Expression of low density lipoprotein receptor-related protein in renal interstitial fibrosis

AIM: To understand the expression of low density lipoprotein receptor-related protein (LRP) in tubulointerstitial fibrosis of unilateral ureter obstruction (UUO) rats. METHODS: The localization of LRP within kidneys were assessed by immunohistochemical staining, the protein level of LRP and connective tissue growth factor (CTGF) in kidney were analysised by Western blot. RESULTS: The location of LRP positive-staining and the protein level of LRP were in the same tendency with CTGF, the level of LRP had strong correlationship with the level of CTGF (r=0.786, P＜0.01); The expression of LRP was positive correlated with tubular-interstitial injury index (r=0.800, P＜0.01). Enalapril treatment downregulated LRP level at 9 days time point. CONCLUSION: The expression of LRP was increased according to the progression of interstitial fibrosis in UUO rats, implicating that LRP may act as receptor of CTGF to mediate the profibrotic effect of CTGF.     

Expression of TGF-β1 and apoptosis in myocardium of diabetic rats

AIM: To study the pathophysiological mechanism of cardiomyopathy, the expression of TGF-β₁ and apoptosis in myocardium of diabetic rats were investigated. METHODS:The diabetes models were established by single intravenous injection of streptozotocin (50 mg/kg) in rats. By the method of immunochemistry, the expression of TGF-β₁ in the cardiomyocytes was detected as the index to evaluate the degree of fibrosis. The method of TUNEL was used to measure the cardiomyocyte apoptosis as the index to explore its importance in process of diabetic cardiomyopathy. RESULTS:① The weight of diabetic rats was apparently lower than that in the rats before the diabetic model was built (P<0.01), and the increase in weight in diabetic rats within three month was less than that in normal group. ② Compared with control group, the concentration of blood glucose was continually elevated during the experiment. ③ The expression of TGF-β₁ in the diabetic cardiac muscle was much more than that in normal group (P<0.01). ④ The apoptosis of myocardium measured by the method of TUNEL was apparent in the diabetic groups than that in normal one (P<0.01). However, no significance was detected in the different courses of diabetic groups. CONCLUSIONS:The apoptosis might play an important role in leading the diabetic cardiomyopathy to heart failure. The expression of TGF-β₁ in the myocardium of diabetic rats was more than that in normal and had an increasing trend in the procession of diabetic cardiomyopathy. TGF-β₁ might be a significant factor in diabetic myocardium fibrosis. Apoptosis might play an important role in the initial stage of diabetes, which promotes the diabetic cardiomyopathy to heart failure.     

Increased expression and possible roles of nicotinamide N-methyltransferase in pancreatic islets of STZ-induced diabetic monkeys

AIM: To verify and localize the expression of nicotinamide N-methyltransferase (NNMT) in pancreas of streptozotocin(STZ)-induced diabetic monkeys and understand its important role in β-cell destruction in the pathogenesis of diabetes. METHODS: Through an olig-microarray gene chip, NNMT was identified as the most obviously up-regulated genes in pancreas of STZ-induced diabetic monkeys versus controls. Semiquantitative RT-PCR and Western blotting were performed to verify the differential expression at mRNA and protein level respectively. Then the cellular localization of NNMT expression within pancreas was identified by immunohistochemical(IHC) staining.RESULTS: An obvious high expression of NNMT at both mRNA and protein levels was shown in pancreas of STZ-induced diabetic monkeys compared to that of controls. Further localization of the protein by IHC staining in pancreas specimens showed that its altered expression was restricted to central islets, most of which were β cells.CONCLUSION: Expression of NNMT is increased in islets of STZ- induced diabetic monkeys, which infers that NNMT might participate in the process of β cell damage in diabetes probably through the mechanism of energy metabolism disturbance.     

Microvessel density and expression of vascular endothelial growth factor in glomeruli of diabetic mice

AIM: To investigate the relationship between microvessel density (MVD) and expression of vascular endothelial growth factor (VEGF) in glomeruli of diabetic mice. METHODS: Streptozotocin-induced diabetic mice as well as the control mice were involved in this study for 6 weeks. The body weight and blood glucose level of the mice in each group were weekly measured at certain time point. The morphological changes of the kidney were observed under light microscope, and the diameter, perimeter and area of the glomeruli were detected by an image analysis system. The expression of CD34 and VEGF in glomeruli was examined by immunohistochemistry method, and MVD and VEGF index were also calculated. RESULTS: In comparison with the control mice, the blood glucose level was significantly increased,and the body weight was decreased in diabetic mice(P<0.01). The diameter, perimeter and area of glomeruli in diabetic mice were significant greater than those in control mice (P<0.05). Increased expression of CD34 and VEGF in the glomeruli of diabetic mice was observed. Glomerular MVD of diabetic mice was significantly higher than that of the controls (P<0.01), and was positively correlated with the VEGF index (r=0.9979, P<0.05). CONCLUSION: VEGF may promote the angiogenesis in glomeruli of diabetic mice. The increase in VEGF expression may play a role in the pathogenesis of diabetic nephropathy.     

Expression and modulation of connective tissue growth factor in renal interstitial fibrosis

CTGF, a member of the CCN family of immediate early genes, is a recently discovered profibrotic growth factor, which is involved in many pathophysiologic procedures. CTGF acts as a downstream effector of TGF-β acting on interstitial cells to enhance the progression of fibrotic renal diseases. It has been shown that CTGF gene expression can be induced or blocked by some kinds of cytokine and drugs. It is an interesting candidate target for future intervention strategies of renal interstitial fibrosis.     

Effect of benazepril on expression of insulin receptor and its substrate-1 protein in renal tissue cell membrane in diabetic rats

AIM: To study effect of benazepril (an ACE inhibitor) on expression of insulin receptor (IR) and its substrate-1 (IRS-1) protein in renal tissue cell membrane in diabetic rats. METHODS: The rats were randomly divided into following groups: control (n=6),streptozotocin induced diabetic (n=7) and diabetic treated with benazepril (n=7). Body weight, kidney weight and kidney weight/body weight were observed after 4 weeks of treatment. ACE activities in plasma, renal tissue were measured by the fluorimetric assay. The expressions of IR and IRS-1 protein were determined by Western blot analysis in renal tissue cell membrane. RESULTS: After 4 weeks of treatment,benazepril significantly ameliorated kidney hypertrophy in diabetic rats. ACE activities in plasma,renal tissue were reduced by approximately 92.00% and 88.77%,respectively. Western blot analysis showed that the expressions of IR and IRS-1 protein were increased by 2.1 and 1.5 folds in renal tissue cell membrane in diabetic rats. However, benazepril reduced expression of IR and IRS-1 protein by 45.74% and 47.66%, respectively. CONCLUSIONS: Increased the expression of IR and IRS-1 protein might be related to abnormally active glucose metabolism in diabetic rat kidney. Down-regulation of expression of IR and IRS-1 protein might be one of important machnisms of Benazepril nephroprotection on diabetic rats.     

Antagonistic effect of thalidomide on TGF-β1-induced change of CTGF gene promoter-binding proteins in HELF cells

AIM:To investigate the antagonistic effect of thalidomide (THD) on the activation of connective tissue growth factor (CTGF) gene promoter induced by transforming growth factor-β₁ (TGF-β₁) in human embryonic lung fibroblasts (HELF). METHODS:Luciferase reporter vector driven by CTGF gene promoter was used to detect the effects of TGF-β₁ and THD on the activity of CTGF gene promoter, and DNA pull-down assay with CTGF gene promoter as a probe was used to analyze the changes of CTGF promoter-binding proteins under different conditions. RESULTS:TGF-β₁ increased the activity of reporter driven by CTGF gene promoter (P<0.01), while THD significantly inhibited TGF-β₁-induced increase in the reporter activity dose-dependently (P<0.01). At the same time, THD had inhibitory effect on the TGF-β₁-induced change of CTGF gene promoter-binding proteins (P<0.05). CONCLUSION:Regulation of CTGF gene promoter-binding proteins takes part in TGF-β₁-induced activation of CTGF gene promoter, while THD has antagonistic effect on this process.     

Protective effect of catalpol on diabetic rat aorta and its antioxidant mechanisms

AIM：To investigate the protective effect of catalpol on Goto-kakizaki (GK) rat aorta and to explore its antioxidant mechanisms. METHODS：Six-month-old GK rats (n=45) were randomly divided into diabetic model group, metformin (100 mg·kg-1·d-1) group, and high-dose (100 mg·kg-1·d-1), medium-dose (50 mg·kg-1·d-1) and low-dose (10 mg·kg-1·d-1) catalpol groups. The healthy male Wistar rats (n=10) were used as control group. The rats in control and model groups were given a same volume of saline. All reagents were administered by oral gavage for 12 weeks. Blood glucose and lipids were detected by an automatic biochemical analyzer. Serum reactive oxygen species (ROS), malondialdehyde (MDA), superoxide dismutase (SOD) and total antioxidant capacity (T-AOC) levels were detected by commercial kits. The expression of nuclear factor E2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1) in the thoracic aorta was determined by Western blotting. The pathological changes of the thoracic aorta were observed by HE staining. The ultrastructural changes of the thoracic aorta were observed under electron microscope. RESULTS：After catalpol treatment, the levels of blood glucose and blood lipids were decreased significantly, and serum levels of ROS and MDA were significantly decreased, but the activity of SOD and T-AOC were significantly enhanced. The protein expression of Nrf2 and HO-1 in the thoracic aorta were significantly increased, the thoracic aortic lesions indicated by HE staining significantly reduced, and the thoracic aortic damage under ultrastructural observation was attenuated slightly. CONCLUSION：Catalpol effectively protects GK rat thoracic aorta, which may be associated with decreasing blood lipids, reducing oxidative stress and activating Nrf2/ARE/HO-1 signaling pathways     

Changes in serum TGF β1 in type 2 diabetic patients

AIM: To explore the changes in serum TGF β₁ in type 2 diabetes mellitus. METHODS: Forty-five cases type 2 diabetes mellitus patients were divided into three groups according to urine albumim excretion rate(UAER): normoalbuminuria(NA)group and microalbuminuria(MA) group and macroalbuminuria group (Overt DN). Serum TGF β₁, fasting blood glucose(FBG), HbA_1c,BUN,Cr,Ccr,lipidemia were detected in all cases. RESULTS: Serum TGF β₁ in NA, MA and ODN groups was higher than that in control. Serum TGF β₁ was positive correlation with Cr(r=0.390,P＜0.05), LDL(r=0.503,P＜0.01), HbA_1c (r=0.676,P＜0.01), and UARE(r=0.777,P＜0.01). CONCLUSION: Type 2 diabetes mellitus have a higher serum TGF β₁ than controls, serum TGF β₁ was positive correlation with HbA_1c and injury of renal function.     

Changes of potassium channels in thoracic aorta of streptozotocin-induced diabetic mice in early stage

AIM:To investigate the changes of potassium channels in thoracic aorta of streptozotocin-induced diabetic mouse in the early stage of diabetes mellitus.METHODS:The effects of 60 mmol/L KCl, phenylephrine (PE), sodium nitroprusside (SNP) were measured and concentration-response curves to SNP were determined in the presence and in the absence of the inhibitors of potassium channels on the thoracic aortic rings of diabetic and age-matched control mice in vitro. RESULTS:STZ-diabetic mice showed a significant increase in the maximum contractile response and sensitivity of thoracic aorta to 60 mmol/L KCl and PE. The endothelium-independent relaxation response to SNP was increased by diabetes and were decreased significantly by pretreatment of the vessels with 1 mmol/L tetraethylammonium (TEA), 1 mmol/L 4-aminopyridine (4-AP) and 10 μmol/L glibenclamide in diabetes thoracic aorta. Only 4-AP decreased relaxation response to SNP in age-matched control mice. The -logIC₅₀ difference of TEA in thoracic aorta rings of diabetes was significantly higher than age-matched control mice.CONCLUSION:In early stage of diabetes mellitus, the opening or expression of K_Ca channels is significantly enhanced.The opening of K_ATP channels is also enhanced in this stage.     

Role of connective tissue growth factor in airway remodeled in rats induced by repeated bacterial infection

AIM: To investigate the role of connective tissue growth factor (CTGF) in airway remodeling in rats induced by repeating Pseudomonas aeruginosa (PA) infection. METHODS: The rats were intratracheally injected with PA for 12 times to induce chronic lung inflammation. The pathological changes of the trachea and lungs were observed, and the thickness of the trachea wall and vessel wall was measured. At the same time, the methods of immunochemistry and real-time quantitative RT-PCR were used to determine the protein and mRNA expression of CTGF in the lung tissues. The relevance between pathological changes and CTGF expression was also analyzed. RESULTS: From the 4th week, the thickness of the trachea wall and vessel wall in infectious group was larger than that in NS group (P<0.05). At the 16th week, obvious chronic inflammation in all grade bronchi appeared, the trachea walls were thickened and the lumens were narrowed in the infected animals. The expression of CTGF was significantly up-regulated (P<0.01) with positive correlations to the thickness of the trachea wall and vessel wall (r=0.880, r=0.829,P<0.01). CONCLUSION: Airway remodeling in rats is induced by repeated injection of PA. CTGF may play some roles in the pathogenesis of airway remodeling.     

Effect of Ginkgo biloba extract on the expression of connective tissue growth factor in the initiating stage of fibrosis in lungs of rats

AIM: To study the effect of Ginkgo biloba extract (GbE) on the expression of connective tissue growth factor (CTGF) in the initiating stage of pulmonary fibrosis of rats after administration of bleomycin (BLM).METHODS: The expression of CTGF in lungs was detected by Western blotting. The content of hydroxyproline was assayed by the method of chloramines T. The content of malondialdehyde (MDA) in plasma was investigated by colorimetry.RESULTS: On day 14 after administration of BLM, the contents of CTGF in lungs and MDA in plasma in BLM+NS group were higher than those in NS group, respectively (P<0.05; P<0.01). On day 30 after BLM, the contents of hydroxyproline in lungs and MDA in plasma in BLM+NS group were higher than those in NS group, respectively (both P<0.01). Treatment with GbE ameliorated the above changes induced by BLM. CONCLUSION: GbE ameliorates the up-regulation of CTGF in the initial stage of fibrosis in lungs of rats after administration of BLM. GbE prevents the hyperoxidative injury in lungs of rats after BLM, which might be one of mechanisms underling the effect of GbE on CTGF.     

Changes of plasma levels of soluble VEGFR2 and SOD in hypertensive patients and hypertensive diabetic patients

AIM: To explore the changes of plasma levels of soluble vascular endothelial growth factor receptor 2 (sVEGFR2) and superoxide dismutase (SOD) in hypertensive patients and hypertensive diabetic patients. METHODS: In this cross-sectional study, 88 cases were enrolled, which were divided into hypertensive group (n=31), hypertensive diabetic group (n=31) and control group (n=26). Blood pressure was obtained from each participant with mercury sphygmomanometer. The levels of sVEGFR2 and SOD were measured by ELISA. Meanwhile, the levels of plasma glucose, glycosylated hemoglobin A1c (GHbA1c) and lipid profile were detected. RESULTS: The levels of total cholesterol (TC) and body mass index (BMI) were significantly higher in hypertensive group than those in control group (P<0.05). The levels of TC, low-density lipoprotein cholesterol (LDL-C), triglyceride (TG), BMI, waist circumference were significantly higher in hypertensive diabetic group than those in control group (P<0.05). The plasma levels of sVEGFR2 and SOD in both hypertensive diabetic group and hypertensive group were significantly decreased compared with control group (P<0.05), while the mean plasma levels of sVEGFR2 and SOD in hypertensive diabetic group were significantly decreased compared to the hypertensive group (P<0.05). A significantly positive correlation between sVEGFR2 and SOD in the whole study population (P<0.05) was observed. CONCLUSION: The plasma level of sVEGFR2 is decreased in both hypertensive and hypertensive diabetic patients, and more significantly decreased in hypertensive diabetic patients. Decreased SOD level may be associated with to the reduction of sVEGFR2.     

Dynamical observation of the expression of TGF-β1 and MAPK1/3 in the renal tubules of rats with diabetes

AIM: To observe the expression of transforming growth factor β₁ (TGF-β₁), MAPK_1/3 and fibronectin (FN) in the development of renal tubulointerstitial disease. METHODS: Wistar male rats were randomly divided into normal control group, diabetic group of 1week, 2 weeks, 4 weeks and 8 weeks. Diabetic model was induced by peritoneal injection of streptozotocin. Immunohistochemistry was employed to detect the expression of TGF-β₁, MAPK_1/3 and FN in the kidney. TGF-β₁ protein in the renal cortex was checked by Western blot. BG, Scr and UP were analysed by biochemical methods, and the morphological changes in renal tubulointerstitium were also examined under microscopy on sections stained with HE and PAS. RESULTS: The expression of MAPK_1/3 and FN was observed, but not the expression of TGF-β₁ in normal renal tissue. Positive staining of TGF-β₁ was observed in the renal tubulo-interstitium in 1-week diabetic group and thereafter it increased in the course of diabetes. A continuous increase in the expression of MAPK_1/3 and FN was also observed in two - week diabetic rats. Chronologically the expression of TGF-β₁,MAPK_1/3 and FN and the ratio of KW/BW were positively correlative with each other in diabetic animals except one -week diabetic rats. There was also a positive correlation between MAPK_1/3 and FN in l -week diabetic rats. CONCLUSION: Our data suggest that TGF-β₁ appears in the renal tubulointerstitium in early period of diabetes and then its signal is mediated by MAPK_1/3 cascades to accelerate production of FN ,and in turn leads to renal hypertrophy and tubulointerstitial fibrosis.