Effects Of Human Amniotic Mesenchymal Stem Cells On Angiogenesis-related Factors In Kidney Of Diabetic Rats

Objective:Diabetic nephropathy?DN?is one of the most common microvascular complications of diabetes mellitus,and its development is closely related to abnormal expression of angiogenesis-related factors in the kidney.At present,there is a lack of effective prevention and treatment for DN clinically.Stem cell transplantation may be a new strategy for the DN.The existing researches mostly focus on mesenchymal stem cells?MSCs?,while the effects of human amniotic mesenchymal stem cells?hAMSCs?on the renal lesions and angiogenesis-related factors in diabetes are rarely reported.The aim of the study was to assess the changes of renal pathology and angiogenesis-related regulatory factors after the intervention of hAMSCs,and to explore the effect of h AMSCs on renal microangiopathy in type 1 diabetic rats.Methods:HAMSCs were obtained from the placenta amniotic membrane of full-term cesarean section,which were isolated by trypsin-collagenase digestion method.The DN model was induced by intraperitoneal injection with streptozotocin?55mg/Kg?.A total of72 rats were randomly devided into the normal control?NC?group?n=24?,DN group?n=24?,and hAMSCs transplantation?MSC?group?n=24?.The rats in the MSC group were transplanted with h AMSCs via penis vein injection at 3^th and 4^th weeks,respectively.At 6^th,8^th and 12^th weeks,the blood,urine,renal and pancreatic tissue were collected respectively after rats were sacrificed.And the blood glucose,urea,creatinine,24h urine protein,urinary kidney injury molecule-1?KIM-1?,renal and pancreatic pathological changes were observed.MAB1281 was tested by immunofluorescence to evaluate hAMSCs in the renal colonization.The expression of protein and mRNA of VEGF,TSP-1,Ang-1,Ang-2 and Tie-2 in renal tissue were respectively tested by immunochemistry,immunofluorescence or RT-PCR.Results:?1?The levels of blood glucose,urea,creatinine,cystatin C,24h urine protein and KIM-1 in the DN and MSC groups at each time point were increased in varying degrees compared with the NC group.Although the blood glucose level of the MSC group was lower than that of the DN group at the same time,the only significant difference was observed at 12^th weeks?P<0.05?.The level of urea in the MSC group at 6^th and 12^th weeks was lower than that in the DN group?P<0.05?.However,there was no significant difference in serum creatinine and cystatin C between the MSC and DN groups at the corresponding time?P>0.05?.The level of 24h urine protein in the MSC group was lower than that in the DN group at the corresponding time,but there was significant difference only at 12^th weeks?P<0.05?.The level of KIM-1 in the MSC group was also significantly lower than that in the DN group at 6^th and 8^th weeks?P<0.05?,but higher than that in the DN group at 12^th weeks?P<0.05?.?2?The histopathology result showed that the islet structure in the DN and MSC groups was destroyed to some extent,and the infiltration of inflammatory cells in the margins of islets was also observed.The kidney/body weight,mesangial matrix,and number of inflammatory cells were increased.And the basement membrane of the renal tubule thickened to a certain extent.In the MSC group,the abovementioned pathological changes of islets and kidneys were relatively alleviated.?3?MAB1281 was found in renal tissue of the MSC group at each time point by the immunofluorescence staining method,but it was not observed in the DN and NC groups.?4?The protein expressions of TSP-1,VEGF,Ang-1 and Tie-2 in the DN and MSC groups were significantly higher than those in the NC group?P<0.05?.The protein expression of TSP-1 in the MSC group was lower than that in the DN group at the same time?P<0.05?.Although the protein expressions of VEGF and Ang-1 were higher than that of the DN group at the same time,there was significant difference of VEGF expression only at 6^th and8^th weeks?P<0.05?,and the expression of Ang-1 was only significantly different at 8^thh weeks?P<0.05?.The protein expression of Tie-2 was higher at 8^th weeks,lower at 12^thh weeks than that in the DN group?P<0.05?.?5?TSP-1,VEGF,Ang-1,Ang-2 and Tie-2mRNA expression in the DN and MSC groups were all up-regulated than those in the NC group.The mRNA expression of TSP-1 in the MSC group was lower than that in the DN group at the same time,but significant difference was observed only at 8^th week?P<0.05?.The mRNA expression of VEGF in the MSC group was lower than that of the DN group at12^th weeks?P<0.05?.Ang-1 mRNA expression in the MSC group was higher than that of the DN group at the same time,but there was significant difference at 12^th weeks?P<0.05?.However,the exprenssion of Ang-2 and Tie-2 mRNA had no significant difference with the DN group?P>0.05?.?6?Correlation analysis showed that 24h urine protein,KIM-1 and TSP-1 were positively correlated with each other,and so did Ang-1,VEGF,and Tie-2.The 24h urine protein was negatively correlated with VEGF,Tie-2 and Ang-1.TSP-1 was negatively correlated with VEGF,Tie-2 and Ang-1.KIM-1 was positively correlated with urea,but negative correlated with Tie-2.Conclusion:?1?hAMSCs transplantation can help to reduce the blood glucose level in STZ-induced type 1 diabetic rats;?2?hAMSCs can improve the renal microangiopathy in STZ-induced type 1 diabetic rats,and this effect may be related to improving the expression of angiogenesis-related factors in kidney,and decreasing the blood glucose in diabetic rats.