| Diabetes is one of the most serious health hazards which induced microvascular complications that have been the major cause of disabled and death. Although insulin therapy can effectively control glycemia, it has little effetc on preventing or reversing diabetic microvascular disease and its complications. Therefore, it is one of the main directions of diabetes research on how to prevent or reverse diabetic microvascular disease and its complications.Recently studies show that islet transplantation effectively amiliorat diabetic microangiopathy, however, the mechanism is not fully awared. Stem cells have the ability of self-renewal and differentiating into different cells. The transplantation of islets derived from human fetal pancreatic stem cells to treat diabetic nephropathy has been successful in experimental animals which may become an effective method for future clinical treatment of diabetes.Objective:To evaluate the effects of islet transplantation on ameliorating diabetic nephropathy in rats and to explore the relevant mechanisms—the effect of C-peptide or GLP-1on treating diabetic nephropathy.Method:(1) Transplantation of islets derived from human fetal pancreatic stem cells. Human fetal pancreatic stem cells were isolated, cultured and induced into islet-like structure. Islets with excellent in vitro function were transplanted into the liver of rats with diabetic nephropathy through portal vein. Non-treated rats with diabetic nephropathy used as diabetic nephropathy controls; to exclude the effect of blood glucose lowering on diabetic nephropathy, insulin therapy group was established (insulin glargine injection injected subcutaneously,2.5IU/day); Wistar rats used as normal controls. Treatment time was16weeks. Before and every2weeks after treatment, blood glucose,24-hour urine volumn,24-hour urinary albumin and24-hour urinary protein were monitored in each group. After treatment, kidneys of all rats were removed rapidly to observe the general morphology and pathology; Real-time PCR werer used to detect the expression levels of GPSD core proteins. Real-time PCR, Western Blotting and immunohistochemistry were used to detect the expression of RAGE, PKC, PKA, iNOS and SOD-1in glomerular.(2) C-peptide or GLP-1treatment. ALZET osmotic pumps infused with C-peptide dilution or GLP-1dilution were implanted into abdominal of GK diabetic rats with diabetic nephropathy. C-peptide were infused at the constant rate of50pmol· kg-1· min-1; GLP-1were infused at the constant rate of15pmol· kg-1· min-1; vehicle treated group (ALZET osmotic pump were infused with2ml0.9%saline embedded intraperitoneal,0.9%Salt is also the dilution of C-peptide and GLP-1); insulin-treated group (nsulin glargine injection was injected subcutaneously,2.5IU/day); normal Wistar rats were used as controls. Treatment time was12weeks. Before and every2weeks after treatment, blood glucose,24-hour urine volumn,24-hour urinary albumin and24-hour urinary protein were monitored in each group. After treatment, kidneys of all rats were removed rapidly to observe the general morphology and pathology; Real-time PCR werer used to detect the expression levels of GPSD core proteins. Real-time PCR, Western Blotting and immunohistochemistry were used to detect the expression of RAGE, PKC, PKA, iNOS and SOD-1in glomerular.(3) cAMP or H89treatment. GK rats with diabetic nephropathy has been injected cAMP (5mg/kg) or H89(5mg/kg) intraperitoneally twice daily for2weeks. Before and every week after treatment, blood glucose,24-hour urine volumn,24-hour urinary albumin and24-hour urinary protein were monitored in each group. After treatment, the kidneys from all groups were removed to observe the general morphology and renal pathology.Results:(1) Islets derived from stem cells were sensitive to the stimulation of high concentration of glucose in vitro. Immunohistochemical staining showed that the transplanted islets in liver express human insulin, human C-peptide and human glucagon. At the end of treatment, the blood glucose of islet transplantation group was8.88±2.06mM (P<0.001), the blood glucose of insulin treatment group was8.53±1.43mM (P<0.001) were significantly lower than diabetic nephropathy group (30.83±2.33mM). The24-hour urine volumn of islet transplantation group was49.17± 12.42ml,24-hour urinary albumin was11.78±2.18mg,24-hour urine protein was362.20±75.24mg, which were significantly lower than diabetic nephropathy rats.The renal function of insulin treatment were not significantly different from the diabetic nephropathy group (P>0.05).Islet transplantation improve the podocyte morphology, partially restored the expression of core protein of GPSD, reduced the thickness of glomerular basement membrane and mesangial hyperplasia; however, insulin treatment do not have the effect on remodeling glomerular structure.(2) C-peptide treatment or GLP-1treatment can improve diabetic kidney function in GK rats:at the end of treatment,24-hour urine volume (23.57±4.48ml),24-hour urinary albumin (237.16±51.18μg),24-hour urine protein (209.17±44.42mg) of GK rats with C-peptide treatment was significantly lower than the rats treated with vehicle; the24-hour urine volume (19.57±3.84ml),24-hour urinary albumin (279.36+56.11μg),24-hour urine protein (154.17±34.27mg) of GK rats with GLP-1treatment was significantly lower than the rats treated with vehicle; the latter's24-hour urine volume; however, insulin treatment has little effect on reverse diabetic nephropathy, the24-h urine volume,24-h urinary albumin and24-h urinary protein continues to rise, and there were not differences between the insulin treatment group and the vehicle treatment group (P>0.05). C-peptide treatment or GLP-1treatment can improve podocyte morphology, reduce the thickness of GBM and mesangial hyperplasia, and partially restore the core protein expression of GPSD; insulin therapy does not have the effect on ameliorate renal structure in rats with diabetic nephropathy.(3) cAMP and H89treatment had little effects on reducing glycemia and24-hour urine volume in rats with daibtic nephropathy. However, after cAMP treatment, the24-hour urinary albumin (1203.27±287.64μg),24-hour urine protein (468.33±37.29) was significantly lower than vehicle treated rats (P<0.001); after H89treatment, the24-hour urinary albumin (3002.59±269.07μg),24-hour urine protein (701.16±48.73mg) was significantly higher than vector treatment of diabetic GK rats (P<0.001). Pathological examination showed that the GBM of vehicle treated rats (504.25±77.92nm) was significntly thickened compared with the normalrats (125.77±27.13nm), and the podocytes showed fusion or deletion; cAMP treatment can reduce the GBM (224.31±62.37nm) which was sifnificantly reduced compared with the vehicle treated rats (P<0.001) and improve the podocytes morphology; the GBM of H89treatment (745.37±86.55nm)was significantly higher than vehicle treated rats (P <0.001).Conclusion:Transplantation of islet derived from human fetal pancreatic stem cells has therapeutic effects on diabetic nephropathy which may be related to the secretion of endocrine hormones other than insulin such as C-peptide, glucagon and so on. |
PDF Full Text Request