The Protective Effects Of Adiponectin In Diabetic Nephropathy And Its Related Mechanism

Diabetic nephropathy(DN) is one of the major complications of diabetes mellitus, it's characteristic with mesangial expansion,extracellular matrix deposition,basement membrane thickening of glomeruli and glomerulosclerosis.The pathogenesis of DN is unkown,it associates with altered glomerular hemodynamics,metabolic disorder,oxidative stress,cytokines and hereditary susceptibility.Diabetic nephropathy has worse outcomes and is more difficult to be treated than nephropathy caused by other diseases, the reasons are unclear.Resent studies have shown that the increased secretion of cytokines and over expression of extracellular matrix(ECM) protein play the important roles in the progression of diabetic nephropathy.Among them, transforming growth factor (31 (TGF-β1)is an important cytokine, which directly or indirectly promote the components of ECM, leading to renal fibrosis in patients with diabetes.Resently,more evidences show that oxidative stress (OS) plays an key role in the development of diabetic nephropathy. Overproduction and less consumption of reactive oxygen species(ROS) resulting in oxidative stress,ROS itself can attack lipids, proteins and DNA, leading to kidney damage.However the mechanisms by which ROS production induce kidney damage need to be addressed.Human adiponectin(ADPN) is a novel adipose-derived adipocytokines.In humans,adiponectin is the most abundant gene transcript proteins in adipose cells. It is also known as complement-related protein (Acrp30) or 28KD collagen binding protein (GBP28).The human adiponectin gene is encoded by apM1 mRNA, located on 3q27,a total length of 16Kb.It is composed by three exons and two introns. ADPN has been shown to have anti-atherosclerosis and anti-inflammatory properties as well as regulation of insulin-sensitizing metabolic effects and vascular protective properties.In 1995,Scherer et al first cloned and identified ADPN.Recently, many researchers confirmed that anti-oxidation properties also have been attributed to adiponectin. On the other hand,clinical studies have demonstrated that plasma concentrations of ADPN are closely related with progression of diabetic nephropathy. Furthermore,circulating adiponectin levels were negatively correlated with proteinuria in type-2 diabetics in early stage,which shows that endothelial dysfunction is associated with low circulating adiponectin. The urinary and serum adiponectin increased in patients with advanced diabetic nephropathy,these data suggest that ADPN may be through regulation of anti-inflammatory,,anti-atherosclerosis to reduce kidney damage and delay the progression of diabetic nephropathy.However,little is known about the protective mechanism of adiponectin on diabetic nephropathy.Therefore, in the present study, we observed the effects and mechanism of recombinant globular adiponectin (gAd) on the generation of TGF-β1,ROS and endothelial NO Synthase (eNOS) in vivo and in vitro.The specific aims are addressed in there thesis.The objective,methods,result and conclusion are listed here. Chapter one Studies of the effects of Adiponectin on oxidative stress and the expression of TGFβ1 in HMCs and involved Cell Signal PathwayObjective:To investigate the effects of adiponectin on high glucose induced ROS,eNOS and TGF-β1 expression in human mesangial cells(HMCs), and study the role of Adenosine Monophosphate Kinase (AMPK) signal pathway in adiponectin actions.Methodes:AdipoR mRNA expression were detected in cultured HMCs by RT-PCR. Cell proliferation was assessed using the MTT assay method.HMCs were incubated with 30mM high glucose and different concentrations of globular adiponectin (gAd,3-10ug/ml)for 24h and 36h to investigate the dose dependent effect.Then the cells were randomly assigned into four groups:the control group, the high glucose group(30mM,HG),HG+adiponectin group, HG+adiponectin+AMPK inhibitor(araA) group.The generation of ROS was detected by fluorescence probe, eNOS and TGF-β1 expression were determined using RT-PCR and Western blot respectively.Furthermore,to define the involved signal pathway we incubated the HMCs with gAd for 0-30minute to detect the expression of AMPK and phosphorylated AMPK by Western blot.Result:1)The expression of AdipoR were exhibitted in HMCs,and the AdipoR2 expression was more than that of AdipoRl for 3.5fold; 2)Adiponectin significantly inhibited cell proliferation in a dose-and time-dependent manner;3)Treatment of HMCs with adiponectin resulted in a time-dependent increased phosphorylation of AMPK;4) Compared to HG group, Cells treated with gAd showed high glucose-induced ROS release inhibited, the expression of eNOS up-regulated and the expression of TGF-β1 decreased (p<0.05).The effects of gAd were partly blocked by AMPK inhibitor araA.Conclusions:Adiponectin inhibits cell proliferation and inhibits generation of ROS induced by high glucose in HMCs.Furthermore, adiponectin also stimulates eNOS activity and down regulates the expression of TGF-β1.The mechanism is partly through activation of AMPK signal passway mediated by AdipoRl. Chapter two Construction of eukaryotic expression plasmid pIRES2-EGFP-gAd and analysis its expression in rat kidneyObjective:To construct the eukaryotic expression plasmid pIRES2-EGFP-gAd, and observe the expression of gAd in rat kidney.Methodes:The gAd cDNA fragments were obtained by PCR from pET/gAd(Plasmid which contains a full length cDNA of gAd).Then, the PCR product was linked with T-vector and translated into JM109.It was digested by two restrictive endonuclease,then the gAd cDNA was collected and recombined with eukaryotic expression vector pIRES2-EGFP by using gene recombination technique.The recombined plasmid pIRES2-EGFP-gAd was transfected into normal rat kidney with Lipofectamine Transfection Reagent by intraperitoneal injection. The kidney tissues were collected at different time points (24h,48h,96h,7d) after injection,gAd/GFP green fluorescence protein expression was determined by fluorescence microscopy and Western Blot respectively.Result:1)The pIRES2-EGFP-gAd expression plasmid was constructed successfully.2) The gAd/GFP green fluorescent protein was detected at the glomeruli and tubular in the 24th hour after injection and the fluorescence intensity became stronger in the 48th hour. The level of fluorescence protein expression became gradually weakened in the 7th day. In Western Blot test, same results were observed. Conclusions:The pIRES2-EGFP-gAd gene was expressed in rat kidney successfully by Lipofectamine Reagent with intraperitoneal injection.In the experiments we fused a green fluorescent reporter gene of the EGFP gene in the 3'end of gAd, not only retained the biological activity of gAd but also could easy to detect protein expression in gene therapy.These provide experimental basis for further investigating of adiponectin on diabetic nephropathy. Chapter three In vivo studies the role of adiponectin on the renal protective effect in diabetic nephropathyObjective:To investigate the reno-protective effect of adiponectin on renal damage in streptozotocin(STz)-induced diabetic rats and its mechanisms.Methodes:Diabetes mellitus models were induced by high-lipids and high-sucrose feeding plus STZ intraperitoneal injection. The recombinant plasmid of pIRES2-EGFP-gAd was induced into rat models mediated by transfection reagent with intraperitoneal injection.32 wistar rats were randomly assigned into four groups:the normal control group(NC);diabetes group without any therapy(DM);the diabetes group treated with pIRES2-EGFP-gAd(DA) and the diabetes group treated with pIRES2-EGFP(DP).After corresponding treatments for 8 weeks,blood glucose,HbA1c and urinary microalbumin(UMA) were measured.The kidneys were collected to test the generation of reactive oxygen species (ROS).The renal pathologic changes were observed by light microcopy.The mRNA expression of eNOS and TGF-β1 were determined by real-time PCR;the protein of eNOS,TGF-β1 and p-AMPK were assessed by immunohistochemical method.Result:UAER and generation of ROS were increased in DM group as compared with control group(P<0.05),while there was no significant differences in UARE among the DM,DA,DP groups(P>0.05).Blood glucose level,HbA1c and generation of ROS were decreased in DA group as compared with group DM(P<0.05).Compared with control group,there were glomerular hypetrophy, mesangial expansion, basement thickening,tubular epithelial cells cavitation and exfoliation, and mononuclear lymphocyte infiltration in DM group.While these changes were ameliorated in gAd transfection group. The expression of eNOS and p-AMPK in DM group were decreased, the expression of TGF-β1 was increased compared with control group(p<0.05),but in gAd transfection group the expression of eNOS and p-AMPK increased compared with DM group(p<0.05),and the expression of TGF-β1 was even lower than DM group(p<0.05).Conclusions:Adiponectin has reno-protective effect on diabetic rat kidney.The mechanisms of it are correlated with stimulating the AMPK signal passway, inhibiting the generation of ROS,relieving oxidative stress,up-regulating the expression of eNOS in renal tissues of diabetic rats;on the other hand,it also can down-regulate the expression of TGF-β1,reduce the accumulation of ECM in the renal interstitial.