| Objective:To explore the protective effect of Exosomes derived from human urine-derived stem cells (USCs-Exo) on kidney in type 1 diabetic nephropathy (DN) rats and the mechanism.Methods:1. Isolation, culture and identification of USCs. Exosomes in USCs conditioned medium were obtained using ultracentrifugation. The USCs-Exo fraction was assessed by transmission electron microscopy. Western blotting was performed to confirm the identity of the USCs-Exo by the presence of the specific surface proteins CD9, CD63 and CD81.2. Diabetes was induced by a single injection of streptozotocin (STZ) Control rats received citrate buffer. SD rats who met the criteria for diabetes were assigned randomly to treated or untreated. The rats of USCs-Exo treatment diabetic group were injected weekly Exo.3. The random blood glucose was detected every week. Twenty-four-hour urine specimens were obtained from all rats every 4 weeks for the measurement of creatinine levels and albumin concentration. Results of urinary albumin were normalized to urinary creatinine levels and expressed as urinary microalbumin-to-creatinine ratio (UACR). The serum creatinine and urea nitrogen was detected. Twelve weeks after injection of Exo, rats were sacrificed, and kidneys were obtained. The kidneys were stained with Periodic Acid-Schiff to observe pathophysiological changes, including glomerular mesangial matrix proliferation. Real time PCR and Western blotting was used to assess the expression of mRNA and protein of Bc1-2ã€Bax and Caspase3, respectively.4. Human podocytes were cultured to differentiate completely in vitro. Then they were divided mainly into six groups according to treatment:normal glucose (NG), mannitol control (NG+Ma), high glucose (HG), treatment group in USCs-Exo (5ug/ml, 10ug/ml and 50ug/ml) for 72h. Flow cytometry was used to detect the apoptosis of PDC binding Annexin V-FITC/PI. Analysis of apoptosis-related caspase-3 protein expression was analyzed by western blotting. Real time PCR was used to assess the expression of synaptopodin mRNA.Results1. USCs and USCs-Exo provided enough raw materials for further experiments:Cell colonies were observed in the USCs cultured plates approximately 5 to 7 days after initial plating. USCs were confirmed using light microscopy to verify a fibroblast-like morphology. The fibroblast-like cells had a robust proliferation capability and reached 80%-90% confluence after 16 days. After several passages USCs retained the elongated morphology. We used flow cytometer to detect the surface antigen expression. The results demonstrated that the USCs were positive for CD29, CD90, CD73 and CD44 antigen and negative for CD34 and HLA-DR. USCs could differentiate into osteoblasts and adipocytes. The morphology of USCs-Exo was observed under transmission electron microscopy, and its size was measured by using Nano Sight analysis. The results showed that USCs-Exo was 30-120 nm spherical vesicles. Western blotting showed that exosomal markers, including CD9, CD63 and CD81 were expressed in USCs-Exo.2. USCs-Exo could attenuate UACR and reduce glomerular mesangial proliferation, decrease tissue cells apoptosis, improve renal function, and delay the occurrence and development of DN:urine volume and UACR in diabetes rats increased significantly in the Normal group (P<0.05), which early met the diagnostic DN criteria. While Diabetes + USCs-Exo treatment group compared with Diabetes group UACR increased less and more slowly.3. Glomerular volume and mesangial area increased; within the glomeruli obvious apoptosis, the Bcl-2 gene and protein expression significantly reduced, Bax and Caspase3 increased significantlv. But above changes in Diabetes+ USCs-Exo treatment group were obviously improved (P< 0.05).3. USCs-Exo could prevent the apoptosis and the injury induced by high glucose in HPDC:in HPDC of HG group, compared with NG group, the rates of apoptosis at 72h were increased dramatically(vs.NG, P<0.05), the protein expressions of caspase3 were also increased significantly(P<0.05), the expression of synaptopodin mRNA in HG was found to decrease (P<0.05), On the contrary, in PDC of HG group, compared with HG group USCs-Exo reduced the rate of apoptosis and the expression of synaptopodin mRNA with a dose-dependent manner(P<0.05), decreased the apoptosis-associated protein expressions of caspase3.Conclusion1. USCs were ideal seed cells has a good application prospect. USCs-Exo extracted from USCs-CM expressed specificity of relatively conservative protein evolution. Meanwhile, USCs-Exo could protect HPDC injuried by HG.2. USCs probably suppressed the podocytic apoptosis in vivo and in vitro to protect the kidney. |
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