Experimental Study Of Bone Morphogenetic Protein-7-modified Mesenchymal Stem Cells On IgA Nephropathy

Background and Objective:IgA nephropathy (IgAN), its features including recurrent macroscopic haematuria or microscopic hematuria, mesangial proliferation, deformation or split of basement membrane and IgA-deposition in the mesangial area, is one of the most common primary glomerulonephritis in our country. Nearly 25% of IgA nephropathy patients progress to end-stage renal disease (ESRD) over a 20-25 year follow-up period. Up to now, no specific treatment has been defined, the treatment for IgAN using glucocorticoid, endoxan, tripterygium wilfordii and ACEI isn't specfic. New therapeutic approaches to inhibit IgA-deposition in the mesangial area and improve the function of glomeruli need to be researched. Recently, several studies have reported that bone marrow mesenchymal stem cells (BMSCs) transplantation has the potential to treat renal diseases. BMSCs are the adult stem cells and exhibit many intriguing characteristics: easily obtaining and culturing, long-term survival in vivo, easily carrying exogenous gene, having the capability of differentiating into multiple cell types. Some studies have showed that BMSCs can differentiate into renal cells in the injured renal tissue to contribute to renal repair. BMSCs, as a valuable tool for cell therapy, have potential to restore damaged renal tissues and improve renal function.Bone morphogenetic protein-7 (BMP-7), a member of the TGF-βsuperfamily, can promote mesenchymal-to-epithelial transition, induce the differentiation and development of mesangial cells, epithelial cells and endothelial cells, counteract TGF-β1 mediated pro-fibrotic effects in the mesangial cell, reduce TGF-βinduced ECM protein accumulation. BMP-7 can inhibit renal fibrogenesis and has a protective effect on the kidney.The study was designed to assess the therapeutic potential of mesenchymal stem cells transplantation for IgA nephropathy and whether BMP-7- modified mesenchymal stem cells deliver to the kidney could result in biologically significant functional recovery. In this study, we first established an IgAN model in BALB/c mice, then injected GFP or BMP-7 modified BMSCs into the IgAN mice. We observed the distribution and differentiation of MBSCs in glomeruli and the resumption of renal function, and investigated whether BMP-7- modified BMSCs deliver to the kidney could result in biologically significant functional recovery. The method will offer reference for the treatment of IgAN.Methods:1. BMSCs were isolated according to standard techniques using density gradient centrifugation and adherent culture. Cells morphology were observed with phase contrast microscopy,cells were identified by phenotypic protein examination with flow cytometry and immunochemistry staining. The ability of the BMSCs to differentiate into osteocytes and adipocytes was tested with alkaline phosphatase, Alizarin Red S and Oil Red O.2. A recombinant adeno-associated virus vector encoding BMP-7 (pAAV-BMP-7) was constructed with the technique of molecular cloning. The recombinant expression plasmid or pAAV-GFP were co-transfected of HEK-293 cells with pHelper and pAAV-RC by calcium-phoshate precipitation method. Recombinant AAV viral particles were used to infect bone marrow mesenthymal stem cells. The expressions of BMP-7 or GFP of infected cells were observed by immunocytochemical staining or by fluorescence microscopy.3. IgA nephropathy model of mice were established and were randomly divided into three groups which were control, GFP-BMSCs transplantation, BMP-7-BMSCs transplantation. GFP or BMP-7 mediated-BMSCs were injected into IgA nephropathy mice via the tail vein. At 7d, 21 d and 56d, mice were sacrificed and kidneys were collected. The distribution of BMSCs was observed by immunohistochemistry staining or laser scanning confocal microscope. GFP-positive BMSCs were observed the coexpression of desmin or CD31 by laser scanning confocal microscope. BMP-7 levels of kidney were detected by western blotting. Serum urea nitrogen, creatinine levels, 24 hour urine protein and histologic appearance were assessed to analyze the effect of GFP-BMSCs or BMP-7-BMSCs transplantation for IgA nephropathy.Results:1. Primary cells demonstrated fibroblast colony-forming units by phase contrast microscopy. In later passages, MSCs contained two morphologically distinct cell types: spindle-shaped cells and large flat cells. However, most cells were spindle-shaped. Cells were tested with flow cytometry. BMSCs expressed the antigens CD29, CD44, CD90 and CD166, They were negative for CD34 and CD45. Immunochemistry staining results showed that significant expression of vimentin in BMSCs, but E-cadherin was negative. When cultured in osteogenic medium for 14d, ALP stain for cells were positive, cells began to mineralize their matrix and were positive for Alizarin Red S with the time. They were also able to differentiate into adipocytes, oil droplets were stained red by Oil Red O after cultivation in adipogenic medium for 2w.2. rAAV-GFP and rAAV-BMP-7 was produced successfully. BMSCs were detected by BMP-7 immunocytochemistry method or observed with fluorescence microscopy after transfection. The gene expression efficiency in BMSCs was about 70%.3. IgAN was produced by continuous oral immunization with 0.1% bovine serum albumin (BSA) in the drinking water, followed by three daily intravenous injections of SEB. Mice showed proteinuria, IgA-deposition in the mesangial area with immunohistochemical staining, swelling of glomerular and mesangial proliferation.4. GFP-positive cells were visible in the glomeruli under laser scanning microscope at 7 d after GFP-BMSCs transplantation, at 56 d the number of GFP-positive cells increased, in addition, GFP-positive cells were present in the periglomerular space, and the interstitium. A few of GFP-positive cells expressed desmin or CD31 with immunocytochemistry.Histological examination showed that BMP-7 gene was expressed in the glomeruli after transplantation of BMP-7-BMSCs. Western blot showed the BMP-7 level of kidney of IgA nephropathy mice with BMSCs transplantation was significantly greater than no transplantation. The level of kidney injected with BMP-7-BMSCs was significantly greater than that in rats treated with GFP-BMSCs and the level was increasing from 7 to 56 d. We assessed IgA-deposition in the mesangial area. After transplantation of BMSCs, mesangial depositions of IgA by immunofluorescent staining was weak. The Serum urea nitrogen, creatinine levels and 24 hour urine protein were decreased by BMSCs transplantation, the level of BMP-7-BMSCs transplantation was lower significantly than GFP-BMSCs transplantation at 56d.Conclusions:1. High purified BMSCs can be obtained by the combination of gradient centrifugation and adherent culture. These cells show typical morphology and the mesenchymal stem cell phenotype, they also could be induced to differentiate to adipocytes and osteocytes in special conditions.2. rAAV-BMP-7 and rAAV-GFP was constructed successfully, viral titer is 3-4×1010IU/ml. Transduction of BMP-7 or GFP gene to BMSCs by rAAV is safe and high efficient.3. BMSCs can migrate in the glomeruli and a few of BMSCs can differentiate into mesangial cells and endothelial cells. After transplantation of BMSCs, renal function of IgA nephropathy mice recover obviously. BMP-7-BMSCs transplantation is more beneficial to recover than simple BMSCs transplantation.