| Objective: Diabetic nephropathy (DN), a common complication of diabetes, is one of the main cause of end stage renal disease. Overt diabetic nephropathy has no effective treatment at present. Adult stem cells (ASC) have the capability of self-renew and multipotential differentiation like other stem cells. However, ASC has the advantages of easy to obtain, low oncogenic risk and less ethics dispute compared with embryonic stem cells, so it has wide clinical prospect. Lots of studies have confirmed that ASC can reduce renal damage and promote the regeneration of renal cells, but there are few studies focus on the effects of ASC on DN. The treatment effects are different because of the different methods to establish DN model. This research is aimed to observe the therapeutic effects of human Adipose-derived mesenchymal stem cells (hAD-MSCs) on the rat model of 1 type diabetic nephropathy and investigate its possible mechanism.Methods: 1. Animal model: Male SD rats were injected intraperitoneally with STZ (65mg/kg) 2 weeks after single-nephrectomy. Blood sugar was detected 1, 2 and 4 weeks later. The rats whose blood sugar greater than 16. 7mmol/L were successful 1 type diabetic model. Protamine zinc insulin (2-4IU every day, subcutaneous injection) was given to maintain blood sugar at 16.7-33.3mmol/L. From the model was established, the weight of rats was measured every two weeks, blood sugar and 24h urine protein were detected every four weeks. 2. Groups:①single-nephrectomy group ( NX ) ,②DN model group ( NX+STZ ) ,③hAD-MSCs treatment group( NX+STZ+hAD-MSCs). 3. hAD-MSCs therapy: 12 weeks after STZ injection, the rats of NX+STZ+hAD-MSCs group were given hAD-MSCs by tail intravenous injection (5×106), once every four weeks, five times of continuous. NX+STZ rats were injected with an identical volume of PBS. 4. Evaluation Index:①32 weeks after STZinjection was the end of this research.②Kidney and pancreas tissues were collected. Kidney weight/ body weight ratio was recorded. A part of tissues was rapidly frozen in liquid nitrogen for further molecular biological detection, another part of tissues was embedded in the OCT and stored in liquid nitrogen for frozen section examination and the other tissues was fixed in formalin and embedded with paraffin.③The morphologic changes of pancreas were observed, the expression of insulin and glucagon were detected by double immunofluorescence labeling technique and laser scanning confocal microscopy. The pathological changes of renal tissues were examined by Periodic Acid-Schiff Staining and semiquantitative analysis. The expression of synaptopodin was evaluated by immunofluorescence assays.④The expression of synaptopodin was also determined by western blot.⑤Hochest and CFSE labeled hAD-MSCs were given to the rats by tail intravenous injection. The colonization of hAD-MSCs was observed in the kidney, heart, lung, liver, spleen and pancreas 1h, 2h, 4h, 6h, 12h, 48h, 7d after injection by laser scanning confocal microscopy.Results: 1. Rat model of diabetic nephropathy was established by using STZ injection combined with single-nephrectomy: Compared with the single-nephrectomized rats of the same age, 32 weeks DN rats have lower body weight(577.9±60.6g, 429.1±44.7g), urine protein(41.6±19.3mg, 369.4±193.6mg)and kidney weight/ body weight ratio(4.11±0. 68×10-3, 10.63±3.86×10-3) of them were significantly increased. The renal pathological manifestations of DN rats included glomerular hypertrophy, mesangial matrix expansion, glomerulosclerosis, vacuolar degeneration in renal tubular epithelial cells, protein cast, tubular ectasia, focal tubule atrophy, inflammatory cell infiltration. The expression of synaptopodin, a marker of podocyte, was reduced obviously. The pathological manifestations of pancreas were islet atrophied and decreasing in number of the pancreas islet. 2. Therapeutic effects of stem cells on DN and pancreas injury:①There was no obvious difference in blood sugar (34.1±5.6 mmol/L, 30.8±7.4 mmol/L)and insulin expression between NX+STZ group and NX+STZ+hAD-MSCs group. The dosages of insulin in the two groups were the same(4IU).②Compared with NX+STZ group, 24h urine protein of the rats in NX+STZ+hAD-MSCs group was reduced significantly ( 369.44±193.65 mg, 156.12±85.92mg)(P<0.05).③Kidney weight/ body weight ratio was markedly decreased(10.63±3.86×10-3, 8.01±2.39×10-3)(P<0.05), Mean Glomerular Area(4.19±0.54μm2×103, 3.41±0.44μm2×103)(P<0.05)and the area of inflammatory cell infiltration and interstitial fibrosis(4.80±1.81%, 2.32±0.84%)(P<0.05) were decreased, kidney injury index was also significantly reduced.④Immunofluorescence and western blot analyses showed the expression of synaptopodin in podocyte was up-regulated. 3. Results of tracing study: 1h to 48h after hAD-MSCs therapy, a little hAD-MSCs were found in the lung, spleen and kidney of the rats, but none labeled cell was found later.Conclusion :This research established a stable rat model of 1 type diabetic nephropathy. Intravenous administration of hAD-MSCs repeatedly could decrease urine protein of DN rats, reduce glomerular hypertrophy and podocyte lesions, lessen inflammatory cell infiltration and interstitial fibrosis. The protective effects of hAD-MSCs on renal lesions were independent on the colonization and differentiation of stem cells and blood sugar. |
PDF Full Text Request