Dysregulation Of Extracellular Matrix Turnover In Diabetic Nephropathy And Protective Effect Of Irbesartan

Objectives: As a severe and most frequent complication of diabetes mellitus (DM), diabetic nephropathy is stated to be one of the most common causes of end-stage renal disease. Diabetic nephropathy is characterized by accumulation of extracellular matrix (ECM) and basement membrane thickening in the glomerulus. The components and volumes of ECM are determined by the dynamic balance between matrix synthesis and degradation. It has been gradually received that the reduced degradation of matrix plays an important role in the pathogenesis of diabetic nephropathy. Matrix metalloproteinases (MMPs) are the major physiologic determinants of ECM degradation and turnover in the glomerulus. With view to collagen IV mainly depositing in glomerular mesangium and basement membrane, MMP-2 (gelatinase A), which degradate collagen IV, is hypothetically quite associated with diabetic nephropathy. A number of literature support this hypothesis but with controversy on it. So far, there is little data available on the role of MT1-MMP (activator of pro-MMP-2) in the development of diabetic nephropathy.Connective tissue growth factor (CTGF) is thought to be a critical downstream mediator of TGF-βwith more simple pro-fibrotic effects. The role of CTGF on ECM dysregulation in diabetic nephropathy has not been thoroughly investigated.The renin-angiotensin system (RAS) is a pivotal contributor to the progress of diabetic renal disease. RAS blocking with type 1 receptor blockers (ARB) has been clearly demonstrated to have positive outcomes on diabetic complications. The precise mechanism of renoprotective effect of irbesartan (angiotensin-receptor 1 blocker) has not been fully understood.The imbalance between production and degradation of ECM induced by TGF-β1 is a main event in the progress of tissue fibrosis. Smads are important protein mediators in TGF-β1 signal transduction pathways. Owing to the marked similarity in their structure it was not possible to respectively study the role of Smad2 or Smad3 in TGF-β1 induced cellular responses in the past. RNA interference (RNAi) is an accurate and potent gene-silencing method, which can offer researchers a powerful tool to selectively silence genes.The present studies were performed to examine the expression of MMP-2, TIMP-2, MT1-MMP and CTGF in the renal tissue of STZ-induced diabetic model as well as glomerular mesangial cells (GMCs) cultured by high glucose medium. We also observed the effects of irbesartan on diabetic rats as well as high-glucose cultured GMCs and investigated the different role of Smad2 and Smad3 in TGFβ1-induced fibrotic responses by knocking them down respectively in GMCs.Methods:1. Diabetic model and examination of MMP-2, TIMP-2, MT1-MMP and CTGFUninephrectomized male Wistar rats were randomly divided into three groups: control group (n=18), DM group (n=18) and DM+Irb group (n=6). The rats of DM and DM+Irb group received a single intraperitoneal injection of STZ dissolved in 0.1mol/L sodium citrate buffer (pH 4.5) at a dose of 65mg/kg. The rats 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 48 hours of the injection. The rats in the DM+Irb group were treated with irbesartan (average dose of 50mg.kg-1.d-1) for 12 weeks. The rats of control and DM group were treated with the same volume of normal saline. Six rats from control or DM group were respectively sacrificed at week 4, 8, 12 after STZ injection. All rats in DM+Irb group were also sacrificed at week 12. The renal cortex were removed and used for histopathology, immohistochemistry, western blot and reverse transcription and polymerase chain reaction (RT-PCR). 2. Cell culture and examination of MMP-2, TIMP-2, MT1-MMP and CTGFGlomerular mesangial cells were cultured at 37℃in 95% air and 5% CO2 in DMEM media containing 10% fetal calf serum, 100U/ml penicillin and 100μg/ml streptomycin. Confluent mesangial cells were divided into four groups: LG group (media containing 5.5mM glucose), HG group (media containing 30mM glucose), LG+M group (media containing 5.5mM glucose and 24.5mM mannitol), HG+Irb group (media containing 30mM glucose and irbesartan). Cells cultured in the mixture of 5.5 mM glucose and 24.5 mM mannitol were used as an osmotic control. The incubation time extends to 24h, 48h, 72h and 96h. At the end of incubation, the supernatant and cells were collected. The expression of MMP-2, TIMP-2, MT1-MMP and CTGF in the cells were assessed by immunochemistry, immunofluorescence, western blot and RT-PCR. ELISA was used to detect the level of collagen IV in supernatant.3. RNA interference and examination of MMP-2, TIMP-2, MT1-MMP and CTGF in GMCs.In this study RNA interference was used to achieve selective and specific knockdown of Smad2 and Smad3. After optimizing transfection conditions the preliminary experiment was performed to determine the most effective siRNA of Smad2 or Smad3 and make sure that siRNA treatment results in selective and specific knockdown of respective Smad protein. After siRNA transfection GMCs were treated with either vehicle (0.1%BSA) or TGF-β1 (2ng/ml) for 48h under serum-free condition. Cellular MMP-2, TIMP-2, MT1-MMP and CTGF were assessed by Wertern blot and RT-PCR.Results:1. The expression of MMP-2, TIMP-2, MT1-MMP and CTGF in the renal tissue of the diabetic rats①The diabetic rats showed slightly enlarged glomeruli at week 4. Thickened glomerular basement membrane and expanded mesangium were observed with tubular epithelial vacuolar degeneration, minor focal atrophy as well as inflammatory cell infiltration were also observed at week 8 and week 12.②Compared with those of control group immunohistochemical positive staining for MMP-2 decreased notably in glomeruli at week 4 but without obvious changes in tubuli of diabetic kidney. The positive stainings for MMP-2 decreased both in glomeruli and tubuli from 8 weeks to 12 weeks in diabetic rats. The control rats showed weak stainings for TIMP-2 and CTGF either in glomeruli or in interstitium whereas markedly enhancing positive signals were detected in diabetic rats from 4 weeks to 12 weeks.③The Western blot and RT-PCR results for TIMP and CTGF are consistent with the immunohistochemical results. The expression and activity of MMP-2 were decreasing from 4 weeks to 12 weeks in diabetic group versus control group. The expression of CTGF was correlated with MMP-2/TIMP-2 negatively (r=-0.7158, p<0.01).④The morphological and functional impairment of the kidneys induced by STZ was ameliorated in irbesartan group. Pro-MMP-2 and active MMP-2 as well as MT1-MMP were up-regulated in irbesartan group, while on the contrary the expression of TIMP-2 and CTGF was down-regulated in irbesartan group.2. The expression of MMP-2, TIMP-2, MT1-MMP and CTGF in GMCs cultured in high glucose medium①The immunochemical positive stainings for MMP-2, TIMP-2, CTGF appeared in cytoplasm. The immunofluorescence positive stainings for MT1-MMP were mainly anchored at the cell membrane of GMCs.②High-glucose incubation resulted in down-regulation of MT1-MMP and up-regulation of TIMP-2 and CTGF along with the increasing secretion of Col IV. All of those regulations mediated by high-glucose are of time-dependent manners. The regulation of MMP-2 seems a little complicated. Either mRNA or protein of MMP-2 slightly increased after 24h-incubation in high-glucose media, then the expression of MMP-2 decreased up to 96h. The changes of active MMP-2 were consistent with changes of pro-MMP-2.③Immunochemistry and immunofluorescence showed decreased expression of TIMP-2 and CTGF, meanwhile increased expression of MMP-2 and MT1-MMP in irbesartan treatment group versus high-glucose stimulating group.3. The different role of Smad2 and Smad3 in TGFβ1-induced fibrotic responses in GMCs①Smad2 and Smad3 siRNA treatment resulted in approx. 80% reduction in band density of respective Smad protein. The knockdown of respective Smad proteins was selective, as Smad2 protein levels were reduced by Smad21 siRNA, but not by Smad33 siRNA. Similarly, Smad3 protein levels were reduced by Smad33 siRNA and not by Smad21 siRNA. Neither of the transfections had any effect on Smad4 protein levels.②TGF-β1 treatment for 48h resulted in about 60%-80% decrease in MMP-2 expression. The TGF-β1-induced down-regulation of MMP-2 was prevented by Smad2 knockdown, but not by smad3 knockdown. TGF-β1 and Smad siRNA had the similar effects on the expression of MT1-MMP.③TGF-β1 treatment for 48h resulted in significant increasing expression of TIMP-2. Inhibition of either Smad2 or Smad3 resulted in partial reduction of TGF-β1-induced TIMP-2 expression. This induction was abolished by simultaneous knockdown of both Smad2 and Smad3.④TGF-β1 treatment for 48h resulted in 4-5-fold increases in CTGF protein expression as well as 6-7-fold increases in its mRNA expression. This induction was markedly attenuated in the presence of Smad3 knockdown. However Smad2 knockdown had no effect on TGF-β1-induced up-regulation of CTGF.Conclusion:①Studies in vivo as well as in vitro reveal that the decrease of expression and proteolytic activity of MMP-2 are accompanied with the decreased expression of MT1-MMP as well as the increased expression of TIMP-2. Dysregulation of MMP-2 might be one of the causes of ECM accumulation which in turn results in diabetic glomerularsclerosis.②The increased expression of CTGF and its correlation with MMP-2/TIMP-2 suggest that CTGF might inhibit the degradation of ECM in DN.③Renoprotective effects of irbesartan might be partially by recovering the tilting balance between synthesis and degradation of ECM.④TGF-β1-induced decreases in expression of MMP-2 and MT1-MMP were Smad2-dependent, whereas increases of CTGF expression were Smad3-dependent. Increases of TIMP-2 expression were dependent on both Smad2 and Smad3.