Study On The Effect And Mechanism Of Mesenchymal Stem Cells Overexpressing ACE2 On Kidney In Diabetic Rats

Background and ObjectiveDiabetic nephropathy(DN)is a devastating microvascular complication of diabetes mellitus,which is the leading cause of end-stage renal disease(ESRD)worldwide and brings a huge economic burden to the society and families.The main pathological changes in DN are initially renal hypertrophy and increased extracellular matrix(ECM)components such as FN and collagen I,followed by increased glomerular filtration rate and increased urinary protein excretion.It will develop into glomerulosclerosis and tubulointerstitial fibrosis,and finally progress to ESRD if not treated in time.While oxidative stress and inflammation play an important role in promoting the occurrence and development of DN by upregulating the expression of TGF-β and ECM in renal mesangial cells.The renin-angiotensin system(RAS),especially local renal RAS activation,plays an essential role in the progression of DN through an increased level of angiotensin(Ang)II generated by the angiotensin-converting enzyme(ACE).Ang II exerts a variety of effects via its type 1 receptor(AT1R),including vasoconstriction,sodium retention,cell proliferation and apoptosis,inflammation and oxidative stress.These effects,acting either alone or synergistically,may lead to glomerular fibrosis,enhanced glomerular permeability and overt albuminuria.Although RAS inhibition is currently the standard therapy in DN,there is only an incomplete response in terms of reduction of proteinuria,improvement of kidney histology and prevention of disease progression,indicating a need to find alternative and/or complementary methods as newer therapeutic approaches for the downregulation of the RAS.Angiotensin-converting enzyme 2(ACE2)is a catalytically active homolog of ACE that degrades Ang II into Angl-7 and mitigates the detrimental effects of Ang II in DN animal models.ACE2 is highly expressed in the normal kidney,but its expression downregulated in rats with DN.Pharmacological inhibition of ACE2 or knockout of ACE2 in diabetic mouse models worsens glomerular injury and promotes albuminuria,indicating that the decrease in ACE2 is one of the major factors contributing to the pathogenesis of DN by permitting Ang II accumulation.Accordingly,therapies aimed at increasing ACE2 expression might attenuate DN.Mesenchymal stem cell(MSC)-based gene therapy is a novel therapeutic approach for several diseases,including acute kidney injury.Furthermore,our previous studies have shown that MSCs delivered intravenously can exert beneficial effects in regulating blood glucose,reducing albuminuria,ameliorating glomerular fibrosis,and modulating immune balance in STZ-induced DN.It is remarkable that MSCs can also be used as a vector to transmit protective genes via overexpression of transgenic genes in damaged sites,which could not only enhance its therapeutic effect but also promote local tissue repair.Therefore,the combination of MSCs and the protective gene ACE2 may be a potential therapeutic strategy for the treatment of DN.In the present study,we explored the effect of ACE2-modified MSCs on kidney of DN in vitro and in vivo and investigated the underlying molecular mechanism.Methods1.Cell studiesBone marrow-derived MSCs and glomerular mesangial cells(GMCs)were isolated,cultured and characterized,and bmMSCs were labeled with GFP in vitro(MSCsGFP).We transfect ACE2 lentiviral plasmid(LV-ACE2)to MSCs and GMCs at a multiplicity of infection(MOI)of 80 and 20,which were named MSC-ACE2 and GMC-ACE2,respectively.The expression of ACE2 mRNA and protein in MSCs,MSC-ACE2,GMCs and GMC-ACE2 were detected by real-time RT-PCR and Western blot(WB),respectively.The expression of ACE2 protein in MSCs,MSC-ACE2,GMCs and GMC-ACE2 cell culture medium was detected by ELISA kit.GMCs were co-cultured indirectly with the stem cells(MSCs,or MSC-ACE2),and then stimulated with Ang Ⅱ.To investigate the effect of MSCs and ACE2 over-expression on GMCs after Ang Ⅱ stimulation,cells were divided into five groups as below:GMCs cultured by normal media was determined as normal control group;GMCs stimulated by Ang Ⅱ(Ang Ⅱ-GMC);GMC-ACE2 stimulated by Ang Ⅱ(AngⅡ-GMC-ACE2);the co-culture of GMCs and MSCs stimulated by Ang Ⅱ(AngⅡ-GMC-MSC);the co-culture of GMCs and MSC-ACE2 stimulated by Ang Ⅱ(AngⅡ-GMC-MSC-ACE2).After stimulation,the expression of collagen Ⅰ,FN,TGF-βmRNA and protein in GMCs was detected by real-time RT-PCR and WB,and the expression of Smad2/3 and phosphorylated Smad2/3 in GMCs was detected by WB.The production of ROS in GMCs was detected by DHE fluorescence staining.The superoxide dismutase(SOD)activity and the malondialdehyde(MDA)in cell culture medium were measured by xanthine oxidase method and thiobarbituric acid method,respectively.The contents of T-GSH and GSSG were determined by detection kit.Real-time RT-PCR and ELISA were used to detect the mRNA and protein expression of IL-6,IL-1β,TNF α,IL-18 and MCP-1 in GMCs and culture medium to evaluate the inflammatory state of cells in each group.ELISA method was used to detect ACE2 and Ang1-7 protein in cell culture medium of GMCs.Real-time RT-PCR and WB were used to detect the mRNA and protein expression of ACE,ACE2,MasR,AT 1R,AT2R in GMCs of each group.2.Animal studiesTo make clear the protective effect of MSCs overexpressing ACE2 on the kidney of DN rats and its potential mechanism.MSC-ACE2 were transplanted to STZ-induced diabetic rats via tail vein.Meanwhile,diabetic rats recevied MSCs or no treatment were as control.We measured physical,biochemical and morphological parameters were after eight weeks.We detected blood glucose,urinary albumin excretion,Ccr and renal mass index by laboratory mathods and assessed the glomerular sclerotic injury by PAS staining;We measured the localizations of TGF-β、FN、collagen I、MCP-1、GFP in kidney tissues by immunohistochemistry;And the mRNA expression of TGF-β,FN,collagen I,MCP-1,IL-6,IL-1β,TNF α,IL-18,ACE,Ang II,ACE2,AT1R,AT2R and MasR in renal tissue was detected by real-time RT-PCR,and the protein expression of TGF-β,FN,collagen I,Smad2/3 and phosphorylated Smad2/3 in renal tissue was detected by WB.The protein levels of ACE,Ang II,ACE2 and Angl-7 in serum and the protein expression of IL-6,IL-1β,TNF a,IL-18,ACE,Ang II,ACE2,Angl-7,AT1R,AT2R and MasR in kidney tissue were detected by ELISA.In order to reflect the level of oxidative stress,the expression of ROS in frozen sections of kidney was detected by chemiluminescence method.The of SOD activity and the MDA in cell culture medium were determined by xanthine oxidase method and thiobarbituric acid method,respectively.The contents of T-GSH and GSSG were determined by detection kit.Results1.Protective effect of MSCs overexpressing ACE2 on GMCs stimulated by Ang II1.1 Culture and identification of MSCsBone marrow-derived MSCs were adherent cultured with a spindle-shaped morphology,and the nucleus is located in the center.They were able to differentiate into adipogenic and osteogenic cells,positive for CD29,CD90,CD44,and negative for CD34,CD45,CD11b.1.2 Construction and identification of MSC lines overexpressing ACE2The optimal concentrations of LV-ACE2 for transfecting MSCs and GMCs were MOI of 80 and 20.Three days after gene transfer,the ACE2 mRNA and protein expression detected by real-time RT-PCR and WB were significantly increased in MSC-ACE2 compared with MSCs by 294.80-and 5.72-fold,respectively.We measured the concentration of ACE2 protein in the culture medium using an ELISA kit.The results demonstrate that MSCs modified by a lentiviral vector to express the ACE2 gene could secrete soluble ACE2 protein into the culture medium at levels approximately 3.61-fold higher than the MSCs alone.The ACE2 mRNA and protein expression of GMCs were significantly increased by 100:80-and 2.43-fold compared with GMCs,respectively.The ACE2 expression level in the culture medium of GMC-ACE2 was about 2.16-fold higher than the GMC group alone.There was no significant difference in adipogenic and osteogenic differentiation of MSCs with or without gene transduction.1.3 Effect of MSC-ACE2 on fibrosis in GMCs stimulated by Ang IIThe expression of ECM protein components including collagen I and FN,the expression of TGF-β and the ratio of phosphorylated Smad2/3 to total Smad2/3 was significantly upregulated after Ang II stimulation,but the expression was significantly downregulated after the intervention with MSCs and/or ACE2,with much lower levels in the MSC-ACE2 group compared with MSCs or ACE2 intervention alone.1.4 Effect of MSC-ACE2 on oxidative stress in GMCs stimulated by Ang IIThe SOD activity and the ratio of GSH to GSSG were significantly lower,whereas MDA content increased after Ang II stimulating than that in the normal group.In contrast,the MSCs treatment and/or ACE2 gene transfer increased the SOD activity,the ratio of GSH to GSSG and decreased the MDA content compared with the Ang II-GMC group.Besides,MSCs and/or ACE2 treatment markedly reduced Ang II induced oxygen free radicals production,as revealed by DHE staining.Importantly,Ang II-GMC-ACE2 group had the highest SOD activity and the ratio of GSH to GSSG,the lowest MDA content and DHE staining with statistical significance compared with other two groups.1.5 Effect of MSC-ACE2 on inflammation in GMCs stimulated by Ang IIThe expression of MCP-1 and inflammatory cytokines IL-6,IL-1β,TNF a and IL-18 increased significantly after Ang II stimulation,while MSCs intervention group and ACE2 gene transfection group could improve the inflammatory state in GMCs.MSC-ACE2 intervention group was superior to MSC and/or ACE2 alone in improving inflammation.1.6 Effect of MSC-ACE2 on RAS in GMCs stimulated by Ang IIWe did not detect the expression of ACE in GMCs by real-time RT-PCR and WB.The ACE2 and Angl-7 level in culture medium was much higher in the Ang II-GMC-ACE2 and Ang II-GMC-MSC-ACE2 groups than in the other three groups.The level of ACE2 and Angl-7 in the Ang II-GMC-MSC-ACE2 group was the highest and was significantly different from the levels in the other groups.After Ang II stimulation,the protein level of ACE2 in GMCs significantly decreased in the Ang II-GMC group,but significantly increased in both the Ang II-GMC-ACE2 and Ang II-GMC-MSC-ACE2 groups.The level in the Ang II-GMC-MSC-ACE2 group was much higher than that in the Ang II-GMC-ACE2 group.There was no significant difference in the protein levels of the Mas receptor among the five groups.The AT1R level was significantly higher in Ang II-stimulated groups than in the GMC group,but there was no obvious difference among the four experimental groups.Compared with the normal group,the expression of AT2R was downregulated after Ang II stimulation,but with higher expression in the Ang II-GMC-ACE2 and Ang II-GMC-MSC-ACE2 groups than in the other two groups.2.Effect of MSC-ACE2 on kidney of diabetic rats2.1 MSCs localization in kidneys with diabetesImmunohistochemistry for GFP demonstrated that at 24 hours after transplantation,a considerable number of MSCsGFP were present in the kidney around the glomeruli and near vessels of MSCsGFP-treated rats with diabetes,but at 8 weeks after transplantation,the number was greatly reduced.2.2 Effect on blood glucose and renal functionRats with diabetes showed increased blood glucose,24-hour urinary albumin excretion,Cer and renal mass index at 16 weeks after STZ injection compared with these indicators in normal control rats.The transplantation of MSCs or MSCs-ACE2 after the induction of hyperglycaemia significantly ameliorated these indexes.Moreover,compared with the MSCs treatment,the MSC-ACE2 intervention significantly decreased the 24-hour urinary albumin excretion.2.3 Effect of MSC-ACE2 on glomerular fibrosis and ECM protein expression in diabetic ratsKidneys of DN group rats exhibited profound ECM deposition in the mesangium as revealed by PAS staining and imnunohistochemistry,MSCs and MSC-ACE2 treatment remarkably ameliorated these pathological abnormalities.The expression of TGF-β,FN and collagen I in kidney was significantly decreased in MSC and MSC-ACE2 group,and the therapeutic effect of MSC-ACE2 is better than that of MSCs.Real-time RT-PCR and WB analysis showed the same trend,and the ratio of phosphorylated Smad2/3 to total Smad2/3 was the same as that of TGF-β.2.4 Effect of MSC-ACE2 on oxidative stress in diabetic ratsThe activity of SOD and the ratio of GSH to GSSG in renal tissue of DN group were significantly lower than those of normal group,while the production of ROS and MDA increased significantly.Compared with DN group,MSCs and MSC-ACE2 treatment improved oxidative stress,showing a significant increase in SOD activity and the ratio of GSH to GSSG and a decrease in ROS and MDA content,while MSC-ACE2 group was superior to MSC group.2.5 Effect of MSC-ACE2 on inflammation in diabetic ratsThe expression of MCP-1 and inflammatory cytokines IL-6,IL-β,TNF α and IL-18 increased significantly in renal tissue of DN group,while MSCs and MSC-ACE2 treatment improved the inflammatory state in DN rats,and MSC-ACE2 group was superior to MSC group.2.6 Effect on the changes in RAS in diabetic ratsIn the kidneys of rats with diabetes,the expression levels of ACE,Ang Ⅱ,AT1R and AT2R were upregulated,while the expression levels of ACE2,Angl-7 and MasR were downregulated.MSCs transplantation had no effect on the changes in RAS,which were not significantly different from those in the DN group.However,MSC-ACE2 transplantation dramatically upregulated the expression of ACE2,Ang1-7 and AT2R and downregulated the expression of ACE and Ang Ⅱ.Notably,however,it had no effect on the expression of ATIR and MasR.In the serum of rats with diabetes,the expression levels of ACE,Ang Ⅱ,ACE2,and Ang1-7 detected by ELISA were all upregulated compared with those in normal control rats.MSCs transplantation also had no effect on the changes in RAS in serum.MSC-ACE2 transplantation had no effect on ACE but dramatically upregulated the expression of ACE2 and Ang1-7 and downregulated the expression of Ang Ⅱ.Conclusion1.MSCs transduced with ACE2 gene by lentiviral vector had a high transduction efficiency and overexpressed enzymatically active ACE2 protein.2.MSCs overexpressing ACE2 reduced oxidative stress and inflammatory response,down-regulated TGF-β/Smad signal pathway,and alleviated fibrosis of GMCs induced by Ang II,which was better than that of MSCs or ACE2 alone.3.MSCs modified with ACE2 gene localized to the injured kidney.Compared to MSC alone,it had advantages in improving local oxidative stress and infflammation,down-regulating TGF-β/Smad signal pathway and improving renal fibrosis.4.This effect was mainly due to the local overexpression of ACE2 protein which leaded to the decrease of Ang II expression and the increase of Angl-7 in the kidney.These findings demonstrated that MSCs modified with ACE2 therapy had additional benefits on the progression of diabetic nephropathy by inhibiting renal RAS activation.