| Backgrounds:Diabetic nephropathy（DN）is the most common microvascular complication of type 2 diabetes mellitus.Traditionally,DN is thought to be characterized by glomerular lesions,but more and more studies have shown that glomerular injury may not be the determining factor in the progression of DN,whereas damage to the tubules is present in the early stages of DN and is closely associated with proteinuria,deterioration of renal function,and the long-term prognosis of the kidneys.Therefore,abnormalities in renal tubular structure and function are critical links in the development and progression of DN,and exploring the mechanisms of its injury and protective measures are of great significance in the prevention and treatment of DN.Human umbilical cord mesenchymal stem cells（hUCMSCs）have been recognized as the preferred MSCs for transplantation due to their low immunogenicity,strong regenerative ability and high paracrine potential,etc.However,the study of hUCMSCs for DN is still in the primary stage,and the specific mechanism of their action still needs to be explored in depth.Our research group’s previous study found that hUCMSCs can improve oxidative stress in the kidney by activating nuclear factor-E2-related factor 2（Nrf2）.And oxidative stress is throughout the ferroptosis,moreover,iron metabolism,lipid metabolism and glutathione synthesis during ferroptosis are all regulated by Nrf2,so targeting Nrf2 to regulate ferroptosis may be a new mechanism for hUCMSCs to treat renal diseases.However,the relationship between hUCMSCs,Nrf2,and ferroptosis is still lack of research in the field of DN.Therefore,the present study focused on exploring whether hUCMSCs could regulate ferroptosis in type 2 diabetic kidneys by activating Nrf2 and thus improve DN,for which we conducted the following studies.Chapter 1 Protective effects of hUCMSCs on type 2 diabetic kidneyObjectives:To clarify the protective effects of hUCMSCs on type 2 diabetic mice kidneys and high glucose and high fat-stimulated human renal tubular epithelial cells.Methods:1.In vivo studies,male wild-type（WT）C57BL/6J mice were randomly divided into 4 groups:control（Ctrl）,hUCMSC（MSC）,diabetes mellitus（DM）,and diabetes mellitus+hUCMSC（DM/MSC）groups.DM group and DM/MSC group mice were subjected to high-fat diet combined with streptozocin to induce type 2 diabetes mellitus model.About 3 months after the success of the model,the urinary albumin-to-creatinine ratio（UACR）of diabetic mice was significantly elevated.According to the previous research basis of our group,mice in the MSC group and DM/MSC group were given1*106 hUCMSCs tail vein injection treatment,once in 7 days,for a total of 3 injections,and the samples were taken 7 days after the last treatment and used for the detection of various indexes.First,the physiological and chemical indices of blood and urine of mice in each group were compared;changes in the morphology of mouse kidneys were observed by PAS,toluidine blue staining and transmission electron microscopy;Western Blot（WB）was performed to detect the protein levels of renal tubular injury markers,i.e.kidney injury molecule-1（KIM-1）and neutrophil gelatinase-associated lipidocalin（NGAL）protein levels.2.In vitro experiments,we applied high concentrations of glucose and palmitate to stimulate human renal tubular epithelial cells to construct a model of cell injury.The experiments were divided into 4 groups:control group（Ctrl）,hUCMSC group（MSC）,high glucose and high fat group（HG/P）,and high glucose and high fat+hUCMSC group（HG/P/MSC）.In the MSC and HG/P/MSC groups,human tubular epithelial cells were co-cultured with hUCMSCs via transwells.Cell viability was detected by CCK-8,and protein levels of KIM-1 and NGAL were detected by WB.Results:1.In vivo experiments,compared with the Ctrl group,mice in the DM group showed a decrease in body weight and a significant increase in blood glucose,renal hypertrophy index,UACR,blood creatinine,and blood urea nitrogen（P<0.05）;hUCMSCs treatment improved the levels of glucose,body weight,UACR,and blood creatinine of the mice（P<0.05）,but had no significant effect on renal hypertrophy index and blood urea nitrogen（P>0.05）.2.In vivo experiments,compared with the Ctrl group,mice in the DM group showed glomerular hypertrophy,increase in the mesangial matrix,fusion of epithelial cell foot processes,thickening of the glomerular and tubular basement membranes,detachment of tubular epithelial cells,and tubulo-interstitial fibrosis,whereas the above pathology in the DM/MSC group was significantly alleviated compared with that in the DM group.In comparison with the Ctrl group,the levels of renal tubular injury factor KIM-1 and NGAL protein were significantly higher in the DM group of mice（P<0.0001）,whereas they were significantly lower in the DM/MSC group compared with the DM group（P<0.001）.3.In vitro experiments,CCK-8 and WB results showed that 30m M glucose acting for 24h combined with 300μM palmitate acting for 6h could successfully construct a high glucose and high fat stimulated human renal tubular epithelial cell model,and2*105 hUCMSCs/ml was the most suitable co-culture dose.Compared with the Ctrl group,KIM-1 and NGAL protein expression was increased in the HG/P group（P<0.001）,but compared with the HG/P group,the protein level of KIM-1 and NGAL were decreased（P<0.001）in the HG/P/MSC group.Conclusions:1.In vivo administration of hUCMSCs can attenuate histological damage and functional abnormalities in the kidneys of type 2 diabetic mice;2.HUCMSCs in vitro intervention can alleviate the injury of human renal tubular epithelial cells stimulated by high glucose and high fat.Chapter 2 Mechanism of hUCMSCs regulating renal ferroptosis in type 2 diabetes mellitus through activation of the Nrf2 signaling pathwayObjectives:To clarify that hUCMSCs can regulate ferroptosis in type 2 diabetic kidneys by regulating iron metabolism,lipid metabolism,and antioxidant pathways,and is may achieved by activating the Nrf2 signaling pathway.Methods:1.Using the in vivo and in vitro models from Part I,mitochondrial morphological changes were observed by transmission electron microscopy in renal tubules of mice and in high glucose and high-fat stimulated human renal tubular epithelial cells,respectively,and changes in the expression of relevant indexes on the iron metabolism,lipid peroxidation and antioxidant pathways in the ferroptosis pathway were assessed by WB,Prussian blue staining,divalent ferric iron fluorescence probes,immunohistochemistry and immunofluorescence techniques.2.In vivo and in vitro experiments were performed to investigate whether hUCMSCs affect the Nrf2 signaling pathway by detecting protein expression changes of Nrf2 and its downstream factors,HO1 and NQO1,in diabetic mouse kidneys and high-glucose and high-fat-stimulated human renal tubular epithelial cells by WB.In vivo experiment,the nucleoprotein and cytoplasmic protein in mouse kidney tissue were extracted,and the expression level of Nrf2 in the nucleoprotein and cytoplasmic protein was measured by WB;in vitro experiments,the expression and localization of Nrf2 protein in human renal tubular epithelial cells were observed by laser confocal microscopy.Results:1.In vivo experiments,mitochondrial atrophy,increased membrane density,a small number of cristae breaks were seen in the renal tubules of mice in the DM group compared with the Ctrl group,but the mitochondrial volume,cristae structure and membrane density of mice in the DM/MSC group were significantly improved compared with those in the DM group.Evaluating iron metabolism indexes,compared to the Ctrl group,the DM group had increased renal iron content（P<0.01）,decreased FTH1 protein levels（P<0.01）,and increased TFR1 protein levels（P<0.0001）,whereas the DM/MSC group had decreased iron content,increased FTH1 protein,and decreased TFR1 protein compared to the DM group（P<0.05）.Detecting lipid peroxidation indexes,MDA content,protein levels of 4HNE and ACSL4 were elevated in the DM group compared to the Ctrl group（P<0.05）,but hUCMSCs significantly reduced MDA content,protein levels of 4HNE and ACSL4 compared to the DM group（P<0.05）.Antioxidant capacity was detected,and protein expression of GPX4 and x CT was decreased in the DM group compared with the Ctrl group（P<0.01）,whereas protein expression of GPX4 and x CT was elevated in the DM/MSC group compared with the DM group（P<0.05）,and the GPX4 protein was expressed predominantly in the renal tubules.2.In vitro experiments,compared with the Ctrl group,mitochondrial atrophy,membrane density increase,mitochondrial outer membrane rupture and ridge fracture were observed in the HG/P group,but compared with the HG/P group,mitochondrial ridge and outer membrane structure were intact and membrane density recovered in the HG/P/MSC group.Measuring the iron metabolism indexes,compared to the Ctrl group,the HG/P group showed a significant increase in Fe2+fluorescence intensity,a decrease in FTH1 protein,and an elevation in TFR1 protein（P<0.0001）,whereas the HG/P/MSC group showed a weakening of the Fe2+fluorescence intensity,an elevation in the level of FTH1 protein,and a decrease in the level of TFR1 protein when compared to the HG/P group（P<0.01）.Assessment of lipid peroxidation products,the HG/P group showed significantly increased ROS fluorescence brightness and increased4HNE and ACSL4 protein levels compared with the Ctrl group（P<0.01）,but in the HG/P/MSC group,ROS fluorescence intensity,4HNE and ACSL4 protein levels were significantly decreased compared with the HG/P group（P<0.05）.Evaluating the antioxidant capacity,the levels of GPX4 and x CT proteins were reduced in the HG/P group compared to the Ctrl group（P<0.0001）,whereas the levels of the above proteins were elevated in the HG/P/MSC group compared to the HG/P group（P<0.001）.3.In vivo experiments,the protein levels of Nrf2,HO1 and NQO1 were significantly decreased in the DM group compared with the Ctrl group,while the protein levels of these three were significantly elevated in the DM/MSC group compared with the DM group（P<0.05）;in vitro experiments,the protein expression of Nrf2,HO1 and NQO1 was significantly lower in the HG/P group compared to the Ctrl group,while the protein content of all three was significantly higher in the HG/P/MSC group compared to the HG/P group（P<0.05）.In vivo experiments,the content of nuclear Nrf2 protein（n Nrf2）was significantly increased in the DM/MSC group compared with the Ctrl and DM groups（P<0.01）,while the difference of cytoplasmic Nrf2 protein（c Nrf2）was not significant among the groups（P>0.05）;in vitro experiments,Nrf2 protein was mainly expressed in the cytoplasm in the HG/P groups,while Nrf2 protein was increased in the HG/P/MSC group compared to the HG/P group and was mainly clustered in the nucleus.Conclusions:1.HUCMSCs ameliorate renal injury in type 2 diabetes in vivo and in vitro by modulating the ferroptosis pathway;2.HUCMSCs can regulate ferroptosis by regulating Nrf2 expression and activating the Nrf2 signaling pathway.Chapter 3 The critical role of Nrf2 in the regulation of ferroptosis by hUCMSCs to ameliorate type 2 diabetic nephropathyObjectives:To clarify the central role of Nrf2 in hUCMSCs to regulate ferroptosis pathway and ameliorate diabetic kidney injury.Methods:1.In vivo experiments,apply Nrf2-knockdown(Nrf2-/-)male mice in the same background as the above WT mice to construct an animal model of type 2 diabetes.The experiments are divided into 5 groups:WT control group（WT Ctrl）,Nrf2-/-control group(Nrf2-/-Ctrl),Nrf2-/-hUCMSC group(Nrf2-/-MSC),Nrf2-/-diabetes group(Nrf2-/-DM),Nrf2-/-diabetes+hUCMSC group(Nrf2-/-DM/MSC).The construction of the diabetes model,the dose and duration of treatment with hUCMSCs,etc.were consistent with the first part of in vivo experiments.Comparison of physiological and biochemical indices of Nrf2-/-mice in each group;the morphological changes in the kidneys of mice were observed by PAS,toluidine blue staining and transmission electron microscopy;protein expression of renal tubular injury factor were detected by WB;the expression levels of relevant indicators in the ferroptosis pathway were evaluated by transmission electron microscopy,WB,and immunohistochemistry;the expression of proteins on the Nrf2 pathway in the kidneys of Nrf2-/-mice was assessed by WB.2.In vitro experiments,human renal tubular epithelial cells were transfected with Nrf2 siRNA,and the experiments were divided into 8 groups,i.e.,the control group（Ctrl）,hUCMSCs group（MSC）,the high-glucose and high-fat group（HG/P）,the high-glucose and high-fat+hUCMSCs group（HG/P/MSC）,the control+Nrf2 siRNA group（s-Ctrl）,hUCMSCs+Nrf2 siRNA group（s-MSC）,high glucose and high fat+Nrf2siRNA group（s-HG/P）and high glucose and high fat+hUCMSCs+Nrf2 siRNA group （s-HG/P/MSC）.Changes in the expression of ACSL4,FTH1 and GPX4 proteins,key indicators on the ferroptosis pathway,were detected by WB.Results:1.In vivo experiments,there were no statistically significant differences in blood glucose,body weight,UACR and serum creatinine between mice in the WT Ctrl group and the Nrf2-/-Ctrl group（P>0.05）.However,the Nrf2-/-DM group showed elevated blood glucose,decreased body weight,and increased UACR,and serum creatinine in its mice compared with the Nrf2-/-Ctrl groups.After treatment with hUCMSCs,the above indexes were significantly improved in the Nrf2-/-DM/MSC group of mice compared with the Nrf2-/-DM group（P<0.05）.Comparing WT and Nrf2-/-diabetic mice,hUCMSCs improved body weight,blood glucose,UACR,and serum creatinine by 12%,22.8%,24.5%,and 36%,respectively,in the former,whereas they improved body weight,blood glucose,UACR,and serum creatinine by 11.6%,18%,14.3%,and12%,respectively,in the latter.In addition,Nrf2-/-and Ctrl groups did not show significant abnormalities in renal morphology,however,knockdown of the Nrf2 gene exacerbated renal injury in diabetes mellitus,and mice in the Nrf2-/-DM group were seen to have marked glomerular basement membrane thickening,fused peduncles,significant increases in mesangial cells and the matrix,detached renal tubular epithelial cells,enlarged lumen and deposition of collagen fibers in tubular interstitiu.In contrast,in the Nrf2-/-DM/MSC group,compared with the Nrf2-/-DM group,the glomerular mesangial cells and the matrix were significantly reduced in mice,and there was a tendency for the thickness of glomerular basement membrane to be reduced,but the difference was not statistically significant（P>0.05）,and obvious detachment of renal tubular epithelial cells and deposition of tubular mesenchymal collagen fibers were still seen.Detection of renal tubular injury proteins revealed that KIM-1 and NGAL protein levels were significantly higher in the Nrf2-/-DM group compared to the Nrf2-/-Ctrl group（P<0.0001）,whereas treatment with hUCMSCs down-regulated the protein expression of KIM-1（P<0.01）but did not have a significant effect on the protein level of NGAL（P>0.05）.2.In vivo experiments,the mitochondrial morphology of the WT Ctrl group and the Nrf2-/-Ctrl group was integrity,but in the Nrf2-/-DM group,mitochondrial atrophy,increased density of mitochondrial membrane and rupture of the outer mitochondrial membrane could be seen in comparison with the Nrf2-/-Ctrl group,however,the Nrf2-/-DM/MSC group showed a slight improvement in mitochondrial membrane density compared with the Nrf2-/-DM group,but significant mitochondrial atrophy and rupture of the outer mitochondrial membrane were still visible.There was no significant difference in iron content,TFR1 and FTH1 protein levels between the WT Ctrl group and the Nrf2-/-Ctrl group,but compared to the Nrf2-/-Ctrl,the Nrf2-/-DM group had increased iron content and TFR1 protein levels（P<0.01）and decreased FTH1 protein levels（P<0.0001）,but the Nrf2-/-DM/MSC group compared to the Nrf2-/-DM group,there was no significant difference in the above iron metabolism indexes（P>0.05）.MDA content,4HNE and ACSL4 protein levels were not significantly different in the WT Ctrl and Nrf2-/-Ctrl groups,but all three were elevated in the Nrf2-/-DM group（P<0.01）,and no significant decrease in all three was seen after treatment with hUCMSCs compared to the Nrf2-/-DM group（P>0.05）.GPX4 protein expression was significantly down-regulated in the Nrf2-/-Ctrl group and the Nrf2-/-DM group compared with the WT Ctrl group（P<0.0001）,whereas there was no significant difference in the GPX4 protein level between the Nrf2-/-DM/MSC group and the Nrf2-/-DM group（P>0.05）.In addition,the x CT protein level was significantly decreased in the Nrf2-/-DM group compared with the Nrf2-/-Ctrl group,but there was no significant change in the x CT protein level in the Nrf2-/-DM/MSC group compared with the Nrf2-/-DM group（P>0.05）.The protein expression of Nrf2,HO1 and NQO1 was significantly suppressed in the Nrf2-/-Ctrl group and the Nrf2-/-DM group compared to the WT Ctrl group,and the proteins of Nrf2,HO1,and NQO1 were still low-expressed after the intervention of hUCMSCs（P>0.05）.3.In vitro experiments,after Nrf2 siRNA transfection,compared with the Ctrl group,the ACSL4 protein content was elevated in the s-Ctrl group,but the difference was not statistically significant（P>0.05）,but compared with the s-Ctrl group,the ACSL4 protein expression was elevated in the s-HG/P group（P<0.001）,whereas no significant decrease was seen in the s-HG/P/MSC group（P>0.05）;in comparison with the s-Ctrl group,the s HG/P group FTH1 protein was down-regulated（P<0.0001）,whereas there was no significant difference in FTH1 protein content between the s-HG/P/MSC and s-HG/P groups（P>0.05）;compared with the Ctrl group,GPX4 protein expression was significantly suppressed in all groups after Nrf2 siRNA transfection,whereas hUCMSCs were unable to up-regulate the protein level of GPX4（P>0.05）.Conclusions:1.Knockdown of the Nrf2 gene weakened the protective effect of hUCMSCs against DN and also exacerbated renal injury in type 2 diabetes;2.Deletion of the Nrf2 gene significantly attenuated the inhibitory effect of hUCMSCs on ferroptosis,and Nrf2 has a critical role in the regulation of ferroptosis by hUCMSCs.In summary,we conclude the full article:1.HUCMSCs can ameliorate histologic damage and functional abnormalities in the kidney of type 2 diabetic mice;2.HUCMSCs can attenuate the damage of high glucose and high fat-stimulated human renal tubular epithelial cells through paracrine effects;3.HUCMSCs can inhibit ferroptosis in type 2 diabetic kidneys in vivo and in vitro by regulating iron metabolism,inhibiting lipid peroxidation and improving antioxidant capacity;4.HUCMSCs may regulate ferroptosis by modulating Nrf2 expression and activating the Nrf2 signaling pathway;5.Knockdown of the Nrf2 gene not only attenuated the protective effect of hUCMSCs on DN,but also aggravated renal injury in type 2 diabetes;6.Knockdown of the Nrf2 gene nearly eliminated the inhibitory effect of hUCMSCs on ferroptosis,and Nrf2 has a critical role in the regulation of ferroptosis by hUCMSCs. |
PDF Full Text Request