Experimental Study On The Correlation Between Renal Stem / Progenitor Cell Injury And Renal Fibrosis In Internal Medicine

Part I The role of depletion of renal Progenitors in the development of renalfibrosis in a rat remnant kidney modelBackground Recent studies demonstrate renal stem/progenitor cells exist in maturemammalian kidney and participate in regeneration of podocytes and tubular epithelia afterkidney injury. Subtotal nephrectomy cause development of renal fibrosis, involvingglomerulosclerosis and tubulointersticial fibrosis, which were characterized by podocyte damageand tubule atrophy respectively. This study hypothesized that depletion of renal progenitor existand play a key role in the development of renal fibrosis in the remnant kidney model.Methods Kidney specimens were obtained from the rats underwent5/6nephrectomy.Morphologic analysis was based on HE-staining. The analysis of CD133,Pax-2,Podocin andKi67expression on/in renal cortex cells was performed by flow cytometry. The level ofTGF-β1,VEGF and AngiotensinII(AngII) in renal cortex were measured by western blotting andenzyme linked immunosorbent assay (ELISA) respectively. The expression of AngII type1receptor (AT1R), AT2R and Pax-2mRNA on/in CD133+cells was evaluated by RT-PCR.Result The level of AngII and TGF-β1were upregulated in cortex of remnant kidneyscompared with normal kidneys, as well as increased sign of AT1R mRNA expression onCD133+cells. Going with gradual downregulation of Pax-2gene expression in CD133+cells, theproportions of CD133+Pax-2+cells and Podocin+Ki67+cells in cortex cells of remnant kidneysunderwent progressive decrease with development of glomerulosclerosis.Conclusion Subtotal nephrectomy induces nephrogenous AT1R-mediated activation of RASand upregulation of TGF-β1level, which cause downregulation of Pax-2gene expression, thuslead to reduction of renal progenitors and deficiency of podocyte regeneration, suggesting thatdepletion of renal progenitors exist in the procession of remnant kidney and maybe play a pivotalrole in the pathogenesis of renal fibrosis. Part II The different impacts of subtotal nephrectomy and ischemia-reperfusion injury on renal stem cells/niche of rats and their significance forkidney repairObjective To observe and compare the impacts of subtotal nephrectomy and ischemia-reperfusion injury on renal stem cells/niche of rats, to explore the significance of renal stemcells/niche for renal repair and the possible cause that the prognosis of renal function in the ratswith acute renal failure or chronic renal failure was disparate. Methods Rats of CRF or ARFmodel underwent5/6nephrectomy and renal artery ligation and reperfusion respectively, rats incontrol group underwent sham operation.The collections of blood and random urine was maderegularly to measure the serum creatinine，blood urea nitrogen and24hour urine protein. Kidneyspecimens were obtained at the set time for HE staining and fluorescence staining. Expression ofCD24,CD133and Podocin were detected by immunofluorescence. Reverse-transcriptionpolymerase chain reaction (RT-PCR) analysis was performed to quantify the expression of TGF-β1,Notch2, HGF, BMP7and Pax-2mRNA in renal non-cortex and the expression of PodocinmRNA in renal cortex.Correlation among the expression of Pax-2mRNA, Podocin mRNA andglomerulosclerosis index were test. Results The rats of two models underwent typical courseof ARF or CRF in renal pathology and function respectively, glomerulosclerosis index in CRFgroup increased gradually with time. Compared with the sham group, CD24+CD133+cells ofthe rats in ARF group showed no significant change in quantity and distribution; While the ratsin CRF group show gradual reduction of CD24+CD133+cells. The expression of podocin inglomerulus decrease temporarily and recruit finally in ARF group,but decrease gradually after5/6nephrectomy. Compared with sham operation group, expression of TGF-β1, Notch2mRNAin renal non-cortex was increased in CRF group, while the expression of HGF, BMP7mRNA inrenal non-Cortex of rats in ARF group were increased. Beween the expression of Pax-2mRNAin non-cortex and the expression of Podocin mRNA in renal cortex, there was positivecorrelation in CRF group,while they both have negative correlation with glomerulosclerosisindex. Conclusion Ischemia-reperfusion injury make no obvious impairment to renal stemcells or their niche; Following by renal stem cell niches being progressively injured in structureand function, subtotal nephrectomy reduce renal stem cells and cause podocytes to repair incompetently, that might be the main pathogenesis that chronic renal failure had poor prognosis. Part III The protection of mesenchymal stem cell to Pax-2+cells in theremnant kidey of rats and their therapeutic mechanisms for chronic renalfailureObjective To observe the impact of subtotal nephrectomy on Pax-2+cells in the remnantkidney of rats and the curative effect of mesenchymal stem cells, to explore the mechanism thatMSCs alleviate chronic renal failure. Methods The rat remnant kidney model was produced inFemale Sprague-Dawwley rats by5/6nephrectomy, MSCs were obtained from male SD rats andwere intravenously administrated to rats in Treat-group in2×105one time every4weeks for12weeks, and same volume NS to Rats in Model group synchronously, no treat to rats in contolgroup that underwent sham operation. The collections of blood and24hour urine was maderegularly to measure the serum creatinine,blood urine nitrogen and24hours urine protein.Kidney specimens were obtained at the set time, HE staining for calculating GSI and TII andimmunohistochemstry for calculating Pax-2+cell in renal cortex were performed.Reverse-transcription PCR analysis was performed to quantify the expression of Pax-2, TGF-β1,HGF and BMP7mRNA in renal tissue. Results Rats in model-group show progressiveglomerulosclerosis, tubulointersticial fibrosis and renal dysfunction, those all were alleviated inTreat group. Compared with Sham-operation group, the expression of TGF-β1mRNA wasgradually increased with time after operation(p<0.05), while the number of Pax-2+cell and theexpression of Pax-2and HGF mRNA both were increased transiently on day7(p<0.01),and thensoon decreased and reached control levels after days30in Model group; Compared with Modelgroup, the expression of TGF-β1mRNA was decreased(p<0.05) while the number of Pax-2+celland the expression of Pax-2, HGFand BMP7mRNA after day30was increased in Treatgroup(p<0.05). Conclusion subtotal nephrectomy cause the increase of TGF-β1mRNAexpression in renal tissue, MSCs treatment can reduced the expression of TGF-β1mRNA and increased the expression of Pax-2, HGFand BMP7mRNA, and protect Pax-2+cell, thenameliorate renal fibrosis of remnant kidney, such might be the main mechanism that MSCsprevent and treat chronic renal failure.