Effects Of Advanced Glycation End Products On The Expression Of TTG And Its Role In Diabetic Nephropathy

Advanced glycation end products (AGEs) are increased obviously in the condition of diabetes under the hyperglycemia, even if the blood sugar returned to normal, AGEs also continue to exist and to harm the organism. Contents of AGEs in the animal and the human diabetic kidney are significantly increased. And these may provoke the accumulation of extracellular matrix (ECM), including both increased synthesis and/or decreased degradation, that play an important role in the pathogenesis of diabetic nephropathy. The decrease of degradation is an important link to ECM accumulation. AGEs may be responsible for excessive accumulation of ECM in glomeruli of diabetic kidneys and play an important role in the pathogenesis of diabetic nephropathy. In vivo, intravenous injection of AGE-modified rat albumin to heathy rats resulted in glomerular hypertrophy, mesangial sclerosis, and excessive matrix synthesis. We have reported that AGEs can inhibit the activities of the plasmin system and matrix metalloproteinase, which are two kinds of protease system in kidney. Tissue transglutaminase (tTG) can resist ECM degradation through modified the ECM protein. There is a new idea to explore the role of AGEs in the pathogenesis of DN through the decrease of degradation.tTG is a Ca2+ dependent transamidation enzyme. After activated, tTG may cause a majority of ECM protein cross-linking including fibronectin, laminin and collagen, which form fast structure to resist degradation. Under diabetes condition, the expression and activeness of kidney tTG increase and those are in positive correlation with ECM. Whether there is a relationship between tTG change and AGEs or not, its role in the occurrence of diabetic nephropathy is currently unknown. This project will research the relationship between the expression and activity of tTG and AGEs with the diabetes animals and the non-diabetes ones. Simultaneously it will analyze and discuss the molecular mechanism between tTG and AGEs with MC and NRK-52E in vitro. About rats studies, in vivo, includes two parts. Diabetes was induced by a single intraperitoneal (i.p.) injection of streptozotocin (STZ) (55 mg/kg, dissolved in ice cold 10 nM citrate buffer, pH 4.4). And then the diabetes animals were dibided into two groups namely the diabetic control (n=10) and the aminoguanidine treated (40 mg/kg, i.p., n=10), along with those groups, there was one age matched control group (n=10) also. The second part, healthy rats were given tail vein injections with either AGE-modified (AGEs Group) or native RSA 100 mg/kg (RSA Group) or AGEs followed by abdominal injection of aminoguanidine 40 mg/kg ( AGEs+AG group ). In vitro, the effects of AGEs to mesangial cells (MC) and renal tubular epith- elial cell (NRK-52E) on different concentration were detected. The research methods employed in the present study include ELISA, Western Blotting, immunohisto- chemistry, RT-PCR, as so on. We want to investigate the following problems:①We will elucidate the effects of AGEs on tTG activities and the expression in STZ-induced diabetic rat kidney, find the relationship between tTG, AGEs and ECM.②We hope to clear the effects of AGEs on the activities and the expressions of tTG, to analyze the relationship between tTG and the kidney injury by AGEs-induced in AGEs- modified rat kidney.③To reseach the effects of AGEs on the expressions of tTG in MC and NRK-52E, and to analyze the interaction between tTG and the contents of ECM.④To clarify the interaction between tTG and TGF-β1 and CTGF. The study was designed to identify the mechanism of AGEs and tTG and ECM, and provide experimental basis for prevention and cure of diabetic nephropathy.The main results are as follows:1. The effects of AGEs on structure and function of glomeruli and tubuli renalesDM group compared with control group, their BG, GSP, Ser and 24 hour urine protein excretoty rate increased obviously. Histologically, glomerular basement membrane segmentally thickened, PAS positive substance excessively deposited, FN and ColⅣcontents increased in DM group rats. While AGEs formation inhibitor AG treatment significantly reduced histology change. Level of AGEs in serum and renal cortex were positive correlation with the expression of tTG in renal cortex (r=0.611, P<0.05) in each groups. Compared with RSA group, PAS positive substance excessively deposited, ECM contents increased, level of tTG protein and tTG mRNA increased obviously in AGEs group. This suggest there is a link between AGEs and tTG.Though AGEs (0~ 400mg/L) cannot stimulate mesangial cell proliferation, it (50~ 400mg/L) can stimulate renal tubular epithelial cell proliferation. Compared with RSA group, AGEs can accelerated tTG and FN and ColⅣcontents of cell medium in AGEs-treated groups. FN and ColⅣcontents decreased after MDC addition. Those were the same results in the study of mRNA. It suggest tTG can effect the degradation of ECM contents.2. The effects of AGEs on tTGCompared with control group, the expression of tTG protein increased in DM group. Compared with native control group and RSA group, tTG content increased ob- viously in AGEs group, it also increased (no statistics significance) in AGEs+AG group. We found the same condition in mRNA level of tTG. AGEs can increase tTG expre- ssion in renal cortex. ECM contents were positive correlation with the expression of tTG mRNA (r=0.911, 0.872) with AGEs-treated. In vitro, these were proved yet. ECM accumulations were decreased with MDC addition. It suggest tTG can partake ECM accumulations.3. tTG and ECM dynamic balance in diabetic nephropathyThe expression of tTG protein was similar with that of tTG mRNA in rat renal cortex. The expression of FN and Col IV mRNA were positive correlation with tTG mRNA. It suggest ECM accumulation is because of resisting the degradation depended on their crosslinking by induction of tTG. The expression of tTG mRNA increased and the protein level of tTG and FN and ColⅣdecreased significantly by AGEs (400 mg/L) in mesangial cell. It was not happened in renal tubular epithelial cell. We guess that high protein level of tTG would inhibit the excessive expression of tTG mRNA. This suggest there is a link between tTG and ECM.4. The interaction of tTG and TGF-β1 and CTGF with AGEs-treatedCompared with control group, the expression of TGF-β1 and CTGF protein increased significantly in DM group, unobviously in AG group. But the expression of TGF-β1 mRNA decreased compared with control group. The expression of growth factor protein and mRNA increased obviously with AGEs injection in AGEs group. In vitro, TGF-β1 and CTGF mRNA should work at the earlier period of AGEs- producted. It must be some type of regulation among tTG and TGF-β1 and CTGF.The main conclusions are:1. AGEs increases obviously in the condition of diabetes, and AGEs can change the structure of kidney , lead to dysfunction of glomeruli filter membrane. AGEs can induce the expression of tTG accentuation and ECM accumulation.2. Intravenous injection of AGE-modified rat albumin to healthy rats resulted the expression of tTG accentuation in kidney.3. tTG may induce ECM accumulation by increasing the levels of protein and gene.4. TGF-β1 and CTGF can stimulate tTG secretion, the latter which induce ECM accumulation.The main creative results:tTG can resist ECM degradation through modified the ECM proteins. There is a new idea to explore the role of AGEs in the pathogenesis of DN through the decrease of degradation.