| Objective: Diabetic nephropathy (DN) is a serious complication of Diabetes mellitus (DM), which has huge harm, can cause end-stage renal failure (ESRF) without active treatment finally. Nowadays DN is the most common etiological factor of clinic renal failure in the United States and the Europe, accounting for 40% of dialysis patients of ESRF. In the year of 2001, a inves- tigation about the hospitalized patients of 30 provinces and autonomous regi- ons in our country, found that the prevalence rate of DN was 33% approxi- mately. So it becomes a hot topic for extended nephrologists to research the pathogenesis as well as the methods of preventing and treating DN.According to recent reports, the pathogensis of DN are considered to be related with many factors, such as glycometabolism disorder, non-enzymatic glycation, polyols pathway activation, protein kinase C activation, lipid disorders, kidney hemodynamic changes caused by hypertension, oxidative stress, activesubstance of blood vessel, cytokines, genetic factors and so on. The pathological features of DN is glomerulus hypertrophy. The histopathological background of DN are the glomerular mesangial cell(GMC) proliferation, extracellular matrix accumulation, these can lead to high glomerular filtration and proteinuria. The ultimate consequences are glomerular sclerosis and tubulointerstitial fibrosis. Both increased synthesis and decreased degradation are responsible for ECM accumulation, such as cytokines or growth factors that promote GMC proliferation, GMC hypertrophy and matrix secretion increased. At the same time, extracellular matrix degradation decreases. At present we already know three main enzymes participate in ECM degradation: (1)matrix metalloproteinases(MMPs); (2)serine protease; (3)cysteine protease. MMPs can degrade collagenⅣ,Ⅴ andⅥin ECM. Plasminogen activator inhibitor-1 (PAI-1) is a pivotal inhibitor for serine protease to degrade ECM. They are directly involved in the dynamic imbalance of ECM accumulation.Megsin is a mesangium-predominant gene, belongs to the serine protease inhibitor superfamily, located in 18q21.3, which encodes protein N-terminal variable reactive site loop (RSL) binding with the serine protease, and shows activity of a serine protease inhibitor. Studies have found that megsin gene and protein are up-regulated markedly in IgA nephropathy, which prompted that megsin as a member of the serpin family, must be involved in certain pathophysiological activities of GMC. DN as a kind of disease whose main pathological changes are GMC proliferation and extracellular matrix accumulation, it is not clear that how the megsin gene participate in the pathogenesy process of DN and which is the effective pathway. In ordre to reveal the mechanism of DN, it is very important to investigate megsin's roles on extracellular matrix synthesis and degeneration in the GMC.In this study we constructed transfection megsin CD-1 mice model of diabetes and made the megsin gene overesxpress or inhibiting expression by using RNA interfered technology in mice renal tissue or mesangial cells in vitro, then observed the expression changs of megsin,extracellular signal-regulated protein(pERK1/2),transforming growthfactor-β1(TGF-β1)and collagen IV from the whole, cell and molecular levels, in order to research the possible effect of megsin on cell signaling pathways and extracellular matrix synthesis in the pathogenesis of DN; Investigating the effect of megsin on extracellular matrix degeneration in the presence of high glucose by the expression change of extracellular matrix metalloproteinase inducer(EMMPRIN),MMP-9 and collagen IV; we also observed the effect of pioglitazone on megsin, MMP-2, collagen IV and PAI-1 in the presence of high glucose by drug interference, which provide a new idea to further elucidate the role of megsin in the pathogenesis of DN and the prevention and treatment of DN. Part 1: Sixty clean healthy male CD-1 mice were operated by using unilateral nephrectomy. After 2 weeks of uninephrectomy, 45 of them were given a intraperitoneal injection of streptozotocin (at a dose of 150mg/kg STZ dissolved in 0.1mol/L sodium citrate buffer, pH 4.5) to induce diabetes. The diabetic mice randomly divided into three groups: a diabetic group administered an injection by tail vein of pCMVsport6.1 megsin cDNA plasmid once a week(group D,n=15), a diabetic group(Group B, n=15), a diabetic group administered a pCMVsport6.1 control plasmid (Group C, n=15) and the non-diabetic mice just with unilateral nephrectomy as control group (Group A, n=15). Experiment lasted for 12 weeks, at the end of week-2, 4, 12, urine, blood samples were collected to test blood glucose (BG), serum creatinine (Scr), kidney weight / body weight ratio (KW / BW) , urinary protein (UP). Kidney tissues were collected at the end of week-2, week-4 and week-12. The expression of megsin, PDGF-BB, pERK1/2, TGF-β1 and collagen IV in the kidney tissues were respectively detected by immunohistochemistry staining and Western blotting. The cell strain of mice GMC that express megsin stably was established and cultured in the presence of high glucose for 48 hours(group D), at the same time a low glucose group of mice GMC (group A, 5.5mmol/L D-glucose), a high glucose group(group B, 30mmol/L D-glucose), high glucose transfected a pBAsi mU6 Neo megain siRNA control plasmid group(group C), high glucose transfected a pCMVsport6.1 megsin cDNA plasmid group(group D), high glucose transfected a pCMVsport6.1 megsin cDNA plasmid and UO126 group (group E) were established. The cells and supernatant were collected to extract protein at 12, 24 and 48 hours after incubation. The expression of megsin, PDGF-BB, pERK1/2, TGF-β1 and FN in cells were measured by immunocytochemistry staining and the protein expression of megsin, pERK1/2, PDGF-BB, TGF-β1 of total protein in each group were detected by Western blotting. The concentrations of type IV collagen in cell supernatant were detected by radio-immunity.Part 2: The preparation of animal models is the same as the first part. Established a diabetic CD-1 mice group administered an injection by tail vein Methods: of megsin siRNA-express-plasmid once a week(group D, n=15), at the same time, a diabetic group(group B, n=15), a diabetic group administered a control plasmid (group C, n=15) and the non-diabetic mice just with unilateral nephrectomy as control group (group A, n=15) were established. Experiment lasted for 12 weeks, at the end of week-2, 4, 12, blood samples, urine, and kidney tissues were collected to test blood glucose (BG), serum creatinine (Scr), kidney weight / body weight ratio (KW / BW) and urinary protein (UP). Kidney tissues were collected at the end of week-2, week-4 and week-12. The expression of megsin, PDGF-BB,pERK1/2, TGF-β1and collagen IV in the kidney tissues were respectively detected by immunohistochemistry staining and Western blotting. The mouse mesangial cells were cultured in vitro, using liposomes as carrier to transfection megsin siRNA plasmid transiently, cultured in high glucose for 48 hours(group D), and established a low glucose group(group A, 5.5mmol/L D-glucose),a high glucose group(group B, 30mmol/L D-glucose),high glucose transfected a pBAsi mU6 Neo megain siRNA control plasmid group(group C). The cells and supernatant were collected to extract protein at 12, 24 and 48 hours after incubation. The expression of megsin, PDGF-BB, pERK1/2, TGF-β1 and FN in cells were measured by immunocytochemistry staining and the protein expression of megsin, PDGF-BB, pERK1/2, TGF-β1 of total protein in each group was detected by Western blotting. The concentrations of type IV collagen in cell supernatant were detected by RIA (the results of total protein with correction).Part 3: The cell strain of mice GMC that express megsin stably were established and cultured in the presence of high glucose for 48 hours(group D); using liposomes 2000 as carrier to transfect megsin siRNA plasmid transiently, cultured in high glucose for 48 hours(group E), a low glucose group(group A, 5.5mmol/L D-glucose),a high glucose group(group B, 30mmol/L D-glucose), high glucose transfected a control plasmid group(group C) were established. The cells and supernatant were collected to abstract protein at 12, 24 and 48 hours after incubation. The expression of megsin, EMMPRIN and MMP-9 in cells was measured by immunocytochemistry staining and the protein expression of megsin, EMMPRIN and MMP-9 of total protein in each group was detected by using Western blotting. The concentrations of type IV collagen in cell supernatant were detected by RIA (the results of total protein with correction).Part4: Mouse mesangial cells were cultured in 96-well culture plate. MTT assay was used to investigate the effect of low glucose, high glucose and pioglitazone on proliferation of mesangial cell. Mouse mesangial cells were cultured in 6-well culture plate and 25cm2 plastic culture flask and divided into three groups: a low glucose group(group A, 5.5mmol/L D-glucose), a high glucose group(group B, 30mmol/L D-glucose))and high glucose+ pioglitazone group(group C). The cells were harvested to abstract total protein and collect supernatant at 12,24 and 48 hours after incubation. The expression of megsin, EMMPRIN, MMP-2 and PAI-1 in cells was measured by immunocytochemistry staining and the protein expression of megsin, EMMPRIN, MMP-2 and PAI-1 of total protein in each group was detected by using Western blotting. The concentrations of type IV collagen in cell supernatant were detected by RIA (the results of total protein with correction).Results:Part1:1. animal in vivo experiment: Compared with the group A, the KW/BW, Scr and UP began to increase significantly from week 4 in the mice of group B and group C (P<0.01), and group D increased more significantly (P<0.01); The change above-mentioned was even more significant at week 12. Light microscopy showed that glomeruli was slightly enlarged, mesangial expansion increased from week 2 in the mice of group B and group C; PAS stain showed that positive erythro-stain mass was increased, Masson stain showed collagen fiber was increased. The change was more significant in mice of group D compared with the group B and group C; the above changes were even more significant at week 12. The results of immunohistochemistry and Western blot showed that compared with Group A, the expression levels of megsin in the kidney tissue of CD-1 mice enhanced in group B, C, D(P<0.01), compared with group B, C, the expression was more significant in group D(P<0.01). With the extension of time, the expression levels of megsin in the kidney tissue was increased gradually, peaked at week-12, which has a significant difference (p<0.01) compared with each time point. The expression PDGF-BB, pERK1/2, TGF-β1, FN and collagen IV were higher in group B, C, D at week 2(p<0.01), which has a significant difference (p<0.01) compared with group A, Compared with group B, C, the expression was more significant in group D(P<0.01). With the extension of time, the expression levels of index obove-mentioned in the kidney tissue was increased gradually, peaked at week-12. The levels of megsin, PDGF-BB, pERK1/2, TGF-β1 at all time points and in each group in Western blot was at equal with the result in immunohistochemistry.2 In vitro mesangial cell culture: The results of immunocytochemistry showed that since hour-12, the protein levels of megsin, PDGF-BB in mice mesangial cells began to increase in high glucose group(group B) and control plasmid group (group C), peaked at hour-48, there was a significant difference compared with group A(P<0.01), there was a significant difference compared the two group in each time point. After the transfection of megsin plasmid(group D), the expression of megsin and PDGF-BB increased significantly in all time point with the extension of time, there was a significant difference compared with group B(P<0.01). After the interference of U0126(group E), we discovered the expression level of megsin, PDGF-BB did not decrease significant comparing with group D in all the time points. From the start of hour-12, the expression of pERK1/2, TGF-?1 and FN began to increase in high glucose group(group B) and control plasmid group(group C), with the extension of time, the expression increased gradually, peaked at hour-48, there was a significant difference compared with group A(P<0.01), the expression of the three, there was a significant difference between the two group in each time point(P<0.01). After the transfection of megsin plasmid(group D), the expression level of pERK1/2, TGF-β1 and FN increased with the extension of time in each time point significantly, there was a significant difference compared with group B(P<0.01). After the interference of U0126(group E), we discovered the expression level of the three decreased significant comparing with group D in all the time points. The result of the protein level of megsin, PDGF-BB, pERK1/2, TGF-β1 was the same with Western blotting. The concentrations of type IV collagen started to rise in group B and group C increased with the extension of time in each time point significantly, peaked at hour-48, there was a significant difference compared with group A(P<0.01); comparing with group B and group D at the same time, the concentration of collagen type IV increased with the extension of time, there was statistical significance about the difference(P<0.01); while comparing with group D, the concentration in group E decreased obviously, the difference was there was statistical significance(P<0.01).Part2:1 In vivo animal experiment: Compared with group A, the mice of group B, group C and group D occurred polydipsia, polyuria and polyphagia at 4 or 5 days after being injected to STZ. The KW/BW, Scr, UP ratio started to increase from week-2 in the mice of group B and group C, group D was lower than group A, but there was no significant difference (P>0.05); Compared with group A, KW/BW, Scr, UP ratio began to increase significantly from week 4 in the mice of group B (P<0.01), and group C increased more significantly (P<0.01); while the KW/BW, Scr, UP ratio in the mice of group D was lower when compared with group B, the change above even more significant at week-12. Light microscopy showed that glomeruli was slightly enlarged, glomerular basement membrane was thickened and mesangium matrix was expanded in mice of group B and group C at week 2, the change less significant in mice of group D compared with group B; the above change even more significant at week 12.The results of immunohistochemistry and Western blot showed that compared with group A, the levels of megsin,PDGF-BB, pERK1/2, TGF-β1 and collagen IV were higher in Group B from week 2(p<0.01); after group D transfected megsin siRNA, the levels of megsin, PDGF-BB, pERK1/2, TGF-β1 and collagen IV were lower in group D than group B, there has a statistical significance (p<0.01); With the extension of time, the levels of megsin, PDGF-BB, pERK1/2, TGF-β1 and collagen IV in group B further enhanced compared with group A (p<0.01), peaked at week-12, these indexes in group D lower than group B (p<0.01). The index above at all time points has no significant difference between group B and group C (P>0.05).2 In vitro masangial cell culture: The results of immunocytochemistry and Western blot showed that since hour-12, the protein levels of megsin, pERK1/2, PDGF-BB, TGF-β1 and FN in mice mesangial cells began to increase in group B than group A, peaked at hour-48, there was no significant difference between group C and group B (P>0.05); The expression level and protein level of megsin, PDGF-BB, pERK1/2, TGF-β1 and FN decreased in group D comparing with group B ,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 group B than in group A, peaked at hour-48(P<0.01), there was no significant difference between group B and group C (P>0.05); while comparing with group B, the concentration of collagen type IV in group D decreased obviously, the difference has significant statistical significance (P<0.05).Part3: The results of immunocytochemistry and Western blot showed that since hour-12, the expression and protein level of megsin in mice mesangial cells began to increase in group B than group A(P<0.01), peaked at hour-48, there was no significant difference between group C and group B (P>0.05); the expression and protein level of megsin increased more obviously in group D, comparing with group B(P<0.01). The expression and protein level of megsin in group E decreased obviously than Group B and group D (P<0.01); From the start of hour-12, the protein level of EMMPRIN and MMP-9 decreased in group B than group A, there was a significant difference(P<0.01), peaked at hour-48, there was no significant difference between group B and group C (P>0.05); the expression and protein level of EMMPRIN and MMP-9 decreased more obviously in group D than group B, there was a significant difference (P<0.01); the expression and protein level of EMMPRIN and MMP-9 increased more obviously in group E compared with group B and group D (P<0.01); from the start of hour-12, the concentrations of type IV collagen (cell total protein concentration correction)started to rise in group B than in group A, peaked at hour-48 (P<0.01), there was no significant difference between group B and group C (P>0.05); but comparing with group B, the concentration of collagen type IV in group D was much higher(P<0.01), while comparing with group B and group D, the concentration of collagen type IV in group E decreased obviously, the difference was significant(P<0.01).Part4: The results of immunocytochemistry and Western blot showed that the expression and the protein level of megsin and PAI-1 in mice mesangial cells increase in group B than group A at hour-48(P<0.01). The expression and the protein level of megsin and PAI-1 decreased in group C compared with group B (P<0.01). The protein level of EMMPRIN and MMP-9 decreased in group B than group A (P<0.01). The protein level of EMMPRIN and MMP-9 increased obviously in group C compared with group B, there was a significant difference(P<0.01). The concentrations of type IV collagen (cell total protein concentration correction) rose in group B than in group A (P<0.05) , comparing with group B, the concentration of collagen type IV in group C decreased obviously (P<0.01).Conclusions:1 The experiments in vivo and in vitro have shown that the expression of megsin in the glomerular strengthen at diabetic state. It was equal with mesangial proliferation and extracellular matrix accumulation. It suggests that megsin plays an certain role in the pathogenesis of renal lesion in diabetic mice.2 Over-expressed megsin may promotes the process of accumulation of extracellular matrix in diabetic mice renal tissue or mesangial cells. The mechanism of that on the one hand may achieved by PDGF-BB-induced activation of ERK signal pathway, the expression of TGF-?1 increased, the expression of FN and the concentrations of type IV collagen increased eventually; on the other hand, over-expressed megsin may inhibits the expression of EMMPRIN and MMP-9 in diabetic mice renal tissue or mesangial cells, resulting in reduced degradation of extracellular matrix, which participated in the pathogenesis of renal lesion in diabetic mice.3 Megsin siRNA and pioglitazone can decrease the expression and protein level of megsin in diabetic mice renal tissue or mesangial cells, also decrease the synthesis and increase the degradation of extracellular matrix by inhibiting the transduction of ERK signal pathway or promoting the synthesis of MMPs, then delays the pathological process of extracellular matrix accumulation, which provides a new idea and theoretical foundation for prevention and treatment of early diabetic nephropathy.
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