasudil group (13.56±2.19mmol/L vs13.36±1.81mmol/L, P>0.05).There were no significant differences in SABP among three groups (P>0.05).6HE stainingIn control group, the structure of kidney glomerulus was normal and thekidney tubules epithelial cells which were rich in cytoplasm lined up in order.However, the kidney glomerulus hypertrophy, mesangial region extension,increased matrix and the kidney tubules epithelial cells which werestructure-disordering, cytoplasm-lost and foam-like all could be observed indiabetes group. The alterations mentioned above were all obviouslyameliorated in fasudil group.7Content of HYPCompared with control group, the content of HYP in renal tissue wasincreased significantly in diabetes group (0.50±0.05μg/mg vs0.36±0.07μg/mg,P<0.01). The content of HYP in renal tissue was markedly reduced in fasudilgroup compared with diabetes group (0.37±0.04μg/mg vs0.50±0.05μg/mg, P<0.01). There were no significant differences in the content of HYP in renal tissue between fasudil group and control group (0.37±0.04μg/mg vs0.36±0.07μg/mg, P>0.05).8Masson stainingCompared with control group, the deposition of collagen in renalinterstitium was significantly increased in diabetes group (density of collagen:0.148±0.007vs0.076±0.005， P<0.01). However, treatment with fasudilsignificantly reduced the deposition of collagen in the renal interstitiumcompared with that seen in untreated diabetic rats (density of collagen:0.079±0.007vs0.148±0.007, P<0.01). There were no significant differencesin the deposition of collagen in renal tissue between fasudil group and controlgroup (density of collagen:0.079±0.007vs0.076±0.005, P>0.05).9Immunohistochemistry stainingThere was faint expression of TGF-β1in renal tissue in control group.Compared with control group, the protein expression of TGF-β1in renal tissuewas increased significantly in diabetes group (positive staining area:21.4±3.1%vs4.3±1.5%, P<0.01). The protein expression of TGF-β1in renaltissue was markedly reduced in fasudil group compared with diabetes group(positive staining area:5.1±2.3%vs21.4±3.1%, P<0.01). There were nosignificant differences in the protein expression of TGF-β1in renal tissuebetween fasudil group and control group (positive staining area:5.1±2.3%vs4.3±1.5%, P>0.05).10Gene expression in renal cortexCompared with control group, the mRNA expression of TGF-β1andCTGF in renal cortex were significantly increased in diabetes group (P<0.01).The mRNA expression of TGF-β1and CTGF in renal cortex were markedlyreduced in fasudil group compared with diabetes group (P<0.01). There wereno significant differences in the mRNA expression of TGF-β1and CTGF inrenal cortex between fasudil group and control group (P>0.05).11Protein expression in renal cortexCompared with control group, the phosphorylation of MYPT1in renalcortex was significantly increased in diabetes group (P<0.01). The phosphorylation of MYPT1in renal cortex was markedly reduced in fasudilgroup compared with diabetes group (P<0.01). There were no significantdifferences in the phosphorylation of MYPT1in renal cortex between fasudilgroup and control group (P>0.05).Conclusion： Hyperglycemia can activate the Rho/ROCK signalingpathway. This pathway regulates TGF-β1/CTGF pathway in kidney and playsa critical role in extracellular matrix expansion in the mesangial region in DN.Fasudil suppresses renal fibrosis and ameliorates urinary protein in diabeticrats at least in part via the inhibition of TGFβ1/CTGF pathway, and thenephroprotective effects of fasudil are independent of blood pressure andglycemic control. The present study suggests inhibition of ROCK may be anovel therapeutic target for DN. |
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