Beneficial Effects Of Hypoxia Preconditioned Urine-derived Stem Cells On Renal Tissue Repair In Patients With Diabetic Nephropathy

Objective: To compare the biological characteristics between urine-derived stem cell from patients with diabetic nephropathy(D-USC)and Urine-derived stem cell(USC)of healthy people,and to investigate whether effects of proliferation,secretion and apoptosis by hypoxia(3% O2)preconditioned USC can improve kidney damage in diabetic nephropathy.Methods: USC and D-USC were isolated from Urine samples of 10 healthy men aged 20-25 years and 10 male diabetic nephropathy patients.The potential differentiation ability of the cells was examined by the analysis of cell morphology and surface markers via flow cytology and the staining of alizarin red and oil red O,respectively.We also mapped the growth curve to compare D-USC and USC of healthy people proliferative capacity both in normoixa(20% O2)and hypoxia conditions(3% O2).Human angiogenic factor Assay Kit was applied to examine the expression of extracellular secretion factor,performed flow cytology to detect cell apoptosis.Transmission electron microscopy and Western blotting were used to detect the difference of autophagy.Streptozotocin(STZ)-induced type 1 diabetic mice received an injection of USC and Hypoxia-preconditioned urine-derived stem cell(HP-USC)for three times every other week at two weeks after streptozotocin injection.After 6 weeks of last injection,the rats were sacrificed for electron microscopy and histological examination to observe the renal pathological changes.Results:A single,“rice-like” USC and D-USC colony formation was observed on day 5-6 of primary culture.Our results showed that the cells could be cultured in vitro continuously,and that their morphology remained the size of the grains.These cells was shown to expressing mesenchymal stem cell markers(including CD 24,CD 29,CD 73,CD 90,CD 105)and the pericyte markers(CD 146).And these cells did not express hematopoietic stem cell markers(including CD 31,CD 34 and CD 45).It also had bone and lipid differentiation potential;compared with USC,the proliferation of D-USC is impaired,the types of secretory factors are basically the same,but the number is reduced,the rate of apoptosis is increased,and the level of autophagy is higher;Compared with normoxic culture conditions,USC and D-USC can obtain significantly more cell clones under hypoxic conditions,and their value-adding ability and paracrine function are significantly better than those of USC and D-USC under normoxia.Apoptotic phenomena decreased and autophagy decreased.Electron microscopy and histological examination showed that the degree of glomerular injury,inflammation and fibrosis in the USC and HP-USC treatment groups were lower than those in the model group,and the effect of the hypoxia group was better than that of the normoxic group.Conclusion:(1)D-USC were successfully extracted from the urine of patients with diabetic nephropathy,which is consistent with the characteristics of USC.But the ability of proliferation and secretion is impaired,apoptosis is increased,and the level of autophagy is higher compare with USC;(2)3% O2 hypoxic culture can increase the proliferation and secretion of USC and D-USC,inhibit the occurrence of apoptosis,and reduce the level of intracellular autophagy;(3)Hypoxic preconditioning can improve the role of USC in the progression of diabetic nephropathy.