Effects And Mechanism Of The Nuclear Genes Tribble3on MAPK Signaling Pathway In Diabetic Nephropathy

BACKGROUNDDiabetic nephropathy is an important diabetic microvascular complication and the major cause of disability and death, morbidity and mortality are increasing every year. Epidemiological survey in China, the incidence of diabetes DN is about33%, more than7%of patients with renal dysfunction. DN has become one of the main reasons for ESRD (end-stage renal disease, ESRD). Now more and more evidence show that diabetic nephropathy eventually develop into renal fibrosis, but its specific mechanism of not fully elucidated. Therefore, further study of the pathogenesis of diabetic nephropathy, and to explore ways to block the development of diabetic nephropathy has far-reaching significance for the prevention and treatment of diabetic nephropathy.Diabetic nephropathy is an important diabetic microvascular complication and the major cause of disability and death, morbidity and mortality are increasing every year. DN is characterized by albuminuria, glomerular hypertrophy, progressive accumulation of glomerular matrix, culminating in glomerulosclerosis, tubulointerstitial fibrosis, and progressive loss of renal function.Tribbles was first discovered in Drosophila embryogenesis regulation test. TRB3targeted at human chromosome20pl3-pl2.2, and type2diabetes gene is also located in this location, which also prompted TRB3have a natural link with diabetes. Translation product of TRB3contains358amino acids and it is a nuclear protein, the results of experimental immunohistochemical staining consistent with those reported. TRB3as a scaffolding protein in a number of signaling pathways, it also play a role in insulin signaling pathway, so the relationship between TRB3and insulin resistance is also concern at home and abroad. Du K, et al found that, compared with the wild-type mice, the TRB3mRNA and protein expression in liver of db/db diabetic mice were significantly elevated. TRB3expression levels increased in the db/db mouse liver promotes blood glucose and glucose tolerance increased. A study also found the TRB3expression of fibrosis model established by unilateral ureteral obstruction (UUO) was positively correlated with kidney tissue fibrosis, which possibly plays a role in promoting the progress of fibrosis through induction of transformation between epithelial and mesenchymal tissue. Therefore, TRB3may be involved in fibrosis development.OBJECTIVE1. To investigate the expression of TRB3in renal tissue of db/db mice at different stages.2. To evaluate the correlation between the expression of TRB3and renal fibrosis in DN.METHODS12-week-old male db/db mice (n=15) and age-matched control db/m mice (n=15) were used. They were maintained on a normal diet under standard animal house conditions.Random blood glucose and body weight was detected weekly. Animals were divided into three groups and put to death at16wk,20wk and25wk respectively. Levels of WBW, UAE, Scr and BUN were detected before death for one time. Renal tissue was harvested for histologic staining to evaluate the morphologic change, and immunohistochemical staining to detect the expression of TRB3. Real time-PCR and Western blotting were used to quantify the TRB3and TGF-β1expression at mRNA and protein levels respectively.RESULTS1. Metabolic data Random blood glucose of control group remained stable, while the model group hyperglycemia was evident, and glucose levels of model group were elevated(P<0.01). Levels of UAE, Scr and BUN of db/db mice were higher than those of age-matched control groups (P<0.05, P<0.01).2. Pathological staining resultsMice in each group were not found the typical diabetic nephropathy K-W nodular performance. There were significantly increased mesangial matrix expansion and mesangial area widened in the DN groups compared with the control groups of same age. The relative area of mesangium matrix and the content of collage in db/db mice were increased significantly from20wk compared with control group.3. Immunohistochemistry staining of TRB3TRB3expressed in the nucleus of the intrinsic glomerular cells and tubular epithelial cells, and increased in the DN groups.4. The expression of TGF-β1and TRB3in kidney of miceExpression of TRB3and TGF-β1in renal tissue of db/db mice increased from20wk and showed significant difference between two groups at25wk.5. Correlation analysisThe expression of TRB3is positively related with TGF-β1protein (r=0.944, P<0.01) and renal interstitial fibrosis (r=0.857, P<0.05) in DN groups.CONCLUSION1. Kidney malformations glomerular hypertrophy, proteinuria and glomerulosclerosis in db/db mice all have a high degree of similarity with human diabetic nephropathy, which makes the db/db mice having more research value in disease pathogenesis and therapeutic targets compared to other animals.2. DN is characterized by albuminuria, glomerular hypertrophy, progressive accumulation of glomerular matrix, culminating in glomerulosclerosis, tubulointerstitial fibrosis, and progressive loss of renal function.3. TRB3is expressed in the kidney of mice.4. Compared with the age-matched db/m mice, TRB3expression increases significantly in the kidney of db/db mice and has positive correlation with renal fibrosis induced by DN. BACKGROUNDMany studies have confirmed that glomerular sclerosis and interstitial fibrosis are the main pathologic characteristics in DN, especially in the mid-anaphase of DN. Deposition of extracellular matrix (ECM) such as collagens and fibronectin (FN) regulated by TGF-β1is the core mechanism for glomerular sclerosis and interstitial fibrosis. The mesangial cells play important roles in DN, which is responsible for the accumulation of ECM and mesangial expansion, and TGF-β1is the core cytokine leading to the synthesis of ECM, which is responsible for mesangial fibrosis and hypertrophy under diabetic conditions. The major components of ECM proteins collagens types Ⅰ-Ⅳ and their synthesis and immoderate deposition are constantly observed in multifarious renal disease processes affecting humans and experimental animals.MAPK chain is one of the important ways of eukaryotic signal transduction networks, plays a key role in the regulation of gene expression and functional activity in the cytoplasm. MAPK subfamilies includes three classic ways, ERK1/2, JNK and P38MAPK. Studies confirmed that p38MAPK, and JNK activity are greatly enhanced in diabetic nephropathy and the expression level of p38phosphorylation in interstitial cells of tubules may reflect tubulointerstitial injury. This suggests that MAPK signaling pathway are closely related with DN.Tribble3(TRB3) was first discovered in Drosophila, is an important member of the tribbles family, a pseudo-kinase control cell stress, growth and metabolism. With combining in the unphosphorylated Akt TRB3can prevent the latter playing activity and regulate the insulin signaling pathway negatively. The growing body of evidence shows that TRB3and its gene polymorphism have a correlation with insulin resistance, and insulin resistance is a vital pathophysiologic characteristic of type2diabetes. Recent study found that, the interaction of TRB3and Smad3can enhance the activity of TGF-β1signaling pathway. Recently, more clinical studies confirm that the TRB3R84allosteric body is related with chronic kidney disease caused by type2diabetes.Recent studies have shown that, TRB3is an important regulator of MAPKs activity. So we demonstrated that there is increased expression of TRB3in kidney of db/db mouse. The expression of TRB3is positively related with TGF-β1protein and renal interstitial fibrosis. The results of this study suggest that TRB3may promote the accumulation of ECM and enhance renal interstitial fibrosis by increasing the expression of TGF-β1protein, and this is very important in diabetic nephropathy.So we propose that TRB3may have a correlation with diabetic nephropathy and be involved in its renal fibrosis, what’s more, there is very little about TRB3with diabetic nephropathy at home and abroad.OBJECTIVETo investigate the possible role of TRB3in regulating MAPK and TGF-β1/Smad signal pathways and in the proceeding of diabetic nephrology.METHODS1. Mycoplasma-free SV40MES13(murine mesangial) cells were treated with normal glucose (5.5mmol/L), OC (mannose,25mmol/L) and high glucose (25mmol/L) respectively for0h,6h,12h,24h,48h, cells were collected, and both the mRNA and protein level of TGF-β1、TRB3and collagen type Ⅳ(Col-Ⅳ) by real time PCR and ELISA or western blotting.2. After the MMCs were incubated with by normal glucose, OC and high glucose for0h,6h,12h,24h,48h, the activity of ERK1/2-MAPK was measured.3. After blockade of TRB3expression by TRB3siRNA, the MMCs were incubated with high glucose. The mRNA and protein levels of TGF-β1and Col-Ⅳ by glucose, the activity of ERK1/2-MAPK of MMCs at24h were measuredRESULTS1. Our data showed an increase in both the mRNA and protein level of Col-Ⅳ and TGF-β1by MMCs following incubation with high glucose (P<0.01). These effects were not due to the changes in osmotic pressure.2. Extracellular signal regulated kinase1/2(ERK1/2) was activated by high glucose (P<0.01).3. The mRNA and protein expression of Col-Ⅳ and TGF-β1induced by high glucose were all down-regulated after TRB3was blocked by TRB3siRNA (P<0.01).4. After ERK phosphorylation was blocked by PD98059, the mRNA and protein expression of Col-Ⅳ and TGF-β1were significantly decreased (P<0.01). However, the protein expression of TRB3was significantly increased (P<0.01).CONCLUSION1. High glucose increase the synthesis of Col-Ⅳ and TGF-β1through activation of ERK1/2.2. High glucose increase the mRNA level of TRB3, and TRB3play a role in Col-Ⅳ and TGF-β1expression induced by high glucose in MMCs through ERK1/2signaling pathway.3. There may be a feedback regulation between TRB3and some downstream cytokines of ERK1/2-MAPK.