| Objective: Diabetic nephropathy (DN) is the most serious chronic microvascular complications of Diabetes mellitus (DM) and gradually becomes the major cause of end-stage renal failure (ESRF). How to effectively prevent and treat early DN is currently a very hot subject concerned by scholars at home and abroad. The pathogenesis of DN is very complicated.It is considered to be the result of a variety of factors based on the glucose and lipid metabolism disorders caused by high blood glucose.More and more research has found that inflammation and oxidative stress play an important role in the occurrence and development of DN.Glomerular mesangial cell (GMC) is one of the important inherent cells in kidney. GMCs play an important role in maintaining the structure and function of the glomerulus,by producing extracellular matrix, secreting cytokines, swallowing and removing foreign bodies and so on.GMCs are the major target cells to a variety of pathogenic factors and play an important role in the lesions progress.Megsin is a mesangium-predominant gene,and its expression product belongs to serine protease inhibitor (serpin) superfamily. Serpin family is composed of a large number of members, their functions relate to blood coagulation, fibrinolysis, inflammation, cell proliferation, apoptosis, signal transduction, digestive and other systems.Megsin can bind to its substrate plasminogen (plasmin) and inhibit its activity,Which directly or indirectly inhibit the degradation of extracellular matrix.Research has suggested that the expression of megsin increased in IgA nephropathy, DN and rat anti-Thy-1 nephritis model,which demonstrated that megsin is closely related to mesangial cell proliferation and mesangial extracellular matrix accumulation. At present the specific biological function of megsin remains unknown, and its role in the pathogenesis of DN has not been thoroughly investigated. Studies have shown that the p38MAPK signaling pathway can be activated by high blood sugar, inflammatory cytokines, reactive oxygen species and angiotensin II, which is not only involved in cell survival, differentiation and apoptosis, but also has an important role in the inflammation and stress response and it is the intersection or common pathway of cell communication. Studies have found that the expression of monocyte chemoattractant protein-1 (MCP-1) and intercellular adhesion molecule-1 (ICAM-1) increased in DN and which was closely related to the activation of p38MAPK signaling pathway.The overexpression of MCP-1 and ICAM-1 cause a large number of chemotaxis and activation of macrophage and increase renal injury in DN.The levels of MCP-1 and ICAM-1 can be used as indicators to measure the degree of inflammatory response in DN. When DM occurs, the reactive oxygen significantly increase in renal tissue,which lead to the content of lipid peroxidation malondialdehyde (MDA) increase and the activity of total superoxide dismutase (T-SOD) and glutathione peroxidase (GSH-PX) decline so that increase the renal damage. The activation of renin-angiotensin system (RAS) is critical in the pathogenesis of DN. Angiotensin II (Ang II) is the main effector. Therefore, inhibiting the production of AngII and interfering its function is the key to treat DN. Valsartan is the angiotensin II 1 receptor antagonist (AT1Ra),study has found that the protective effect of Valsartan for renal function is independent of the effect to lower blood pressure.We constructed megsin cDNA expression plasmid and megsin siRNA expression plasmid, transfecting them into STZ-induced diabetic mice and cultured mesangial cells, then systematically observed the changes of megsin, p38MAPK signaling pathway, inflammatory cytokines including MCP-1, ICAM-1 and oxidative stress-related indexes and the relationship between them from the whole, cell and molecular levels to discuss the possible roles of megsin in the pathogenesis of early DN. We also observated the effect of valsartan on megsin expression and the activation of p38MAPK signaling pathway in cultured mesangial cells to investigate the mechanism of valsartan in renal protection. We finally explained the roles of megsin in early DN, which may provide an effective way in blocking pathogenic role of megsin in the levels of gene or protein, and may also provide a new idea to further elucidate the molecular pathogenesis of DN and prevent and treat DN.Methods:Part 1: After 2 weeks of uninephrectomy, 48 healthy male CD-1 mice were randomly divided into two groups:control group(CN)and diabetic group(DM).The mice of diabetic group received a single intraperitoneal injection of STZ dissolved in 0.1mol/L sodium citrate buffer(pH 4.5)at a dose of 150mg/kg.The mice of control group only received an injection of the same volume of 0.1mol/L sodium citrate.The model of diabetes was considered to be successful when the blood glucose was≥16.7mmol/L and the glucose in urine was+++～++++after 72 hours of the injection.Six mice from each group were respectively sacrificed at 1,2,4 and 12 weeks after STZ injection.At each time point, blood and urine samples were collected, and kidney tissues were harvested.BG (blood glucose), Scr (serum creatinine), KW/BW (kidney weight/body weight ratio), 24-hour UP (urinary protein) were measured. Partial renal tissues were fixed in 4% paraformaldehyde and embedded in paraffin for light microscopy and immunohistochemistry. Partial renal tissues were fixed in 4% glutaraldehyde for electron microscopy.Light and electron microscopy were used to observe the pathological changes. Immunohistochemistry and Western blot were used to analyze the expressions of megsin, p38MAPK, p-p38MAPK, MCP-1 and ICAM-1.Part 2: The preparation of animal models was the same as the first part. Megsin cDNA and megsin siRNA expression plasmid were constructed.30 male CD-1 mice were divided into five groups:a control group(group A,n=6),a diabetic group(group B,n=6),a diabetic group administered a pCMVsport6.1 control plasmid (group C,n=6), a diabetic group administered a pCMVsport6.1 megsin cDNA plasmid (group D,n=6) and a diabetic group administered a pBAsi mU6 Neo megsin siRNA plasmid (group E,n=6).The mice received a single intravenous dose of 150 mg/kg streptozotocin in citrate buffer to induce diabetes. Group A received the same dose of citrate buffer.Hyperglycemia (≥ 16.7 mmol/L)was confirmed 3 days after STZ administration.Plasmids were then delivered weekly by tail vein injection using the TransIT-EE Hydrodynamic Delivery System each week.Animals were sacrificed at 1,2,4 and 12 weeks.At each time point,urine and blood samples were collected, and kidney tissues were harvested.BG, Scr, KW/BW and 24-hour UP were measured. Partial renal tissues were fixed in 4% paraformaldehyde and embedded in paraffin for light microscopy and immunohistochemistry.Partial renal tissues were fixed in 4% glutaraldehyde for electron microscopy. Light microscopy and electron microscopy were used to observe the pathological changes. The expressions of megsin, p38MAPK, p-p38MAPK, MCP-1 and ICAM-1 were detected by Immunohistochemistry and Western blot.The content of malondialdehyde(MDA) in renal tissues was detected by thiobarbituric acid (TAB) method and the activities of total superoxide dismutase(T-SOD), glutathione peroxidase(GSH-PX) in renal tissues were measured separately by Xanthine oxidase and Dithio-2 nitrobenzoic acid method.Part 3: Mouse mesangial cells were cultured in DMEM/F12 containing 10% fetal calf serum,penicillin(100 U/ml),streptomycin(100 U/ml)at 37°C and 5%CO2. Confluent mesangial cells were divided into five groups: a low glucose group(group A, 5.5mmol/L glucose),a high glucose group(group B, 30mmol/L glucose),high glucose transfected a pCMVsport6.1 control plasmid (group C, 30mmol/L glucose),high glucose transfected a pCMVsport6.1 megsin cDNA plasmid(group D, 30mmol/L glucose) and high glucose transfected a pBAsi mU6 Neo megsin siRNA plasmid (group E, 30mmol/L glucose).When the cells grown to 80% confluence,transplanted the cells to 96-well culture plate, 6-well culture plate and 25cm2 plastic culture flask. Cells were cultured without antibiotics for 24 hours,then were transfected with pBAsi mU6 Neo megsin siRNA plasmid and pCMVsport6.1 control plasmid using lipofectamine 2000 reagent.The cells in 96-well culture plate were washed once with serum-free DMEM/F12 and further incubated for 24 hours to synchronize the cell growth.MTT assay was used to measure the proliferation of mesangial cells at 12, 24 and 48 hours,meanwhile to investigate the effect of megsin on proliferation of mesangial cells in high glucose medium.The cells were cultured in 6-well culture plate and 25cm2 plastic culture flask were harvested to abstract total protein and collect supernatant at 12,24 and 48 hours after incubation.The expressions of megsin, p38MAPK,p-p38MAPK,MCP-1 and ICAM-1 were measured by immunocytochemical staining and western blot.The content of malondialdehyde(MDA) in supernatant was detected by thiobarbituric acid (TAB) method and the activities of Total superoxide dismutase(T-SOD), Glutathione peroxidase(GSH-PX) in supernatant were measured separately by Xanthine oxidase and Dithio-2 nitrobenzoic acid method.The level of collagen IV in supernatant was detected by ELISA.Part 4: Mouse mesangial cells were cultured in 96-well culture plate.The cells were washed once with serum-free DMEM/F12 and further incubated for 24 hours to synchronize the cell growth.MTT assay was used to investigate the effect of valsartan on proliferation of mesangial cell in high glucose medium at different time(12,24 and 48 hours) and different drug concentration(1,10 and 100μmol/L). Mouse mesangial cells were cultured in 6-well culture plate and 25cm2 plastic culture flask and divided into three groups:LG group(5.5mmol/L glucose), HG group(30mmol/L glucose) and HG+Val group(30mmol/L glucose+10μmol/L valsartan). The incubation time extended to 48h. At the end of incubation, the supernatant and cells were collected and total protein were abstracted.The expressions of megsin,p38MAPK, p-p38MAPK,MCP-1 and ICAM-1 were measured by immunocytochemical staining and western blot.The content of malondialdehyde(MDA) in supernatant was detected by thiobarbituric acid (TAB) method and the activities of Total superoxide dismutase(T-SOD),Glutathione peroxidase(GSH-PX) in supernatant were measured separately by Xanthine oxidase and Dithio-2 nitrobenzoic acid method.The level of collagen IV in supernatant was detected by ELISA. Results:Part 1:①Compared with the control group,the KW/BW began to increase from week 2, Scr and 24-hour UP began to increase significantly from week 4 in diabetic mice(P<0.05).②There were obvious morphological changes of renal tissues of diabetic mice at week 2.Light microscopy showed that the number of glomerular inherent cells was increased,glomeruli was slightly enlarged, glomerular basement membrane was thickened and mesangium was expanded in diabetic mice.Electron microscopy showed that irregular thickening of glomerular basement membrane,mesangial expansion and fusion of foot processes in diabetic mice.The above morphological changes of glomeruli were markedly deteriorated at week 12.③Immunohistochemical staining showed that positive stainings for megsin, p38MAPK, p-p38MAPK, MCP-1 and ICAM-1 were observed in renal tissues of both control group and diabetic group. Megsin was predominantly expressed in cytoplasm of mesangial cells. p-p38MAPK was observed in the nuclei of mesangial cells, endothelial cells, podocytes and tubular epithelial cells and p38MAPK was detected mainly in cytoplasm of above cells. The positive stainings for MCP-1 and ICAM-1 were observed in cytoplasm of mesangial cells, endothelial cells and tubular epithelial cells.④Immunohistochemical staining and Western blot analysis indicated that: compared with control group, the expression of megsin was higher after 1 week and the expressions of MCP-1 and ICAM-1were higher after 2 week in diabetic kidneys and progressively heavier with duration of diabetes(P<0.05). The positive staining for p-p38MAPK was begin to increase at week 2(P<0.05), peaked at week 4(P<0.01), and decreased at week 12(P>0.05).The expression of p38MAPK was no statistic difference in each group at different time points(P>0.05).Part 2:①A t week 4, 24-hour UP,Scr and KW/BW were the lowest in group A,the highest in group D,followed by group B,these indexes were significantly lower in group E than in group B, there were significant differences among the four groups(P<0.05). there was no statistic difference between group C and group B (P>0.05).②There were obvious morphological changes of renal tissues of diabetic mice at week 4.Light microscopy showed that the number of glomerular inherent cells was increased, glomeruli was slightly enlarged, glomerular basement membrane was thickened and mesangium was expanded in diabetic mice.Electron microscopy showed that irregular thickening of glomerular basement membrane, mesangial expansion, fusion of foot and disappearing of endothelial cell fenestrae structure processes in diabetic mice. The above morphological changes of glomeruli were markedly deteriorated in group D and were significantly ameliorated in group E.③Immunohistochemical staining and Western blot analysis indicated that the protein levels of megsin,p-p38MAPK,MCP-1 and ICAM-1 were significantly increased in glomeruli of group B than group A(P<0.01), higher in group D than in group B(P<0.05) and the above indicators in group E expressed between group A and group B (P<0.05), there was no statistic difference between group C and group B (P>0.05). The expression of p38MAPK was no significant change in each group (P>0.05).④The concent of MDA increased and the acticities of T-SOD and GSH-PX decreased in group B than in group A(P<0.01), there was no statistic difference between group B and group C (P>0.05). Compared with group B, the concent of MDA was higher and the acticities of T-SOD and GSH-PX were lower in group D (P<0.01), the concent of MDA was lower and the acticities of T-SOD and GSH-PX were higher in group E than in group B (P<0.05).Part 3:①Compared with low glucose,high concentration of glucose could promote the proliferation of mesangial cells and more obvious with time(P<0.01), there was no statistic difference between group C and group B (P>0.05), the proliferation of mesangial cell was more evident in group D and decreased in group E compared with group B in the same time point(P<0.05).②Immunocytochemical staining indicated that the positive stainings for megsin,p38MAPK,MCP-1 and ICAM-1 were detected mainly in cytoplasm of mesangial cells and p-p38MAPK was observed mainly in the nuclei of mesangial cells.Western blot analysis indicated that since hour-12, the protein levels of megsin,p-p38MAPK,MCP-1 and ICAM-1 of mesangial cells began to increase in group B than group A,peaked at hour-48 (P<0.05), there was no statistic difference between group C and group B (P>0.05), but the trend in group D was more obvious, the difference was significant compared with the group B (P<0.05), the expressions of megsin,p-p38MAPK,MCP-1 and ICAM-1 reduced in group E compared with group B(P<0.05). The expression of p38MAPK was no statistic difference in each group at different time (P>0.05).③From the start of hour-12,the concents of type IV collagen and MDA started to rise in group B than in group A,peaked at hour-48(P<0.01),there was no statistic difference between group B and group C (P>0.05).Compared with group B,the concents of type IV collagen and MDA were higher in group D and lower in group E,the difference was significant(P<0.05).The changes of activities of T-SOD and GSH-PX were contrary to the concent of MDA(P<0.01, P<0.05).Part 4:①Compared with low glucose,high concentration of glucose could promote the proliferation of mesangial cells(P<0.01).1,10 and 100μmol/L valsartan could suppress the proliferation of mesangial cells in high glucose(P<0.05). 1μmol/L valsartan had little suppression on proliferation of the mesangial cells exposed to high glucose and the suppression became more stronger at a dose of 10μmol/L(P<0.05),but it showed over suppression at a dose of 100μmol/L(P<0.01).②Immunocytochemical staining and western blot analysis indicated that compared with the LG group,the expressions of megsin,p-p38MAPK,MCP-1 and ICAM-1 were increased in HG group(P<0.01).Compared with the HG group,the expressions of megsin, p-p38MAPK,MCP-1 and ICAM-1 were decreased in valsartan treated group(P<0.01).The expression of p38MAPK was no statistic difference in each group (P>0.05).③The concents of MDA and type IV collagen in supernatants exposed to high glucose were higher than that in low glucose group at 48 hours(P<0.01).After treamented with valsartan,the contents of MDA and type IV collagen was decreased,compared with the high glucose(P<0.05).Compared with the high glucose,the activities of T-SOD and GSH-PX in supernatants were increased in HG group(P<0.01).After treamented with valsartan, the activities of T-SOD and GSH-PX were decreased, compared with the high glucose(P<0.05).Conclusions:1 The experiments in vivo and in vitro have confirmed that the expression of megsin in the glomerular strengthen at diabetic state,consisting with mesangial proliferation, extracellular matrix accumulation and suggesting that megsin plays an important role in the mechanism of the occurrence of diabetic nephropathy.2 Overexpressed megsin may increase the expression levels of p-p38MAPK,MCP-1,ICAM-1 and MDA in renal and mesangial cells,but decrease the activities of T-SOD and GSH-PX so that promote glomerular mesangial cell proliferation and mesangial extracellular matrix deposition, suggesting that the mechanism of megsin promoting the progression of early diabetic nephropathy may be concerned with the activation of p38MAPK signal pathway. However the specific ways have to be further studied.3 Megsin siRNA plasmid and valsartan (Ang II type 1 receptor antagonist,AT1Ra) can decrease the expression of megsin in the gene and protein level and reduce the expression of p-p38MAPK,MCP-1,ICAM-1 and the level of MDA,increase the activities of T-SOD and GSH-PX,then retard the pathological process of mesangial proliferation and extracellular matrix accumulation,which provides a new idea and target for prevention and treatment of early diabetic nephropathy. |
PDF Full Text Request