The Role Of Chemerin In Renal Failure In Patients With Diabetic Nephropathy

Objective:Through the study of related factor expression in rat mesangial cells and diabetic renal dysfunction rat model,we investigated the effect of chemerin on TGF-?1/Smads/CTGF signaling pathway and inflammatory factors,and clarified the effect of chemerin on renal insufficiency function in diabetic nephropathy;at the same time,we analyzed the clinical biochemical indexes of patients with renal failure,and explored the relationship between serum chemerin and renal failure.Methods:1.According to the gene bank of rat chemerin gene sequence,adding two restriction sites of Hind III and Bam H I,pc DNA3.1(+)expression vector and full-length chemerin sequence were simultaneously digested and connected.After that we screened positive clones for identification.2.Rat mesangial cells were randomly divided into five groups: control group,low sugar group,high glucose group,chemerin group and combined intervention group.The rat mesangial cells were transfected with the recombinant expression vector,The m RNA level of TGF-?1/Smads/CTGF signaling pathway were detected by Real-time PCR,and the protein expression were detected using Western blot method for semi quantitative analysis.ELISA method was used to determine the levels of inflammatory factors TNF-? and IL-1.The above-mentioned technique were armed to analyze the effects of chemerin on inflammation and TGF-?1/Smads/CTGF signaling pathway.3.The diabetic rat model of renal insufficiency was made.Diabetic rats with renal insufficiency were divided into normal control group,saline group,diabetic nephropathy group,chemotactic group,and combined intervention group.By injected the constructed expression vector of chemerin into rat kidney,the m RNA level of TGF-?1/Smads/CTGF signaling pathway were detected by Real-time PCR,and the protein expression were detected using Western blot method for semi quantitative analysis.ELISA method was used to determine the levels of inflammatory factors TNF-? and IL-1.The above-mentioned technique were armed to analyze the effects of chemerin on inflammation and TGF-?1/Smads/CTGF signaling pathway.4.112 patients with renal failure and 45 healthy controls were randomly selected.The patients were divided into diabetic kidney disease with renal failure group(DKD)and non diabetic kidney disease with renal failure group(NDKD)according to the etiology of renal failure.ELISA method was used to detect the level of serum chemerin,and to analyze the association between the factor and clinical indexes.Results:1.The eukaryotic expression vector pc DNA3.1(+)-chemerin was successfully constructed.The fragment was confirmed by PCR,double enzyme digestion,which was consistent with gene bank.2.The m RNA and protein expression of TGF-?1/Smads/CTGF pathway including TGF-?1,Smad2,Smad4 and CTGF were increased in high glucose group and chemerin group on rat mesangial cells.The inflammatory factor including TNF-? and IL-1 levels were increased.The levels of TGF-?1/Smads/CTGF pathway and TNF-?,IL-1 were significantly higher in combined intervention group.3.The m RNA and protein expression of TGF-?1/Smads/CTGF pathway including TGF-?1,Smad2,Smad4 and CTGF were increased in the diabetic renal insufficiency group and chemerin groupin.The inflammatory factor including TNF-? and IL-1levels were increased.The levels of TGF-?1/Smads/CTGF pathway and TNF-?,IL-1were significantly higher in combined intervention group.4.Compared with the control group,the serum chemerin level was significantly higher in NDKD group and DKD group,while the level of chemerin in DKD group was higher than that in group NDKD.There was no significant difference in serum chemerin level between male and female patients.After adjusting for age and gender,simple linear regression analysis showed that the serum chemerin level was positively correlated with SBP,BUN,Cr,TG,CRP,and negatively correlated with Ccr.Conclusion:1.The eukaryotic expression vector pc DNA3.1(+)-chemerin was successfully constructed,and laid the foundation for the mechanism study of chemerin.2.Chemerin may up-regulated the level of TGF-?1/Smads/CTGF pathway and inflammatory cytokines to injury the rat mesangial cells.3.Chemerin may cause renal dysfunction by up-regulation of TGF-? /Smad/CTGF pathway and inflammatory factors in rats.4.The level of serum chemerin in DKD patients was significantly increased,and the level of serum chemerin was closely related to renal function.