The Role Of Megsin In The Pathogenesis Of Diabetic Nephropathy

Objective: Diabetic nephropathy (DN) is the most common and serious chronic microvascular complications of Diabetes mellitus (DM). With the increased incidence of DM in the whole world, DN gradually becomes the major or first cause of end-stage renal failure (ESRF). Patients have to rely on dialysis or kidney transplantation to live, which seriously affected the quality of life and brought about a heavy financial burden and stress to the family and the community. How to effectively prevent and treat early diabetic nephropathy is currently a very hot subject concerned by scholars at home and abroad.Pathological features of DN are the glomerular mesangial cell proliferation, hypertrophy, extracellular matrix accumulation, and ultimately occurring glomerular sclerosis. Glomerular mesangial cel(lGMC)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, located in 18q21.3, which encodes protein N-terminal variable reactive site loop (RSL) binding with the serine protease, and plays a serine protease inhibitor (serpin) activity. 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. The dynamic balance between serpin and their substrates is closely related to the normal physiological activities in the body. We speculate that megsin, as a member of the serpin family, participates in certain physical activities of mesangial cells. To discuss megsin's important roles in glomerular mesangial cells was greatly significant to reveal the mechanism of diabetic nephropathy.In this study we observed the megsin expression in clinical renal biopsy sections,renal tissues of diabetic animals and GMCs in vitro. We Constructed megsin siRNA expression plasmid and megsin cDNA expression plasmid, transfected them to STZ-induced diabetic mice and cultured mesangial cells, then investigated the relationship between megsin and mesangial cell proliferation, matrix metalloproteinase (MMP-2)/tissue inhibitor of metalloproteinase (TIMP-2), and Cell cycle inhibitor P27, to discuss the possible roles of megsin in the pathogenesis of early DN. We also observated the effect of hydroxymethyl-Coenzyme A(Ahydroxy-methyl-glutaryl coenzyme A, HMG-CoA) reductase inhibitors (HMG-CoA reductase inhibitor, HCRI) on megsin expression in STZ-induced diabetic rats. 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 or treat DN.Methods:Part 1: 60 diabetes patients in line with the diagnostic criteria for type 2 diabetes of American Diabetes Academy in 1998 and 38 cases of primary nephrotic syndrome were screened out from the department of nephrology in 3rd hospital of Hebei Medical Universty(HBMU) from June 2006 to June 2007.18 cases in diabetic nephropathy and 29 cases in minimal change glomerulopathy were confirmed by renal biopsy and clinical data as research subjects.In addition, five control cases were received from the normal tissues around the resected renal tumors of hospitalized patients in 3rd hospital of HBMU.The expression of megsin in renal tissue of every group was semi-quantitatively analysed by immunohistochemical staining.Part 2: In vivo experiment:after 2 weeks of uninephrectomy, 12-week-old male CD-1 mice were divided into three groups: a non-diabetic control group (A group,n=15), a diabetic group administered a pCMVsport6.1 control plasmid (B group,n=15), and a diabetic group administered a pCMVsport6.1 megsin cDNA plasmid (C group,n=15).The mice received a single intravenous dose of 150 mg/kg streptozotocin in citrate buffer to induce diabetes. A group received the same dose of citrate buffer. Hyperglycemia (>15 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 week-1, week-2 and week-12. At each time point, urine and blood samples were collected, and kidney tissues were harvested. Blood glucose (blood glucose, BG), serum creatinine (serum creatinine, Scr), kidney weight / body weight ratio (Kidney weight / body weight ratio, KW / BW) , urinary protein (Urinary protein, UP) were measured.Kidneys were fixed in 4% paraformaldehyde and embedded in paraffin for light microscopy and immunohistochemistry. The paraformaldehyde-fixed and paraffin-embedded kidney tissues were cut into sections of 2-4μm thick. 2μm sections were stained with Hematoxylin and Eosin (HE). 4μm sections were used for immunohistochemistry staining. HE staining were used to observe the pathological changes. Immunohistochemistry was used to analyze the renal expression of megsin, MMP-2, TIMP-2, P27 and collagen IV. The expression of megsin, MMP-2, TIMP-2, P27 and collagen IV were also detected by Western blotting. In vitro experiments:Mouse mesangial cells were grown in DMEM F12 containing 5% FBS, penicillin (100 U/ml), streptomycin (100μg/ml) at 37°C and 5% CO2. The cells were divided into 4 groups: a normal glucose group (A), a high glucose group (B), high glucose transfected a pBAsi mU6 Neo megain siRNA control plasmid group (C), and high glucose transfected a pCMVsport6.1 megsin cDNA plasmid group (D). When the cells grown to 80% confluence,transplanted the cells to 6-well culture plates (2×106 cells per well). After 24 hours, B group, C group and D goup cells were further cultured in DMEM F12 containing high glucose and A group cells were cultured in normal glucose for up to 48 hours. Cells in 6-well culture plates were collected for protein extraction and the culture medium were collected for Collagen IV measurement at hour-12,hour-24 and hour-48.The levels of megsin,MMP-2,TIMP-2 and P27 in GMCs were measured by Western blot and supernatant Collagen IV were measured by RIA.All of the data was analysed by SPSS15.0 statistics software, P value<0.05 was considered to have a statistical significance.Part 3:The preparation of animal models is the same as the second part. 12-week-old male CD-1 mice were divided into three groups: a non-diabetic control group (A group,n=15), a diabetic group administered a pBAsi mU6 Neo control plasmid (B group,n=15), and a diabetic group administered a pBAsi mU6 Neo megsin siRNA plasmid (C group,n=15).The mice received a single intravenous dose of 150 mg/kg streptozotocin in citrate buffer to induce diabetes. A group received the same dose of citrate buffer. Hyperglycemia (>15 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 week-1, week-2 and week-12. At each time point, urine and blood samples were collected, and kidney tissues were harvested. Blood glucose (blood glucose, BG), serum creatinine (serum creatinine, Scr), kidney weight / body weight ratio (Kidney weight / body weight ratio, KW / BW) , urinary protein (Urinary protein, UP) were measured.Kidneys were fixed in 4% paraformaldehyde and embedded in paraffin for light microscopy and immunohistochemistry. The paraformaldehyde-fixed and paraffin-embedded kidney tissues were cut into sections of 2-4μm thick. 2μm sections were stained with Hematoxylin and Eosin (HE). 4μm sections were used for immunohistochemistry staining. HE staining were used to observe the pathological changes. Immunohistochemistry was used to analyze the renal expression of megsin, MMP-2, TIMP-2, P27 and collagen IV. The expression of megsin, MMP-2, TIMP-2, P27 and collagen IV were also detected by Western blotting. In vitro experiments: Mouse mesangial cells were grown in DMEM F12 containing 5% FBS, penicillin (100 U/ml), streptomycin (100μg/ml) at 37°C and 5% CO2. The cells were divided into 4 groups: a normal glucose group (A), a high glucose group (B), high glucose transfected a pBAsi mU6 Neo control plasmid group (C), and high glucose transfected a pBAsi mU6 Neo megsin siRNA plasmid group (D). When the cells grown to 80% confluence,transplanted the cells to 6-well culture plates (2×106 cells per well).Cells were cultured without antibiotics for 24 hours , then were transfected with pBAsi mU6 Neo megsin siRNA plasmid or pBAsi mU6 Neo control plasmid using lipofectamine 2000 reagent.After 24 hours, B group, C group and D goup cells were further cultured in DMEM F12 containing high glucose and A group cells were cultured in normal glucose for up to 48 hours. Cells in 6-well culture plates were collected for protein extraction and the culture medium were collected for Collagen IV measurement at hour-12,hour-24 and hour-48.The levels of megsin,MMP-2,TIMP-2 and P27 in GMCs were measured by Western blot and supernatant Collagen IV were measured by RIA.All of the data was analysed by SPSS15.0 statistics software, P value<0.05 was considered to have a statistical significance.Part4: The male SD rats(weight about 200+/-10g) were divided into three groups: a non-diabetic control group (NC,n=6), a diabetic group(DC,n=6), and a diabetic group treated with fluvastatin(DF,n=6,2mg·kg-1·d-1,ig). The animals of DC and DF groups received a single intravenous dose of 65 mg/kg streptozotocin in citrate buffer to induce diabetes. NC group received the same dose of citrate buffer.Hyperglycemia (>15 mmol/L) was confirmed 3 days after STZ administration. The animals were sacrificed at 6 week. The urine and blood samples were collected, and kidney tissues were harvested. Kidneys were fixed in 4% paraformaldehyde and embedded in paraffin for light microscopy and immunohistochemistry. The paraformaldehyde-fixed and paraffin-embedded kidney tissues were cut into sections of 2-4μm thick. 2μm sections were stained with periodic acid-Schiff (PAS). 4μm sections were used for immunohistochemistry and immune-double staining. HE and PAS staining were used to observe the pathological changes. Immunohistochemistry was used to analyze the renal expression of megsin,collagen IV and laminin(LN). BG, serum total cholesterol (TC) , triglyceride (TG), serum creatinine (Scr) and urine creatinine (Ucr), creatinine clearance rate (Ccr) and hypertrophy index(the ratio of kidney weight :body weight) were evaluated in every group. All the data were analysed by SPSS15.0 statistics software, P value<0.05 was considered to have statistical significance.Results:Part1:megsin expressed none or only weakly in mesangial area of renal tissue of A group and B group.There wasn't a significant difference between two groups (p> 0.05).But megsin was strengthened significantly in the renal tissues of diabetic nephropathy patients compared with the other two groups(p<0.01).Part2:In vivo experiment:diabetic mice occurred polydipsia,polyuria and polyphagia at 4 or 5 days after being injected to STZ.At week-1,the number of glomerular inherent cells in B group mice was more than in A group(p<0.05) but less than in C group(p<0.05).The results of immunohistochemistry and Western blot showed that the levels of megsin,TIMP-2,P27 and collagen IV were higher than A group(p<0.05),lower than C group(p<0.05) and MMP-2 lower than A group(p<0.05) but higher than C group(p<0.05).At week-2 all of the results were changed more markedly and there was the significant difference among three groups(p<0.05).The number of glomerular inherent cells of group B decreased at week-12, there is no difference compared with A group (p> 0.05), the expression level of P27 dropped, but still higher than the A group (p <0.05), and no difference compared with group C(p> 0.05), the other parameters such as megsin,TIMP-2 and type IV collagen further enhanced,but still weaker than C group,MMP-2 expression level of further lower, but still higher than C group (p<0.05). At week-12 UP, Scr and KW / BW ratio are the lowest in the A group,the highest in the C group,followed by B group,there are significant differences among the three groups(P<0.05).In vitro experiment:Since hour-12,the protein levels of megsin, P27, and TIMP-2 of mesangial cells in groupB began to increase,peaked at hour-48,the level of MMP-2 was the most lowest at hour-48(P<0.05),there was no significant difference between C group and B group (P>0.05),but the trend in D group is more obvious,the difference was significant compared with the B group and C group(P<0.05);since hour-12,the concentration of supernatant collagen IV (cell total protein correction) became higher in B group than in A group,peaked at hour-48(P<0.05),there is no significant difference between C group and B group (P> 0.05),but the concentration of type IV collagen in D group increased most obviously,there is a significant difference compared with the other groups(P <0.05).Part3:In vivo experiment:A similar symptoms of diabetes with the second part was appeared.The number of glomerular inherent cells in B group increased,comparing with A group at week-1,the results of immunohistochemistry and Western blot showed that the expression of megsin,TIMP-2,P27and type IV collagen strengthened, MMP-2's expression decreased.The above-mentioned indicators in C group expressed between A group and B group,there was a significant difference among three groups(p <0.05).The number of glomerular inherent cells, the expression levels of megsin,MMP-2,TIMP-2,P27 and type IV collagen at week-2 were more significantly different,the above indicators in C group expressed between A group and B group,there were significantly difference among the three groups(p<0.05).The number of glomerular inherent cells decreased in B group at week-12,the expression levels of P27 decreased,but still higher than in A group and C group (p<0.05),the expression of other indicators such as megsin, TIMP-2 and type IV collagen further enhanced,the expression level of MMP-2 further reduced,the expression of above-mentioned indicators in C group was between A group B group,there was significant difference among the three groups(p<0.05).UP,Scr and KW/BW ratio were in the lowest in the A group,in the highest in B group,followed by C group at week-12,the differences among the three groups was significant(P<0.05).In vitro experiment:Since hour-12,the protein levels of megsin, P27, and TIMP-2 in mice mesangial cells began to increase in B group than A goup, peaked at hour-48,the level of MMP-2 dropped to the lowest at hour-48(P<0.05),there was no significant difference between C group and B group (P>0.05);the protein level of megsin,P27 and TIMP-2 reduced in D group,comparing with B group or C group,MMP-2 increased, there was a significant difference(P<0.05);from the start of hour-12,the concentrations of type IV collagen (cell total protein concentration correction)started to rise in B group than in A group,peaked at hour-48(P<0.05),there was no significant difference between B group and C group (P>0.05); comparing with C group,the concentration of collagen type IV in D group was lower,the difference was significant(P<0.05). Part4:There was no difference about blood glucose between DC group and DF group at week-6,but higher than NC group (P<0.01);the PAS-positive material on rats glomerular increased mildly at DC group,the number of inherent cells incresed,serum TC,TG,Ccr,UAER,kidney/body weight ratio increased,as well as immunohistochemical results showed that the expression levels of megsin, IV collagen and laminin was higher than at NC group,the above-mentioned indicators in DF group was lower than at DC group, but higher than at NC group,the difference among three groups was significant(P<0.05).Conclusions:I.Clinical and experimental studies have shown 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.II.The experiments in vivo and in vitro have confirmed that over-expressed megsin may increase the expression levels of TIMP-2, P27 in renal mesangial cells,but decrease the expression of MMP-2,suggesting that megsin promotes the mechanism of early diabetic nephropathy and concerned with MMP-2/TIMPs and P27.However the specific ways have to be further studied.III.Megsin siRNA plasmid and HMG-CoA reductase inhibitor can decrease the expression level of megsin in the gene level and protein level, reduce the expression of TIMP-2 and P27 and increase the expression of MMP-2 in renal tissue,then retard the pathological process of mesangial proliferation and extracellular matrix accumulation,which provides a new idea for prevention and treatment of diabetic nephropathy.