Study On The Effect And Mechanism Of Bone Marrow-derived Mesenchymal Stem Cells On Kidney In Rats With Diabetes

Background and ObjectiveDiabetic nephropathy (DN) is the most common cause of chronic kidney disease, with a significant increase in morbidity and mortality in patients with diabetes. Currently available treatments involving strict glycemic and/or blood pressure control can delay, but not eliminate the occurence and development of DN. Therefore, it is necessary to identify new strategies that could specifically target on DN.Recent studies have shown that chronic inflammation play critical roles in promoting the development and progression of DN. Macrophages are key inflammatory cells mediating kidney inflammation in diabetic nephropathy and other types of renal disease. It has been found that macrophages accumulation is substantially increased in kidney tissue and associated with the progression of renal injury and a decline in renal function in experimental and human diabetes. More important, several experimental studies have shown that different therapeutic strategies which can inhibit macrophage infiltration directly or indirectly can prevent the development or ameliorate renal injury in diabetes. All these indicate that macrophage-mediated renal injury plays an important role in the development of diabetic nephropathy.Oxidative stress is a major culprit in DN. Oxidative stress is caused by an imbalance between the production of reactive oxygen species (ROS) and a biological system’s ability to readily detoxify the reactive intermediates or easily repair the resulting damage. When this occurs, oxidation of important macromolecules including proteins, lipids, carbohydrates, and DNA ensues. Glucose is the primary fuel source of ROS generation. High glucose induces ROS and upregulates TGF-β and extracellular matrix (ECM) expression in the glomerular mesangial cells. Exogenous hydrogen peroxide (H2O2) or H2O2continuously generated by glucose oxidase also upregulates TGF-β and fibronectin (FN) expression in mesangial cells. So intensive glycemic control is the main intervention that may decrease oxidative stress. On the other hand, it has been suggested that increases in glucose uptake in the kidney cells during diabetes could lead to high intracellular glucose level, thus increasing the ROS production. Then ROS activate signal transduction cascade and transcription factors, leading to upregulation of genes and proteins involved in glomerular mesangial expansion and tubulointerstitial fibrosis. So except intensive glycemic control, enhancing the ability of these susceptible cells to decrease glucose uptake in hyperglycemia environment may be another intervention that may decrease cellular ROS generation in diabetic kidney. A number of studies have also shown that interventions to prevent membrane localization of glucose transporters (GLUT), particularly GLUT1can benefit in experimental DN.Recent studies have shown that mesenchymal stem cells (MSCs) therapy reduces blood glucose, microalbuminuria and ameliorates glomerular injury. An emerging body of studies have found that MSCs may play specific roles as immunomodulators in a wide array of disease due to their immunomodulatory properties including suppression of T cell proliferation, influencing dendritic cell (DC) maturation and function, supression of B cell proliferation and terminal differentiation, etc. Recently, there is increasing evidence to suggest that the immunomodulatory effects of MSCs on macrophages is also a critical mechanism in the amelioration of inflammation-related disease, such as wounding healing, sepsis, acute myocardial infarction and renal ischemia reperfusion injury. However, the interactions between MSCs and macrophages in the process of DN are poorly understood. MSCs constitutively express SOD1, SOD2, CAT, GPX1enzyme and high levels of glutathione, have the ability to effectively remove peroxide and peroxynitrite, with low sensitivity to deleterious effects of ROS and reactive nitrogen products. Salmon et al also find that MSCs possess the main enzymatic mechanisms to detoxify reactive species and to prevent oxidative damage of the proteome and genome. Thus, MSCs are endowed with the main molecular mechanisms to manage oxidative stress efficiently. But, there is little study to explore the effect of MSCs on oxidative stress in DN and related mechanisms.Based on the backgrounds mentioned above, we tested our strategy by transplanting bone marrow-derived MSCs to streptozotocin-induced diabetic rats to explore the effect of MSCs on macrophage infiltration and oxidtive stress. Meanwhile, we cultured glomerular mesangial cells (GMC) in vitro to further explore the mechanisms underlying the oxidative stress.Methods1. To make clear the effect and mechanism of MSCs on macrophage infiltration in kidney tissue in diabetic rats, we isolated, cultured and characterized bone marrow-derived MSCs, labeled with GFP in vitro. Streptozotocin (STZ)-induced diabetes animals received no treatment or treatment with MSCs (2X106, via tail vein) for two continuous weeks. Eight weeks after treatment, physical, biochemical and morphological parameters were measured. Blood glucose, urinary albumin excretion, creatinine clearance (Ccr) and renal mass index were detected by laboratory mathods; PAS staining were performed to assess the glomerular sclerotic injury; the localizations of ED-1, MCP-1, Collagen I and FN in kidney tissues were measured by immunohistochemistry; expressions of pro-inflammatory cytokines (IL-1β, IL-6and TNF a) and HGF at gene level and protein level were determined by real time RT-PCR and ELISA, respectively.2. To make clear the effect and mechanism of MSCs on oxidative stress in kidney tissue in diabetic rats, we isolated, cultured and characterized bone marrow-derived MSCs, labeled with GFP in vitro. STZ-induced diabetes animals received no treatment or treatment with MSCs (2X106, via tail vein) for two continuous weeks. Meanwhile, rats recevied antioxidant-probucol (PB) or insulin were as control. Eight weeks after treatment, physical, biochemical and morphological parameters were measured. Blood glucose, urinary albumin excretion, Ccr and renal mass index were detected by laboratory mathods; PAS staining were performed to assess the glomerular sclerotic injury; the localizations of FN、Collagen I、TGF-β、GFP in kidney tissues were measured by immunohistochemistry; expressions of TGF-β at gene level were determined by real time RT-PCR; Western blot method was used to detect the expressions of FN、Collagen Ⅰ、TGF-β and GLUT1in diabetic kidney tissue; the MDA content and the total SOD activity of the renal cortical homogenate were measured by the thiobarbituric acid method and xanthine oxidase method, respectively; chemifluorescene method was used to detect the expression of ROS in fresh kidney frozen sections. Cultured GMC in vitro and divided into three groups as below:GMC; co-culture of GMC and MSCs; GMC cultured in MSC-conditioned media (MSC-CM), after high glucose stimulation, the expression of DHE in GMC was detected, the expressions of TGF-β and GLUT1in GMC were detected by western blot method; to assess the effect of HGF on high glucose induced TGF β expression and ROS production in GMC, MSC-CM and fresh medium were preincubated with or without HGF neutralizing antibody (HGF-Ab) before its addition to GMC cultures, after high glucose stimulation, the expressions of TGF-β and DHE were detected by western blot and chemifluorescene method, respectively; to assess the effect of HGF on TGF-β induced GLUT1expression in GMC, after being preincubated with or without HGF-Ab, MSC-CM or fresh medium was added into GMC cultures, after TGF-β stimulation, the GLUT1expression was detected by western blot method.Results1. MSCs transplantation ameliorated glomerular injury in STZ-induced diabetic nephropathy in rats via inhibiting macrophage infiltration.1.1Characterization of rat MSCsBone marrow-derived MSCs were adherent cultured with a typical fibroblast-like morphology. They were able to differentiate into adipogenic and osteogenic cells, positive for CD29, CD90, CD44, and negative for CD34, CD45, CD11b.1.2MSCs tracking At24hours after transplantation, a small amount of MSCsGFP were present in kidney around the glomeruli and near vessels of MSCsGFP-treated diabetic rats. But at8weeks after transplantation, there was only few MSCs found in glomeruli, and not found elsewhere.1.3MSCs ameliorted physical and biochemical parametersAfter MSCs treatment, the blood glucose level, urinary albumin excretion, Ccr were significantly lower compared with the DN group, with the renal mass index being lower in MSC group.1.4MSCs ameliorated glomerular fibrosis, downregulated the expression of ECM proteinKidneys of DN group rats exhibited profound ECM deposition in the mesangium as revealed by PAS staining, MSCs treatment remarkably ameliorated these pathological abnormalities. The expression of FN and Collagen I in kidney as detected by immunohistochemical method were significantly decreased in MSC group.1.5Effect of MSCs on renal MCP-1expression and macrophages infiltrationThe immunostaining for MCP-1was significantly increased in the DN group in glomeruli and tubulointerstitium, but remarkably reduced by MSCs treatment. ED-1positive cells (representing macrophage) infiltrated in the glomeruli of the diabetes animals at16weeks after STZ injection. But the MSCs treatment remarkably suppressed the macrophages infiltration into the glomeruli compared with the DN group.1.6Effect of MSCs on HGF expressionA significant amount of HGF was detected in MSC-CM by ELISA. In diabetic kidney tissues, the mRNA and protein expression of HGF was significantly increased as compared with the normal group, but the level was much higher in MSC group than the DN group.1.7Effect of MSCs on pro-inflammatory cytokinesThe expression of IL-1β, IL-6, TNF a at gene level and protein level as revealed by real time RT-PCR and ELISA respectively were all significantly increased in DN group as compared with the normal group. However, the levels were significantly decreased in MSC group than the DN group.2. MSCs transplantation ameliorated glomerular injury in STZ-induced diabetic nephropathy in rats via inhibiting oxidative stress.2.1MSCs ameliorated blood glucose level and renal functionSixteen weeks after STZ injection, serum blood glucose level was significantly higher in diabetes rats than that in the normal group, but significantly lower in MSC group and Ins group. The urinary albumin excretion and Ccr were significantly lower in the PB group, Ins group and MSC group than in the DN group. The renal mass index was significantly lower in the MSC group than in the DN group, Ins group and PB group.2.2MSCs ameliorated glomerular fibrosis, downregulated the expression of ECM proteinKidneys of DN group rats exhibited profound ECM deposition in the mesangium as revealed by PAS staining, the glomerular sclerosis (GS) was significantly increased, but was dramatically decreased in the PB, Ins group and MSC groups. Moreover, the GS in MSC group was significantly lower than that in PB group and Ins group.2.3MSCs ameliorated oxidative stress, downregulated TGF-β expression in diabetic kidney and GMCAfter MSCs treatment, the SOD activity was significantly increased and MDA content decreased in the renal tissue, ROS fluoresence staining reduced in fresh kidney frozen sections, with no significant difference with PB group. The renal expression of TGF-β mRNA and protein was significantly decreased in MSC group compared with the DN group as measured by real time RT-PCR, immunohistochemical and Western blot assay. MSCs and MSC-CM treatment remarkably reduced reactive oxygen production in GMC, as revealed by DHE staining. The western blot analysis revealed that the protein expression of TGF-β was also significantly down-regulated by MSCs and MSC-CM.2.4Effects of MSCs on GLUT1protein expressionThe plasma membrane localization of GLUT1in GMC as revealed by western blot was significantly down-regulated by MSCs and MSC-CM, compared with the high glucose control group. Meanwhile, the expression of GLUT1in plasma membrane protein of kidney tissue was significantly decreased in MSC group compared with the DN and PB groups.2.5MSCs secreted HGF and effects of MSC-CM on glucose-induced TGF-β expression, TGF-(3-induced GLUT1expression and glucose-induced reactive oxygen productionA significant amount of HGF was detected in MSC-CM by ELISA. Western blot analysis shows that MSC-CM inhibited upregulation of TGF-β expression after high glucose stimulation and HGF-Ab blocked the inhibitory effect of MSC-CM. But MSC-CM preincubated with or without HGF-Ab did not inhibit the upregulation of GLUT1expression in membrabe protein after TGF-β stimulation. MSC-CM inhibited production of reactive oxygen induced by high glucose, and HGF-Ab blocked the inhibitory effect of MSC-CM.Conclusion1. MSCs transplantation played an active role in the treatment of DN, as revealed by reduction of blood glucose level, amelioration of renal function.2. MSCs transplantation ameliorated glomerular fibrosis, downregulated the expression of ECM protein in STZ-induced diabetic nephropathy.3. The mechanism of MSCs-mediated beneficial effects was related to inhibit macrophage infiltration. MSCs treatment ameliorates DN via inhibition of MCP-1expression by secreting HGF, thus reducing macrophages infiltration, down-regulating IL-1β, IL-6, TNF a expression in renal tissue in diabetic rats.4. The mechanism of MSCs-mediated beneficial effects was related to inhibit oxidative stress. The possible mechanisms underlying these effects involved reduced blood glucose level and cellular glucose uptake mediated by GLUT1, thus inhibiting oxidative stress.