Expression of SREBP-1 and SREBP-2 in kidney of type 1 diabetic rats

AIM:To investigate the expression of sterol regulatory element binding protein-1 (SREBP-1) and sterol regulatory element binding protein-2 (SREBP-2) in the kidney of type 1 diabetic rats.METHODS:The triglyceride (TG) and cholesterol content in the kidney of experimental rats were measured by the assay kits and oil red O staining.Furthermore, the expressions of SREBP-1 and SREBP-2 protein were detected by the methods of Western blotting and immunohistochemistry.The analysis of SREBP-1 mRNA was performed by in situ hybridization.RESULTS:Renal triglyceride content was markedly higher in diabetic rats than that in normal rats and the result of oil red O showed that lipid deposited in the renal tubular epithelium.Immunohistochemistry and Western blotting presented similar results that SREBP-1 protein was up-regulated in renal tubular epithelium in diabetic rats.On the other hand, SREBP-2 protein didnt show difference between diabetic rats and normal control rats.In situ hybridization confirmed the increasing of SREBP-1 mRNA in renal tubular epithelium in diabetic rats.CONCLUSION:Above results suggest that altered SREBP-1 may play an important role in the pathogenesis of renal lipid accumulation in type 1 diabetic rats.     

Up-regulation of Snail1 expression in renal tubular epithelial cells through Akt/GSK-3β pathway under high-glucose condition

AIM: To examine the effects of high glucose (HG) on the expression of Snail1 and protein kinase B (Akt)/glycogen synthase kinase 3β (GSK-3β) in primary renal tubular epithelial cells (RTECs). METHODS: The primary RTECs were randomly treated with normal glucose, high glucose or D-mannitol for 30 min~72 h. RT-PCR and Western blotting were used to observe the expression of Snail1, Akt and GSK-3β at mRNA and protein levels in these cells. The primary cultured RTECs were pretreated with LY294002 (a PI3K inhibitor, 25 μmol/L) to observe the specific inhibitory effects of phosphatidylinositol 3-kinase (PI3K) on HG-induced expression of Snail1 protein. RESULTS: Treatment of RTECs with HG resulted in increased mRNA and protein levels of Snail1, Akt1, and phosphorylation of Akt and GSK-3β. LY294002 blocked the HG-induced up-regulation of p-Akt, p-GSK-3β and Snail1 expression at protein level, but no effect of LY294002 was seen on the total protein expression of Akt1 and GSK-3β. HG did not affect the expression of GSK-3β at mRNA and protein levels. CONCLUSION: HG-induced up-regulation of Snail1 may be regulated by Akt/GSK-3β pathway in RTECs.     

Effect of interlukin-22 antibody on diabetic nephropathy via inhibition on Snail1 expression

AIM:To investigate the effect of interlukin-22 (IL-22) on diabetic nephropathy (DN) and its possible mechanism. METHODS:C57BL/6 mice were randomized to normal control (NC) group,DN group, DN+recombinant IL-22 (rIL-22) group and DN+IL-22 antibody (anti-IL-22) group. After successful establishment of diabetes model for 8 weeks, the mice in DN+rIL-22 group and DN+anti-IL-22 group were intraperitoneally injected with rIL-22 (200 μg/kg) and anti-IL-22 (200 μg/kg), respectively, and the mice in NC group and DN group were intraperitoneally injected with 0.1% bovine serum albumin, twice a week for 4 weeks. After the intervention, blood glucose, kidney function, 24 h urine microalbumin (m-Alb) and 24 h urine creatinine (UCr) were measured. The pathological changes of renal tissues were observed under light microscope. The mRNA expression of Snail1 was detected by qPCR. The protein levels of fibronetin (FN) and E-cadherin were determined by Western blot. RESULTS:After the intervention, the ratio of 24 h m-Alb/UCr increased significantly in other model groups compared with NC group (P<0.05). The levels of 24 h m-Alb and 24 h UCr increased significantly in DN+rIL-22 group compared with DN group (P<0.05). However, in DN+anti-IL-22 group, the levels of 24 h m-Alb, 24 h UCr and 24 h m-Alb/UCr ratio were significantly lower than those in DN group and DN+rIL-22 group (P<0.05). The tubular epithelial cell vacuolar degeneration, protein cast formation and glomerular mesangial expansion in the renal tissues from diabetic mice were observed under light microscope. The lesions were more severe in DN+rIL-22 group, but attenuated in DN+anti-IL-22 group. The mRNA expression of Snail1 increased significantly in diabetic mice (P<0.05), but decreased significantly after a 4-week intervention by anti-IL-22 (P<0.05). The expression of FN, an extracellular matrix protein, increased significantly in DN+rIL-22 group (P<0.05). The expression of E-cadherin, an epithelial-mesenchymal transition marker, decreased significantly in DN+rIL-22 group as well (P<0.05). CONCLUSION:IL-22 neutralizing antibody may attenuate microalbuminuria and delay the progression of DN via inhibition of Snail1 expression in the renal tubular epithelial cells.     

Expression of Raf kinase inhibitor protein and p-ERK in renal tissues of diabetic rats

AIM: To investigate the expression of Raf kinase inhibitor protein (RKIP) and phosphorylated extracellular signal-regulated kinase (p-ERK) in the renal tissues of diabetic rats. METHODS: Forty SD rats were randomly divided into normal control group and diabetic nephropathy group. The pathological changes of the renal tissues were observed under microscope with HE and periodic acid-Schiff (PAS) staining. The expression of RKIP and p-ERK1/2 was detected by the methods of immunohistochemistry and Western blotting. RESULTS: Following the extension of duration, the blood sugar of the rats in diabetic nephropathy group increased, with the appearance of proteinuria, increase in kidney weight, increase in renal p-ERK1/2 expression and decrease in RKIP expression as compared with control group. CONCLUSION: The development of diabetic nephropathy may be accelerated by the decrease in RKIP and the increase in p-ERK.     

Influence of Xiaoke keli Ji on glycemia and serum lipoprotein levels in diabetic nephropathy rats

AIM:To observe the changes of glycemia and serum cholesterol and triglyceride in diabetic nephropathy rats and therapeutic effects of Xiaoke Keli Ji.METHODS:3/4 nephrectomy was adopted firstly, three weeks later streptozotocin(STZ) was administered intraperitoneally to establish diabetic nephropathy model in rats. Animals were divided into four groups:model group, Xiaoke Keli Ji treatment group, positive control group and sham group. Changes of serum sugar and serum creatinine, cholesterol and triglyceride were examined at 1, 2, 3, 4, 5 weeks after STZ injection. Renal tissue samples were adopted at 6th week and studied by light microscopy.RESULTS:Model group demonstrated different degree of glomerular sclerosis. Lesions in treatment group were alleviated. Serum creatinine, serum sugar and serum cholesterol were higher at 1, 2, 3, 4, 5 weeks after STZ injection in model group than that of the sham group (P＜0. 05), serum triglyceride was higher at 1, 2, 3, 5 weeks after STZ injection in model group than that of the sham group(P＜0. 05). Xiaoke Keli Ji reduced those changes. Serum sugar was in positive correlation with serum lipoprotein.CONCLUSION:Diabetic nephropathy model was duplicated successfully. High serum sugar may lead to high serum lipoprotein, Xiaoke Keli Ji may treat diabetic nephropathy by reducing serum sugar and serum cholesterol and triglyceride.     

Expression of angiogenic factors in myocardial tissue of diabetic rats

AIM：To observe the expression of angiogenesis factors in the myocardial tissue of streptozotocin-induced diabetic rats. METHODS：The diabetic rat model was induced by intraperitoneal injection of streptozotocin. After 12 weeks, the cardiac function was measured by MPA cardiac function analysis system. The myocardial collagen volume fraction (CVF) was assessed by Masson staining. The capillary vessels was quantified as the ratio of capillary to myocyte (C/M) using CD31 immunostaining. The expression levels of vascular endothelial growth factor (VEGF), angiopoietin (Ang)-1, endostatin and Ang-2 were observed by Western blotting. RESULTS：Compared with normal control group, the left ventricular end-diastolic pressure (LVEDP) was evidently increased (P<0.01), but left ventricular pressure rise maximum rate (+dp／dtmax), left ventricular pressure decrease maximum rate (-dp／dtmax) and the ratio of capillary/myocyte (C/M) were significantly decreased (P<0.05). The CVF and the expression level of endostatin were significantly increased, whereas the expression levels of VEGF and Ang-1 evidently decreased (both P<0.05) in diabetic rats. However, no marked difference in the expression of Ang-2 between the 2 groups was observed (P>0.05). CONCLUSION：Imbalances between the angiogenic factors (VEGF and Ang-1) and anti-angiogenic factors (endostatin) may play an important role in the pathogenesis of diabetic cardiomyopathy.     

Effects of fluctuant high blood glucose on apoptosis in glomerular endothelial cells and renal tubular epithelial cells in diabetic rats

AIM:To explore the effects of fluctuant high blood glucose and stable high blood glucose on apoptosis and the expression of Bax and Bcl-2 in glomerular endothelial cells and renal tubular epithelial cells in diabetic rats. METHODS: 24 SD rats were divided into 3 groups: control group, stable high blood glucose group and fluctuant high blood glucose group. Diabetic rats were induced by intraperitoneal injection of STZ, and the fluctuant high blood glucose animal model was induced by intraperitoneal injection of aspart and glucose at different time points every day. Apoptosis was assessed by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL), and immunohistochemistry was used to detect apoptosis associated gene bax and bcl-2 expression in kidney. RESULTS:After 4 experimental weeks, a significant increase in cell apoptosis, up-regulation of Bax protein expression in kidney tubular epithelial cell and down-regulation of Bcl-2 in glomerular endothelial cell in fluctuant high blood glucose rats were observed compared with stable high blood glucose rats.CONCLUSION: Fluctuant high blood glucose induces more apoptosis in renal tubular epithelial cells than that in stable high blood glucose diabetic rats.     

Snail1/IGF-1 pathway mediates high glucose-induced EMT in renal tubular epithelial cells

AIM: To observe the expression of Snail1 and insulin-like growth factor-1 (IGF-1) in NRK-52E cells induced by high glucose, and to investigate the relationship of Snail1 and IGF-1 in the mechanism of epithelial to mesenchymal transition (EMT) in diabetic kidney disease (DKD).METHODS: The NRK-52E cells were treated with Snail1 siRNA and IGF-1 siRNA after cultured with high glucose medium for 72 h, and divided into control group, high glucose group, non-targeting (NT) siRNA group, Snail1 RNAi group and IGF-1 RNAi group. The cells were harvested at 48 h and 72 h. Real-time PCR was used to detect the mRNA expression of Snail1, IGF-1, E-cadherin and fibronectin (FN), and the protein levels were determined by immunofluorescence staining.RESULTS: Compared with control group, the expression of E-cadherin at mRNA and protein levels declined after stimulation with high glucose (P<0.01), while that of FN was elevated (P<0.01). Meanwhile, the mRNA and protein levels of Snail1 and IGF-1 were markedly increased (P<0.01).The expression of E-cadherin at mRNA and protein levels was improved in Snail1 RNAi group as compared with high glucose group(P<0.01), while that of FN, IGF-1 and Snail1 was significantly down-regulated (P<0.01). The same changes were observed in IGF-1 RNAi group (P<0.01). The protein expression of each factor in NT group had no significant change as compared with high glucose group (P>0.05). Pearson correlation analysis showed a close positive relationship between the expression of Snail1 and IGF-1 protein (r=0.852, P<0.01).CONCLUSION: Snail1 may facilitate DKD development by regulating IGF-1 in the process of EMT.     

Expression of LOX-1 in type 2 diabetic rat tubular interstitial tissues

AIM： To explore the effect of lectin-like oxidized low-density lipoprotein receptor 1 (LOX-1) on type 2 diabetic nephropathy in rats. METHODS：The Sprague-Dawley (SD) rats were randomly divided into control group and model group. The animal model was established by intraperitoneal injection of low-dose streptozotocin (STZ, 30 mg/kg) plus feeding with high-fat/high-glucose diets for one month. Biochemical parameters and 24 h urine album excretion rate (UAER) were monitored. The morphological changes of the renal tissue were examined under microscope with hematoxylin-eosin (HE) and periodic acid-Schiff reaction (PAS) staining. The protein levels of LOX-1 and transforming growth factor β1 (TGF-β1) in the renal tissues were determined by the method of immunohistochemistry. The mRNA expression of LOX-1 and TGF-β1 was detected by real-time polymerase chain reaction. The serum levels of monocyte chemotactic protein-1 (MCP-1), intercellular adhesion molecule (ICAM) and tumor necrosis factor α(TNF-α) were measured by enzyme-linked immunosorbent assay (ELISA). The correlation between LOX-1 and UAER, inflammatory factors and TGF-β1 was analyzed. RESULTS：Compared with normal group, total cholesterol (TC), triglyceride (TG), low-density lipoprotein (LDL) and UAER in model groups markedly increased, high-density lipoprotein (HDL) only increased at 0 week, then decreased at 4 and 8 weeks. The expression of LOX-1 and TGF-β1 at mRNA and protein levels was increased, as well as the concentration of serum inflammatory factors such as MCP-1, ICAM and TNF-α. The obvious relations between LOX-l mRNA with UAER, TNF-α and TGF-β1 were observed (r=0.509, 0.649 and 0.800, respectively, P<0.05). CONCLUSION：LOX-1 is significantly increased in type 2 diabetic rat tubular interstitial tissues and aggravates the dysfunction of renal tubules by releasing inflammatory factors and promoting inflammatory cell infiltration.     

Expression and significance of thrombospondin-1 in myocardium of type 2 diabetic cardiomyopathy rats

AIM: To investigate the expression and significance of thrombospondin-1 (TSP-1) in left ventricular myocardium of type 2 diabetic cardiomyopathy (DCM).METHODS: The rat model of DCM was established by eating a high-fat diet together with injection of low dose streptozocin (30 mg/kg) intrapertoneally.After 12 weeks,the content of collagen was quantified by Masson staining.The mRNA level of TSP-1 was determined by quantification real-time RT-PCR,while the protein level of TSP-1 was analyzed by Western blotting and immunohistochemistry.RESULTS: Compared with the control group,the content of collagen in the DCM group was increased greatly (11.01±3.05 vs 16.92±3.18,P<0.01).The mRNA and protein expressions of TSP-1 were significantly higher than those in control group (0.0089±0.0034 vs 0.0141±0.0037,P<0.05；96.38±16.80 vs 129.98±16.96,P<0.05).In DCM group,the mRNA and protein expressions of TSP-1 showed significantly positive correlations with the levels of fasting blood glucose and collagen (r=0.762,P<0.01; r=0.717,P<0.05; r=0.735,P<0.01; r=0.750,P<0.01).There was a significantly positive correlation of TSP-1 mRNA level with LVEDP (r=0.658,P<0.05).In contrast,there was a significantly negative correlation of TSP-1 protein with LVSP and -dp/dtmax (r=-0.605,P<0.05; r=-0.694,P<0.05).There was a significantly positive correlation of TSP-1 protein with LVEDP (r=0.716,P<0.05).There was a significantly negative correlation of TSP-1 protein with LVSP and -dp/dtmax (r=-0.633,P<0.05; r=-0.669,P<0.05).CONCLUSION: The increased expression of TSP-1 may play an important role in the development of myocardial interstitial fibrosis in DCM.     

Role of focal adhesion kinase expression in renal tissues of type 2 diabetic rats

AIM：To explore the dynamic change of focal adhesion kinase (FAK) in renal tissues of rats with type 2 diabetes mellitus (T2DM), and to investigate its role in the pathogenesis of diabetic nephropathy (DN). METHODS：The rat model of T2DM was established and the diabetic rats were randomly divided into 8-week DM (DM8), 12-week DM (DM12) and 16-week DM (DM16) groups. Meanwhile, normal control (NC) and high-fat high-sucrose control (HC) groups were also established. The protein expression of FAK, transforming growth factor β1 (TGF-β1), extracellular signal-regulated kinase 1/2 (ERK1/2), p-ERK1/2 and fibronectin (FN) was detected by immunohistochemical staining. The protein levels of FAK and p-FAK (Tyr397) were detected by Western blotting. The mRNA level of FAK in the renal cortex was examined by RT-PCR. RESULTS：The expression of FAK protein in renal tubular epithelial cells in DM12 and DM16 groups was significantly higher than that in NC, HC and DM8 groups (P<0.05). Moreover, TGF-β1, p-ERK1/2 and FN protein expression in DM groups was significantly increased compared with NC and HC groups (P<0.05). The levels of FAK and p-FAK (Tyr397) in the renal cortex in DM12 and DM16 groups were significantly up-regulated compared with NC, HC and DM8 groups (P<0.05), and the expression trend of p-FAK in different groups was in accordance with that of total FAK. The FAK protein expression was positively correlated with the expression of TGF-β1, p-ERK1/2 and FN proteins (P<0.01). Compared with NC, HC and DM8 groups, the expression of FAK mRNA increased remarkably in DM12 and DM16 groups (P<0.05). CONCLUSION: There is a possibility that FAK is activated as a downstream effector of TGF-β1 in T2DM, which enhances the expression of FN protein through activating ERK1/2, and therefore plays an important role in the pathogenesis of type 2 diabetic nephropathy.     

Effect of blood glucose control on expression of Smad7 and renal fibrosis in diabetic rats

AIM： To verify the hypothesis that treatment with insulin to control the blood glucose (BG) may relieve or slow down the development of diabetic nephropathy (DN) in diabetic rats by increasing the expression of Smad7. METHODS：The diabetic rat model was established by tail-vein injection of streptozotocin. Sixteen rats were divided into 2 groups. Eight of these animals in diabetes mellitus (DM) group had no treatment. The remaining eight of them in insulin treatment (INS) group were injected with insulin. After 13 weeks, the rats in INS group were given individual treatment with insulin to let the blood glucose level keep within 4 to 7 mmol/L. Meanwhile, 8 rats were used for normal control (NC group). After 16 weeks, the rats were sacrificed to detect the relevant biochemical parameters, and to observe the histophathological changes of the kidney and pancreas. In addition, immunohistochemical staining and Western blotting were employed to detect the protein expression of transforming growth factor β1 (TGF-β1), Smad ubiquitin regulatory factor 2 (Smurf2), Smad7, E-cadherin, α-sooth muscle actin (α-SMA), fibronectin (FN) and collagen I. RESULTS：Compared with NC group, the body weight was significantly reduced in DM group, whereas the body weight in INS group increased gradually. Compared with NC group, the levels of 24 h urine protein (24 h UP), BG and triglyceride (TG) were remarkably increased in DM group. Pathological detection on pancreas indicated that the islet was destroyed. The levels of TGF-β1, Smurf2, α-SMA, FN and collagenⅠ in the kidneys were increased in DM group, and the expression of Smad7 and E-cadherin, which were mainly located in renal tubular epithelial cells, was significantly reduced. Compared with DM group, the levels of 24 h UP and BG were significantly reduced in INS group, and the alleviated renal fibrosis was observed under light microscope. In addition, the protein levels of TGF-β1, Smurf2, α-SMA, FN and collagenⅠ in INS group were decreased compared with DM group, and the expression of Smad7 and E-cadherin was increased significantly. CONCLUSION：Target glucose control with insulin treatment restores the protein expression of Smad7 in the kidney of diabetic rats, reduces the accumulation of extracellular matrix and slows down DN progress. The decrease in TGF-β1 and Smurf2 expression, and the attenuation of Smad7 ubiquitination in renal tissues are the crucial parts in this process.     

Protective effects of Zhenwu decoction on kidney injury of diabetic rats induced by streptozotocin

AIM：To investigate the effects of Zhenwu decoction (ZWD) on the expression of α-smooth muscle actin (α-SMA) and NF-κB in diabetic nephropathy (DN) rats. METHODS：Diabetic rat model was induced by intrape-ritoneal injection of streptozotocin (STZ), and the animals were randomly divided into STZ group (n=22) and STZ+ZWD group (n=23). The normal rats served as control (n=16). All rats were sacrificed on 8 weeks after modeling. Biochemical assay and pathological observation (HE staining and transmission electron microscopy) were used to evaluate the effects of Zhenwu decoction on the renal function and pathological morphology. The body weight, renal index, blood glucose, total urinary protein in 24 h, and superoxide dismutase (SOD), malondialdehyde (MDA),inducible nitric oxide synthase (iNOS) were determined as well. Western blotting was used to observe the effects of Zhenwu decoction on the expression of α-SMA and NF-κB in diabetic nephropathy (DN) rats. RESULTS：Compared with normal group, the renal index, blood glucose concentration, total urinary protein in 24 h, blood urea nitrogen (BUN), serum creatinine (SCr) and MDA were significantly higher and body weight was lower in DN rats (P<0. 05). Pathological examination of the kidneys in DN group showed glomerular hypertrophy, glomerular basement membrane thickening, tubular epithelial cell degeneration, mesangial matrix proliferation, protein cast formation in some renal tubules. The protein expression levels of α-SMA and NF-κB were markedly increased (P<0.05). After ZWD treatment, the level of renal index, total urinary protein in 24 h, BUN, SCr and the expression of α-SMA and NF-κB at the protein level were significantly decreased (P<0.05). The renal histological injury in ZWD group was significantly ameliorated. CONCLUSION：Zhenwu decoction might protect kidney against STZ-induced injury via decreasing the expression of α-SMA and NF-κB.