Experimental Studies Of Metanephric Mesenchymal Cells Transplanting In Adriamycin-Induced Chronic Nephropathy In Rats

OBJECTIVETo search the functional repair of metanephric mesenchymal cells (MMCs) in adriamycin(ADR) induced chronic glomerulopathy in rats.METHOD1.Isolation,cultivation and identification of metanephric mesenchymal cells derived from embryonic rats.Rats were anaesthetized on gestation day 13 and metanephric balstemata were separated from ureteric buds by microdissection.Cells were grown in DMEM containing 10%fetal calf serum at 37℃in a 5%CO₂ atmosphere.The morphology of MMCs were observed by inverted phase contrast microscope,HE staining,and electronic microscope.The surface antigen phenotypes were identified by immunocytochemistry,and the purification was identified by flow cytometry. A growth curve to be used for studying the characteristics of growing.2.Labeling and tracing of metanephric mesenchymal cells derived from embryonic rats.MMCs were infected with green fluorescence protein(GFP) before tail vein injection into ADR nephrosis rats,GFP expression was detected at 1 wk,3 wk,and 5 wk,respectively.Also,4',6-diamidino-2-phenylindole(DAPI) and DAPI-labeled MMCs were injected and detected respectively. 3.Search the differentiation of MMCs in vitro and in vivo,and the functional repair of metanephric mesenchymal cells in ADR induced chronic glomerulopathy in rats.We intravenously transplanted metanephric mesenchymal cells into rats treated with ADR to induce glomerulopathy,a model of CKD,and also induced MMCs differentiation in vitro using medium containing ADR rat serum and homogenates.4.Detect the functional repair of transplants of metanephric mesenchymal cells in adriamycin-induced glomerulopathy in rats.90 Sprague-Dawley female rats weighting 200 - 220 g were randomly divided into three groups:ADR glomerulopathy(ADR group,n = 40):Rats were subjected to ADR via the tail vein over a period of 3 weeks.A single dose of 0.25 mg/100 g body weight was used.ADR glomerulopathy followed by MMCs transplantation(ADR-MMCs group,n = 40):8 weeks after the second ADR administration,animals were transplanted with MMCs. Approximately,5-7×10⁶ MMCs were used per rat.Control(n = 10).All rats were scarified 16 weeks after the second ADR injection.Parameters including 24h urinary protein excretion(UP),serum album(ALB),blood urea nitrogen(BUN),creatinine(Scr),total cholesterol(TCH),renal histology,and collagenâ…£expression in renal were detected in this study.Moreover,matrix metalloproteinases 2(MMP-2) and matrix metalloproteinases 9(MMP-9) expression in the renal were also detected with immunohistochemistry,and quantity analysis of protein and gene was further demonstrated with Western blot or RT-PCR analysis,respectively.RESULT1.MMCs grew adherencely as fibroblast-like,positive for vimentin, nestin,and CD133.Instead,they were negative for cytokeratin(CK),CD34 and Dolichos Biflorus(DB).The flow cytometry showed a single peak of MMCs.2.(1) GFP-transfected MMCs were able to survive and integrate into tubular structures after 1 wk,however,a few GFP-positive MMCs appeared in glomerular tufts.The GFP expression,detected by GFP fluorescence or labeled with an anti-GFP antibody,persisted on 5 wk after transplantation.(2) DAPI-labeled MMCs were also detected to integrate into tubular structures, but during the observation of 5 weeks,DAPI fluorescence was decreased gradually.(3) ADR nephrosis rats injected with DAPI simply were also detected DAPI- positive cell in tubular structures,but DAPI fluorescence was dimmer and significantly decreased during the observation of 5 weeks.3.We detected the induction of an early epithelial phenotype(CK-18) and proximal tubule phenotype(vitamin D receptor) in vitro,and MMC-derived epithelia in proximal tubule and glomerulus in vivo.MMCs transplantation after ADR glomerulopathy significantly increased creatinine clearance rate(Ccr),an indicator of glomerular filtration rate,but had no significant changes in other parameter analysis(24-h urinary protein excretion,serum albumin,total cholesterol,and quantitative analysis of tubulointerstitial injury and glomerulosclerosis).In addition,there is no significant difference of blood urea nitrogen or serum creatinine in the rats with and without ADR administrated.4.Administering two injections of ADR to rats resulted in chronic ADR nephrosis,which displayed severe proteinuria,tubulointerstitial injury and glomerulosclerosis.Compared with ADR group,decreased collagenâ…£and MMP-2 expression,and increased MMP-9 expression were detected in renal in MMCs-ADR group.Collagenâ…£expression was positive correlation with MMP-2,and negative correlation with MMP-9.There were no significant difference between ADR and MMCs-ADR group in levels of UP,TP,ALB, TCH,tubulointerstitial injury score and glomerulosclerosis degree.In addition,just as in part 3,there was no significant difference of blood urea nitrogen or serum creatinine in the rats with and without ADR administrated.CONCLUTION1.The results suggested that the cultured cells were MMCs,they were purified and coincided with stem cells.2.Liposome mediated GFP gene transfer was an efficient labeling in vitro and suitable tracing method for cell differentiation experiment in vivo. MMCs could differentiate into tubule-like cells when transplanted into ADR nephrosis rats. 3.Longer observation should be given in this study.Thus,whether MMCs potentiated functional repair after chronic glomerulopathy in rats is still an area to be further explored.4.Transplants of MMCs contributed to decrease the fibrosis in adriamycin-induced glomerulopathy in rats,and the signaling pathways of matrix metalloproteinases(MMPs) appeared to be involved in these processes.