| BackgroundRenal interstitial fibrosis is a common pathological pathway and irreversible process that eventually leads to end-stage renal disease.Interstitial fibrosis is characterized by the destruction of renal tubules and interstitial capillaries as well as by the accumulation of extracellular matrix(ECM).Till now,the underlying molecular mechanisms of renal interstitial fibrosis are still not fully understood,and there is still no effective treatment strategy to prevent and halt the progression of renal fibrosis.Therefore,it is necessary to explore the molecular mechanisms involved in renal interstitial fibrosis progression and to develop effective therapeutic strategies for prognosis improvement.Adipose-derived stem cells(ADSCs)are a mesenchymal stem cell source with capacities of self-renewal and multipotential differentiation.ADSCs can differentiate into a variety of cell types including adipocytes,chondrocytes,osteoblasts,myocytes,neurocytes,endothelial cells,and other cell types.ADSCs also have the potential to treat a variety of diseases,such as autoimmune-induced diseases,diabetes mellitus,graft-versus-host disease,tracheomediastinal fistulas,and multiple sclerosis.Compared with bone marrow derived stem cells,ADSCs have two main advantages.First,ADSCs are easily accessible in large quantities from subcutaneous adipose tissue with less donor site lesion.Second,ADSCs are nonimmunogenic and avoid the ethical and political concerns compared to use of embryonic stem cell,because they can be derived from autologous fat.These two characteristics make ADSCs a more ideal source for tissue and organ transplantation in regenerative medicine and clinical studies.Recently,several reports had demonstrated that ADSCs therapy could reduce kidney injury and improve kidney function by attenuating inflammation and immune responses.For example,in mice,ADSCs were demonstrated to reduce acute kidney damage through inhibiting the expression of tissue chemokine and cytokine.ADSCs therapy could protect against kidney injury in sepsis syndrome caused by cecal ligation puncture in rats.ADSCs could improve protection against acute kidney ischemia-reperfusion injury by reducing the protein expressions of oxidative stress,apoptosis and inflammatory reaction in rats.However,the impacts of ADSCs on renal interstitial fibrosis have not been elucidated.This study aimed to evaluate the effects of ADSCs treatment on the improvement of renal interstitial fibrosis in a rat model of renal interstitial fibrosis induced by UUO.Furthermore,we investigated whether ADSCs treatment was able to ameliorate EMT and inflammatory response via TGF-β1 signaling pathway in the rat UUO models.Part ⅠADSCs improve tubulointerstitial fibrosis in UUO rats by inhibiting renal tubular epithelial-mesenchymal transition(EMT)ObjectiveTo investigate the effect of ADSCs on EMT and interstitial fibrosis in renal tubular epithelial cells of UUO rats and its possible mechanism.MethodsADSCs were isolated,cultured and identified and labeled with green fluorescent protein(GFP)in vitro.45 male Wistar rats were divided into 3 groups,15 in each group:(1)sham-operated group,(2)UUO group,(3)UUO + ADSCs group.In the sham group,rats had their left ureters exposed and manipulated without ligation.Rats in UUO and UUO + ADSCs group were obstructed left ureter.Rats in UUO + ADSCs group received tail vein transplantation of 1 mL ADSCs(5×106 cells),and rats in UUO and sham group received equal volume Saline at 7 days after operation.To track the ADSCs intrarenal distribution after ADSCs administration,eight rats with UUO received ADSCsGFP transplantation instead of ADSCs.Groups of rats were sacrificed at 14 days after the operation,and blood samples and the obstructed kidneys were collected for tissue analysis.Renal function was evaluated by measuring serum creatinine(Scr)and blood urea nitrogen(BUN)levels with automatic biochemistry analyzer.HE and Masson staining were used to assess the extent of renal interstitial fibrosis.The levels of α-SMA,FSP-1,FN and E-cadherin were measured by immunohistochemistry.The expression levels of a-SMA,FSP-1,FN and E-cadherin in renal tissues were detected by real-time quantitative RT-PCR and Western blot analysis.Results1.ADSCs showed a typical fibroblast-like morphology,and when cultured in appropriate conditions,they displayed potential for adipogenic and osteogenic differentiation.Cells also expressed characteristic stem cells positive markers for CD29,CD90,CD44 and negative markers for CD34,CD45,CD11b.2.ADSCs renal tracing exhibited that a small amount of ADSCsGFP were visible in the kidney around the glomeruli and near vessels of ADSCsGFP-treated rats with UUO at 24 h after transplantation.But eight weeks after transplantation,very few ADSCsGFP were found in the glomeruli.3.There were no significant differences in Scr and BUN between UUO and UUO +ADSCs groups 14 days after the operation(p>0.05).4.HE and Masson staining showed that the kidneys of Sham group were histologically normal,UUO group showed extensive fibrosis,loss of brush border,significant tubular epithelial cell necrosis,tubular dilation,and inflammatory cell infiltration.After treatment with ADSCs,the level of renal interstitial fibrosis in UUO+ADSCs group was significantly lower than that in group UUO(p<0.05).5.The results of immunohistochemistry showed that the expressions of a-SMA,FSP-1 and FN were remarkably upregulated,but E-cadherin was significantly reduced in UUO group compared with the sham group(p<0.05).After 7 days ADSCs administration,the expressions of a-SMA,FSP-1 and FN were significantly reduced,whereas the expression of E-cadherin was increased compared with UUO group(p<0.05).6.ADSCs therapy significantly decreased mRNA and protein levels of a-SMA,FSP-1 and FN,whereas increased the mRNA and protein levels of E-cadherin confirmed by real-time quantitative RT-PCR and Western blot analysis(p<0.05).ConclusionsADSCs therapy could significantly decrease EMT and renal interstitial fibrosis after UUO induction,which may be related to down-regulation of the expressions of a-SMA,FSP-1 and FN,and up-regulation of the expression of E-cadherin.Part IIADSCs improve renal interstitial fibrosis in UUO rats by inhibiting inflammatory responsesObjectiveTo observe the effect of ADSCs on the interstitial inflammatory response of UUO rats,and to investigate the mechanism of ADSCs delaying the progression of renal interstitial fibrosis.MethodsExperimental grouping and tissue collection were the same as in the first part.Expression levels of MCP-1,TLR4,TNF-a,IL-1β and IL-6 from kidney tissues were measured by immunohistochemistry and real-time quantitative RT-PCR.ResultsThe results of immunohistochemistry and real-time quantitative RT-PCR showed that the expressions of MCP-1,TLR4,TNF-a,IL-1β and IL-6 were remarkably upregulated in UUO group compared with the sham group(p<0.05).After 7 days ADSCs administration,the expressions of MCP-1,TLR4,TNF-α,IL-1β and IL-6 were significantly reduced in UUO + ADSCs groups compared with UUO group(p<0.05).ConclusionsADSCs can ameliorate renal interstitial fibrosis in UUO rats,which may be related to the inhibition of the interstitial inflammatory response of UUO rats.Part ⅢThe molecular mechanism of ADSCs in improving renal interstitial fibrosis in UUO ratsObjectiveTo investigate whether ADSCs can inhibit the renal interstitial fibrosis of UUO rats by inhibiting the TGF-β1/Smad signaling pathwayMethodsExperimental grouping and tissue collection were the same as in the first part.Immunohistochemical was used to detect the expression of TGF-β1 protein in renal tissue of each group.Expression levels of TGF-β1 and Smad7 were measured by real-time quantitative RT-PCR.The expressions of TGF-β1,Smad2,p-Smad2,Smad3,p-Smad3 and Smad7 in renal tissues of each group were detected by Western blot.Results1.The result of immunohistochemistry showed that the expression of TGF-β1,was remarkably upregulated in UUO group compared with the sham group(p<0.05).After7 days ADSCs administration,the expression of TGF-β1 was significantly reduced in UUO + ADSCs groups compared with UUO group(p<0.05).2.The results of real-time quantitative RT-PCR showed that the expression of TGF-β1 was remarkably upregulated,but Smad7 was significantly reduced in UUO group compared with the sham group(p<0.05).After 7 days ADSCs administration,the expression of TGF-β1 was significantly reduced,whereas the expression of Smad7 was increased compared with UUO group(p<0.05).3.Western blot showed the expressions of TGF-(31,p-Smad2 and p-Smad3 were remarkably upregulated,but Smad7 was significantly reduced in UUO group compared with the sham group(p<0.05).After 7 days ADSCs administration,the expressions of TGF-β1,p-Smad2 and p-Smad3 were significantly reduced,whereas the expression of Smad7 was increased compared with UUO group(p<0.05).ConclusionsADSCs can improve the renal interstitial fibrosis in UUO rats by inhibiting the TGF-β1/Smad signaling pathway. |
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