| Objective:Diabetic nephropathy (DN) is a major microvascular complication of diabetes (DM), and a leading cause of end stage renal disease (ESRD).The pathogenesis of DN is multi-factorial.There are many mediators in DN, such as advanced glycation end products (AGES),intracellular polyols, angiotensin11, growth factors, and inflammatory cytokines.Of them, TGF-β1is a key mediator in DN. TGF-β1regulates the expression of target genes such as CTGF to echibit its biological activities by activating AGEs/TGF-β1/Smads signaling pathway. Connective tissue growth factor (CTGF),a kind of TGF-β1-mediated early immediate genes, plays an important role in fribrosis as a downstream effector of TGF-β1. Without CTGF, TGF-β could not avtivate effectively, they interract and coordinate in the pathogenesis of DN.It is now well accepted that CTGF is mainly activated via TGF-β1/Smads signaling pathway mediated by AGEs,but the underlying mechanism remain to be elucidated.This study reaches the related factors of DN in diabetic rat model to explore the activity of TGF-β/Smads/CTGF signaling pathway in Diabetes. It also examines the effect of PPAR-γ agonist-pioglitazone which plays a protectic role in the progression of DN.Method:1.Diabetes was induced in Sprague-Dawley rats by a single intravenous tail injection of streptozotocin (STZ,40mg/kg body weight). Experimental animals were randomly divided into normal control group (NC) and diabetic group (DM). The body weight was measured every week, and the blood glucose every two weeks.All the rats were sacrificed after8weeks, a set of biochemical data and indicators related to renal function were detected in each group.At the same time, the liquid nitrogen frozen specimens from renal cortex were taken to use to analyze the level of TGF-β1, CTGF,Smad2,Smad3mRNA by the method of Real-time PCR.The protein expression of TGF-β1and CTGF were determined by immunohistochemistry, then analyzed the result by semi-quantitative method, in order to research the effect of TGF-β1and CTGF in the progression of DN.2.For furtger reseasch of the effect of pioglitazone, we examine the factors related to diabetic function in pioglitazone-treated group(PT,15mg/kg/d),and measure the serum adiponectin by ELISA method in different experimental group. The expression of TGF-β1, CTGF, Chemerin, ChemR23, TNF-α and ICAM-1mRMA were determined by Real-time PCR. TGF-β1， CTGF and Chemerin protein levels were assayed by mmunohistochemistry. Then analyze the results to study the significance of pioglitazone in DN therapy.Result:1. Compared with the normal control group, diabetic rats showed significant increased plasma glucose level and decrease in body weight (P<0.05). The level of BUN,24h urine volume and urinary microalbumin. KW/BW was significantly higher in DM group versus controls (P<0.05), but the level of Scr was significantly lower (P<0.05). TGF-β1and CTGF mRNA expressed in renal cortex were increased obviously in DM group compared with controls, as well as their protein expression,(P<0.05). The expression of Smads also increased in renal cortex of diabetic rats, which Smad3increased significantly (P <0.05), while the Smad2showed no statistically significant difference (P>0.05).2. With Pioglitazone, the body weight increased and serum glucose level decreased compared with diabetic rats. The level of BUN,24h urine volume and urinary microalbumin, KW/BW was significantly lower than the rats in DM group (P <0.05), but there was no obvious effect on the level of serum creatinine (P>0.05). From the ELISA of serum adiponectin (ADPN), its level decreased both in diabetic and Pioglitazone-treated rats, but it is higher in the PT group compared with the rats in DM group (P <0.05). The study also revealed that the TGF-pi and CTGF mRNA and protein expressed in rat renal cortex were decreased with Pioglitazone treatment (P <0.05). The expression levels of chemerin and ChemR23were found to be significantly elevated in diabetic rats compared to control group. After8weeks of treatment, the expression levels of chemerin mRNA showed decrease in the kidney of pioglitazon treated group compared with diabetic group; however, the differences were not significant (P>0.05). In addition, the expression of ChemR23mRNA was significantly decreased by pioglitazone (P＜0.05). The results also indicated that TNF-a and ICAM-1mRNA expression level elevated in renal cotex of diabetic rats (P <0.05), there showed a positive correlation between these inflammatory fators and UMA(TNF-a: r=0.964,P=0.000; ICAM-1: r=0.875,P=0.001) of rats in DM group.Using the Pioglitazone, the expression level of TNF-α and ICAM-1mRNA were significantly decreased (P <0.05).Conclution:1. TGF-β1is a key mediator of fibrosis and regulation the expression of CTGF, which coordinates with TGF-β1to initiate biological activities. Both TGF-β1and CTGF are significantly increased in the early stage of DN, paticipating in the development of DN.2. Kidney damage caused by high blood glucose is mainly induced by TGF-β/Smads/CTGF signaling pathway. The R-Smad composed of Smad2and Smad3plays an important role in the signaling, but Smad3not the Smad2s is the key mediator.3. CTGF acts as the downstream fectors of TGF-β1, expressed in the early stage of DN. As CTGF more specific than other cytokines, it can be used as the indicators of DN early diagnosis with UMA.4. PPAR-gamma agonist pioglitazone can reduce the expression level of renal fibrosis factors TGF-β1and CTGF effectively, and regulate adipocyte differentiation via altering the expression of Chemerin and its receptor, as well as reduce the expression of inflammatory factors, such as TNF-alpha and ICAM-1. Thus, pioglitazone plays a protective role in reducing the renal damage in DM. |
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