Changes Of Renal Tissue Proteomics And Mechanisms Of Early Tubulointerstitial Injury In Rats With Type 2 Diabetes

Objective:To investigate the possible mechanisms of early renal tubulointerstitial lesions with proteomics and the protective effects of the natural glycosaminoglycan(GAG)product Sulodexide on renal tubulointerstitial lesions in type 2 diabetes mellitus(T2DM)rats.Methods:Four-week-old female SD rats were treated with mtraperitoneal injection of low-dose streptozotocin(35 mg/kg)on a high-sugar,high-fat diet(normal feed+10%sucrose+10%lard+10%egg yolk powder)for six weeks to establish the animal model of T2DM.The success of the T2DM model was proved by the blood glucose greater or equal to 16.67mmol/L and clearly insulin resistance detected by the hyperinsulinemic-euglycemic clamp test.The T2DM rats were randomly divided into diabetes group(DM group)and diabetes treated with GAG group(GAG group),the same age of SD rats were served as normal control group(NC group).The sulodexide was intravenously injected at a dose of 10 mg/kg body weight every day for six weeks in the GAG group,and the equal volume of saline was inj ected to the rats of other two groups in the same way.The general state,biochemical indicators,microalbuminuria(mALB),urine N-acetyl-?-D-glueosaminidas(NAG),urine kidney injury molecule 1(KIM-1)were recorded in all the rats.Renal pathological changes were evaluated by light microscope and transmission electron microscope(TEM).The differentially expressed proteins were screened using proteomic technique.The expression of TLR2/4 inflammatory pathways related molecules and the glycocalyx markers-syndecan(SDC)1 and 4 were detected using the techniques of immunohistochemistry,western blot,and real-time PCR.Results:1)The type 2 diabetic rats showed increased water intake and urine output,and inceased kidney index(KI).All of the changes above were improved after treatment with GAG,especially the changes of BW(P<0.01)and KI(P<0.01).2)Compared with the NC group,the levels of urine mALB,NAG and KIM-1 were increased significantly in the DM group.After GAG treatment,the changes of urine mALB,NAG and KIM-1 were ameliorated in the diabetic rats.3)The light microscope showed severely tubular vacuolar degeneration and tubulointerstitial inflammatory cell infiltration in the DM rats.The TEM also showed severely tubular injury,included:the tubular microvilli broke off,the large amount of cytoplasm vacuoles formed,the mitochondria swelling,and the mitochondrial ridge breaks.Meanwhile,the glomerular lesions were mild.All the lesions above were greatly improved after the GAG supplementation.4)The 2824 proteins in renal tissues were successful identification by proteomics in T2DM rats.Compared with the NC group,there were 388 differentially expressed proteins in the DM group,of which 173 were up-regulated and 215 were down-regulated.We found that TLR2/4 inflammatory pathway-associated protein Myeloid Differentiation Factor 88(MyD88),Ras-related C3 botulinum toxin substrate 1(Racl),high mobility group box 1(HMGB1),and NF-?B inflammation-associated protein S100A8 were significantly up-regulated,so the TLR2/4 pathway was selected for further study.5)The expression of TLR2/4 inflammatory pathway related molecules and the CD68 positive cells in the renal tubulointerstitial of the DM group were significantly higher than that in the NC group(P<0.01).After GAG supplementation,the changes of the above were significantly improvedd(P<0.01);The expression of SDC1 and SDC4 were found in the renal tubules,but only the SDC1 expressed different statistically among the groups.Conclusion:1)Tubulointerstitial changes are the main lesions of the kidney in the early stage in T2DM rats.2)Proteomics showed the early renal lesions of the T2DM rats may be associated with the TLR2/4 inflammatory pathway.3)The early renal tubulointerstitial lesions in T2DM rats are closely related to the activation of TLR2/4 inflammatory pathway.4)The glycocalyx markers SDC1 and SDC4 expressed in the luminal and cytoplasm of the renal tubules.The microvilli lesions of the renal tubular in T2DM rats may be associated with the decrease of glycocalyx.5)Supplementation of GAG can significantly reduce the early renal tubulointerstitial lesions in T2DM rats.The mechanism may be related to inhibiting the expression of TLR2/4 inflammatory pathway,reducing the infiltration of tubulointerstitial inflammatory cells,and increasing the expression of renal tubular SDC1,and improving the microvilli lesions of renal tubules.