Effect of rosiglitazone on SREBP-1 and TGF-β1 expressions and accumulation of ECM in renal tubular cells of Wistar rats treated with high fat diet

AIM: To study the effect of high fat diet on the expression of sterol regulatory element biding protein-1 (SREBP-1) and transforming growth factor β1 (TGF-β1) in renal tubular cells and rosiglitazone intervention. METHODS: Wistar rats were treated with high fat diet and rosiglitazone for 3 months. The serum glucose, serum insulin and serum triglyceride were detected. Oil Red O staining was used to observe the renal lipid deposit and Masson staining was for the detection of ECM accumulation. SREBP-1, TGF-β1 and FN protein were determined by the methods of immunohistochemistry and Western blotting. SREBP-1 mRNA was detected by in situ hybridization. RESULTS: Rosiglitazone prevented effectively the increase in serum glucose, serum insulin and serum triglyceride resulted from high fat diet. High fat diet led to lipid droplet formation in renal tubular cells and interstitial ECM accumulation, which was decreased by rosiglitazone treatment. Compared to normal rats, SREBP-1 protein and SREBP-1 mRNA showed high expressions in high fat diet rats that were lowered by rosiglitazone. The precursor segment and mature segment of SREBP-1 protein were decreased by 27.39% and 27.32%. Similarly, the high expressions of TGF-β1 and FN protein in kidney of high fat diet rats were also prevented by rosiglitazone intervention. Compared to high fat diet rats, the expression of TGF-β1 in rosiglitazone treatment rats was lowered by 19.14%. CONCLUSION: Rosiglitazone prevents effectively the over-expression of SREBP-1 and TGF-β1 in renal tubular cells, and decreases lipid accumulation and ECM production in rats fed with high fat diet.     

Concentration-dependent effect of insulin on expression of SREBP-1, FAS and lipid droplet formation in HKC cells

AIM: To investigate the effect of insulin at different concentrations on the expression of sterol regulatory element binding protein-1 (SREBP-1), fat acid synthase (FAS) and lipid droplet formation in human renal proximal tubular epithelial cell line (HKC). METHODS: HKC cells were treated with insulin at concentrations of 0 nmol/L, 1 nmol/L, 10 nmol/L, 100 nmol/L and 200 nmol/L respectively for 6 h. The analysis of SREBP-1 and FAS mRNA was performed by RT-PCR and the protein level of SREBP-1 was detected by Western blotting and immunocytochemistry. Oil red O staining was used to determine the cellular lipid droplet formation. RESULTS: Compared to HKC cells under the condition without insulin treatment (0 nmol/L group), the expression of SREBP-1 and FAS mRNA was significantly increased in HKC cells treated with insulin at concentrations of 10 nmol/L, 100 nmol/L or 200 nmol/L. Furthermore, the highest expression of SREBP-1 and FAS mRNA was observed in 100 nmol/L group. The SREBP-1 protein was located in the plasma of the HKC cells and was significantly upregulated in the cells treated with insulin at concentrations of 10 nmol/L, 100 nmol/L or 200 nmol/L. The results of Western blotting showed that the precursor and mature segments of SREBP-1 protein were increased in the cells of 10 nmol/L group, 100 nmol/L group and 200 nmol/L group, and those in 100 nmol/L group were the highest. The result of oil red O staining showed that the markedly deposited lipid droplet was only observed in 100 nmol/L group. CONCLUSION: The results suggest that insulin at high concentration up-regulates SREBP-1 and FAS, resulting in the formation and deposit of cellular lipid droplet in HKC cells, which may play an important role in the pathogenesis of renal lipid accumulation in metabolism syndrome.     

Effect of phospho-FOXO1 on lipid deposit in high glucose-stimulated human renal tubular cells

AIM:To study the effect of forkhead box O1 (FOXO1) expression and phosphorylation on lipid accumulation in high glucose-stimulated human renal tubular cell line HKC. METHODS:HKC cells were stimulated with high glucose. The total FOXO1, phospho-FOXO1 and sterol-regulatory element-binding protein 1 (SREBP-1) were detected by the methods of immunofluorescence and Western blotting. The cellular lipid deposit was measured by oil red O staining. Moreover, the wild-type FOXO1 vector or mutant FOXO1 vector was transfected into HKC cells followed by the treatment with high glucose. Immunofluorescence, Western blotting and oil red O staining were used to determine the effect of the phosphorylation site mutation on the lipid metabolism in renal tubular cells. RESULTS:No difference in total FOXO1 expression between normal glucose group and high glucose group was observed 48 h after HKC cells were stimulated with high glucose. However, phospho-FOXO1 was significantly increased in high glucose-treated HKC cells. Subsequently, SREBP-1 and cellular lipid deposit were up-regulated. The wild-type FOXO1 vector increased total FOXO1, phospho-FOXO1, SREBP-1 and cellular triglyceride in high glucose-treated HKC cells. However, mutant FOXO1 vector at the phosphorylation site attenuated the effect of high glucose on SREBP-1 and cellular lipid deposit. CONCLUSION: The phosphorylation of FOXO1 is involved in high glucose-induced up-regulation of SREBP-1 and cellular lipid accumulation in renal tubular cells. In addition, the mutation at the phosphorylation site prevents high glucose-induced enhancement of SREBP-1 and lipid deposit.     

Insulin and gliclazide therapies improve liver lipid deposition in type 2 diabetic rats

AIM：To investigate the effect of insulin and gliclazide therapies on the liver fat accumulation in type 2 diabetic rats. METHODS：A high-fat diet plus low-dose streptozotocin was implemented to establish a type 2 diabetic rat model, and the rats were randomly divided into diabetes mellitus (DM) group, diabetic rats treated with insulin (INS) group, diabetic rats treated with gliclazide per os （PO） group, and normal control (NC) group. The diabetic rats in INS group and PO group were given insulin and gliclazide for 3 weeks, respectively. The changes of the liver fatty were evaluated with oil red O staining. Fasting plasma adiponectin concentration was measured by ELISA. The expression of adiponectin receptor 1 (AdipoR1) was detected by real-time PCR. The protein levels of AMP-activated protein kinase (AMPK), phosphorylated AMPK on threonine 172 (Thr172p-AMPK), sterol regulatory element-binding protein 1c (SREBP-1c), phosphorylated SREBP-1c on serine 372 (Ser372p-SREBP-1c), acetyl-CoA carboxylase (ACC), phosphorylated ACC on serine79 (Ser79p-ACC) and immunoglobulin-binding protein (BiP) in the liver homogenate were determined by Western blotting. RESULTS：Compared with the normal rats, in DM group, the presence of cytoplasmic lipid deposits was confirmed by oil red O staining. In INS group, these changes were significantly lower than those in DM group. Similar results were obtained in PO group. Insulin therapy significantly increased the plasma concentration of diponectin and liver tissue levels of AdipoR1 compared with DM group. At the same time, these 2 indicators returned to normal levels after gliclazide therapy. Thr172p-AMPK/AMPK, Ser372p-SREBP-1c/SREBP-1c and Ser79p-ACC/ACC expression ratios were significantly reduced in DM group compared with control values. The expression of BiP was increased on the contrary. After insulin therapy, Thr172p-AMPK/AMPK and Ser372p-SREBP-1c/SREBP-1c were significantly increased, and Ser79p-ACC/ACC and BiP returned to the normal levels. After gliclazide treatment, Thr172p-AMPK/AMPK and Ser372p-SREBP-1c/SREBP-1c returned to the normal levels, the expression ratio of Ser79p-ACC/ACC had no significant improvement compared with DM group, and the expression of BiP significantly declined. CONCLUSION：Both the insulin and gliclazide therapies reduce the lipid deposition in the liver of rats with type 2 diabetes by activating AMPK, but the extent and mechanism are not the same. In insulin therapy, AMPK restrains the expression of SREBP-1c directly, increases the phosphorylation of SREBP-1c, and affects SREBP-1c by inhibiting the endoplasmic reticulum stress. Gliclazide treatment, which has no effect on the lipid oxidation, reduces lipid deposition in the liver only through the phosphorylation of SREBP-1c and the suppression of the endoplasmic reticulum stress.     

Effects of PTEN/AKT/mTOR signalling pathway on regulation of autophagy in renal tissues of diabetic rats

ATM:To observe the expression change of PTEN and autophagy in the renal tissues of diabetic rats, and to explore the regulatory mechanism of PTEN/AKT/mTOR pathway to autophagy in diabetic nephropathy. METHODS: The Sprague-Dawley rats were randomly divided into normal control group and diabetic group (8 in each group). The diabetic rat model was established by injection of streptozotocin. The biochemical and kidney indexes were measured after the model of diabetic nephropathy was successfully established. The expression location of PTEN in the renal tubular epithelial cells was observed by the method of immunohistochemistry. The protein levels of autophagy-related protein LC3, PTEN and PTEN/AKT/mTOR signalling molecules were determined by Western blotting. The mRNA expression of PTEN was detected by real-time PCR. RESULTS: The blood glucose, 24 h urine protein and kidney index in diabetic group were all obviously higher than those in control group. Compared with control group, the protein levels of LC3I and LC3II in diabetic group were obviously decreased. PTEN was mainly located in the renal tubular epithelial cells. Compared with control group, the protein expression of PTEN was significantly down-regulated in diabetic group. Furthermore, the activity of PTEN/AKT/mTOR pathway increased in diabetic nephropathy rats. CONCLUSION: The level of autophagy in renal tissues of diabetic rats is decreased, whereas the activity of PTEN/AKT/mTOR pathway is increased. The level of autophagy may be mediated by PTEN/AKT/mTOR pathway.     

Influence of losartan potassium on renal expression of TGF-β1, CD68 and MCP-1 in type 2 diabetic nephropathy rats

AIM: To observe the effects of losartan potassium on renal expression of transforming growth factor beta 1(TGF-β₁), CD68 and monocyte chemoattractant protein-1 (MCP-1) in type 2 diabetic nephropathy rats for exploring the protective mechanism of losartan potassium on type 2 diabetic rat kidney. METHODS: Thirty Sprague-Dawley rats were randomized into 3 groups: normal control group, model group and treatment group. The morphology of kidney tissues, the renal function, and the change of 24 h urinary protein quantitative index were measured after 15 weeks of treatment, while TGF-β₁, CD68 and MCP-1 expression in kidney cortex was observed by immunohistochemistry. RESULTS: Compared with the normal control rats, the body weight of the rats was lower in other groups, but the levels of blood glucose, triglyceride and cholesterol were higher.The expression of CD68, MCP-1 and TGF-β₁, 24 h urinary protein quantitative index and serum creatinine were higher in model group than those in normal control rats. However, compared with model group, serum creatinine, 24 h urinary protein quantitative index and the expression of CD68, MCP-1 and TGF-β₁ were decreased in treatment group. CONCLUSION: Losartan potassium protects the kidney of diabetic nephropathy rats through inhibiting the expression of TGF-β₁ and MCP-1 in the kidney and restraining macrophage infiltration.     

FGFR2-IIIb D3 extracellular domain inhibits lipid synthesis in keratinocytes

AIM:To investigate the inhibitory effect of extracellular domain of fibroblast growth factor receptor 2 (FGFR2)-Ⅲb D3 on lipid synthesis in HaCaT keratinocyteas. METHODS:The bioactivity of FGFR2-Ⅲb D3 extracellular domain was detected by isothermal titration calorimetry (ITC) and CCK-8 assay. The inhibitory effect of FGFR2-Ⅲb D3 extracellular domain on lipid synthesis in HaCaT cells was analyzed by real-time PCR, Western blot, flow cytometry and oil red O staining. RESULTS:The results of ITC, real-time PCR and CCK-8 assay showed that FGFR2-Ⅲb was highly expressed in HaCaT cells, and FGFR2-Ⅲb D3 extracellular domain specifically bound to FGF7 to inhibit HaCaT cell viability. The results of real-time PCR and Western blot showed that FGF7 significantly up-regulated the mRNA expression of sterol regulatory element binding protein-1c (SREBP-1c), fatty acid synthase (FAS), acetyl-CoA synthase (ACS), stearoyl-CoA desaturase (SCD) and 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR), and the protein levels of p-FGFR, p-AKT and SREBP-1 in HaCaT cells, and these effects were inhibited after the treatment with FGFR2-Ⅲb D3 extracellular domain and AKT inhibitor, which was significantly different from the FGF7 group. and The results of oil red O staining and flow cytometry showed that the lipid content of HaCaT cells was increased after FGF7 induction. After treatment with FGFR2-Ⅲb D3 extracellular domain and AKT inhibitor, the lipid content was decreased (P<0.01). CONCLUSION:FGFR2-Ⅲb D3 extracellular domain inhibits the autophosphorylation of FGFR2-Ⅲb by its competitive binding to FGF7, and then blocks downstream PI3K-AKT signaling pathway, thus resulting in the down-regulation of SREBP-1c and downstream lipid synthases, and inhibiting lipid synthesis in keratinocytes.     

Expression of zinc-finger transcription factor Snail 1 in the kidney of diabetic rats

AIM: To explore the expression of Snail 1 in renal tissues of diabetic rats, and to investigate its contribution to the progression of diabetic nephropathy. METHODS: Streptozotocin-induced diabetic rats were randomly divided into 2, 4, 8, 12, 16, 20, 24 weeks groups and 16 week A, 20 week A and 24 week A groups. A groups were treated with insulin to control blood glucose to normal level from the 13th week. Control groups were set up in age-matched time points. Blood glucose, 24 h urine protein, serum creatinine (Scr) and kidney index of rats were measured. Periodic acid-silver (PAS) staining was used to observe the renal pathological changes. The mRNA and protein expressions of Snail 1 and FN in renal cortex were detected by RT-PCR and immunohistochemical staining, respectively. Western blotting was employed to detect the expression of Snail 1 protein in the renal cortex. RESULTS: The levels of blood glucose, Scr, kidney weight index were increased remarkably in diabetic rats as compared with those in control groups (P<0.05, P<0.01), and decreased remarkably in the insulin-treated rats as compared with those in the diabetic rats (P<0.05, P<0.01). The Snail 1 protein was not detected by immunohistochemical staining in normal renal tissues. However, strongly positive staining was observed in renal tubules of diabetic rats. A time-dependent loss of Snail 1 expression was detected in the kidney in insulin-treated rats. The Snail 1 protein and mRNA of Snail 1 and FN were significantly up-regulated in the diabetic rats as compared with those in controls (P<0.01), while down-regulated in the insulin-treated diabetic rats (P<0.01). A close positive relationship existed between the mRNA expression of Snail 1 and FN (r=0.800, P<0.01). The level of Snail 1 protein expression was positively correlated with blood glucose, urine protein, Scr, kidney index (r=0.877, 0.694, 0.522, 0.875, P<0.01).CONCLUSION: These findings suggest that Snail 1 gene and protein expression are up-regulated in the kidney of rats with diabetes and may be involved in the pathogenesis of diabetic nephropathy.     

Notch1 promotes renal tubulointerstitial fibrosis in renal tissues of diabetic mice by inhibiting autophagy through Akt/mTOR signaling pathway

AIM: To observe the changes of Notch1 expression and autophagy in the renal tissues of diabetic mice, and to explore the regulatory effect of Notch1 on tubulointerstitial fibrosis by inhibiting autophagy in diabetic nephro-pathy. METHODS: The mice were randomly divided into normal control group (db/m mice) and diabetes group (db/db mice), with 8 rats in each group. After 12 weeks of feeding, the mice were sacrificed and the corresponding biochemical indexes were measured. The protein expression of Notch1 in the renal tubular epithelial cells was observed by immunohistochemical staining. The protein levels of Notch1, PTEN, p-Akt (Thr308), Akt, p-mTOR (Ser2448), mTOR, LC3, P62, collagen type Ⅰ (Col-Ⅰ) and collagen type Ⅲ (Col-Ⅲ) were determined by Western blot. RESULTS: Compared with the db/m mice, the blood glucose, glycosylated hemoglobin, serum creatinine, triglyceride and total cholesterol were increased in the db/db mice (P<0.01). Renal tubular epithelial cell vacuolar degeneration, renal tubular expansion and interstitial inflammatory cell infiltration in db/db mouse renal tissues with HE staining were observed. The images of Masson staining showed collagenous fiber-like substance deposition in the glomerular capillaries and renal interstitium, and disarrangement of tubular structure in the renal tissues of db/db mice. The protein expression levels of PTEN and LC3-Ⅱ were decreased (P<0.01 or P<0.05), while the protein levels of Notch1, P62, p-mTOR (Ser2448), p-Akt (Thr308), Col-I and Col-III were increased in the db/db mice as compared with the db/m mice (P<0.01). However, no significant change of total mTOR and Akt proteins between the 2 groups was found. CONCLUSION: Notch1 protein expression was increased, PTEN expression was significantly reduced, Akt/mTOR pathway was activated, autophagy was inhibited, and fibrosis was aggravated in the renal tissues of the diabetic mice.     

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.     

Expression and role of SnoN in kidney tissue of diabetic rats

AIM: To observe the changes of SnoN expression in renal tissues of diabetic rats, and to elucidate the role of co-repressor SnoN protein in the diabetic nephropathy. METHODS: Diabetic nephropathy was induced by intravenous injection of streptozotocin in male Sprague-Dawley rats. The model animals were divided into 2 weeks, 4 weeks and 8 weeks groups randomly. Meanwhile other 3 age-matched normal control groups were set up. The expressions of SnoN, TGF-β₁, Smad2/3, APC were detected by immunohistochemistry staining. The SnoN proteins in renal cortex were detected by Western blotting. Blood glucose, serum creatinine and 24 h urine protein were examined by biochemistry methods. The renal morphology was checked on PAS staining sections under light microscopy. RESULTS: The results of immunohistochemistry and Western blotting showed that the expression of SnoN in diabetic renal tissues at 2 weeks was the same as the normal kidney. There was a significant decrease in DM 4 weeks (P<0.01). A time dependent increases in TGF-β₁, Smad2/3 and APC were detected in the kidney of diabetic mellitus rats. The degree of SnoN down-regulation had close relation with the increases in TGF-β₁, Smad2/3 and APC. CONCLUSION: These results suggest that in the DM, renal increase in TGF-β₁ expression may down-regulate the SnoN expression through APC dependent degradation, indicating a key role in the mechanism of diabetic nephropathy.     

Lipotoxicity of free fatty acid mixture and effect of free fatty acid mixture on lipid metabolism-related genes in L-02 cells

AIM：To investigate the lipotoxicity of free fatty acid (FFA) mixture and the effect of the FFA mixture on lipid metabolism-related genes in L-02 cells. METHODS：A normal human hepatocytes-derived cell line L-02 was treated with 0.5, 1 and 2 mmol/L FFA mixture (oleate and palmitate, 2∶1) for 24 h. The cellular total lipid accumulation was determined after Nile red staining by confocal laser scanning microscopy and flow cytometry. Intracellular triglyceride (TG) content was measured using an enzymatic kit. The viability of L-02 cells was determined by MTT assay and the apoptosis-inducing effect of FFA mixture was evaluated by annexin V/PI staining. The levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in the culture medium were detected by ALT and AST kits. The mRNA levels of lipid metabolism-related genes, adipose differentiation-related protein (ADRP) and sterol regulatory element-binding protein 1 (SREBP-1), were examined by quantitative real-time PCR. RESULTS：All the different concentrations of FFA mixture increased intracellular lipid accumulation and TG content in a dose-dependent manner. FFA mixture at concentration of 1 mmol/L increased intracellular TG by 2.6 folds, which matched with the change in non-alcoholic fatty liver disease patients. Treatment for 24 h with 0.5 mmol/L and 1 mmol/L FFA mixture did not trigger apparent cell death and apoptosis, while treatment with 2 mmol/L FFA mixture resulted in a marked decrease in cell viability and induced early and late stages of apoptosis in L-02 cells. The levels of ALT and AST in the culture supernatant had no significant difference between control group and FFA treatment group. Treatment with 1 mmol/L FFA mixture up-regulated the expression of ADRP and SREBP-1 by 2.660 and 2.758 folds, respectively. CONCLUSION：FFA mixture induces the hepatic steatosis and 2 mmol/L FFA mixture causes mild cells damage in L-02 cells. The up-regulation of ADRP and SREBP-1 may be involved in FFA-induced hepatic steatosis.     

Protective effects of riboflavin on diabetic nephropathy in STZ-induced diabetic rats

AIM: To explore the protective effects of riboflavin on the kidney in streptozotocin (STZ)-induced diabetic rats. METHODS: Male Sprague-Dawley rats were randomly divided into 3 groups: normal control group, diabetic model group and riboflavin-treated group. Diabetes was induced by a single injection of STZ (dissolved in 0.01 mol/L citrate buffer, pH 4.5, 65 mg/kg, ip) in rats. The biochemical methods were used to measure the contents of urine protein and malondialdehyde in the kidney, and the activities of superoxide dismutase (SOD) and catalase (CAT) in serum and renal tissues. Furthermore, the protein expression of TGF-β1 and plasminogen activator inhibitor-1(PAI-1) in renal cortex was detected by Western blotting. The morphological changes of renal tissue were observed under microscope.RESULTS: Compared to the diabetic model group, riboflavin significantly increased the activities of SOD and CAT (P<0.01) in the serum and renal tissues, and decreased the contents of urine protein and MDA (P<0.01) in the renal tissues in riboflavin-treated group. The levels of TGF-β1 and PAI-1 in the renal cortex were dramatically decreased in the treated diabetic rats compared to the diabetic model rats (P<0.01).CONCLUSION: Riboflavin inhibits the protein expression of TGF-β1 and PAI-1 in renal tissue of STZ-induced diabetic rats. Riboflavin may alleviate the pathologic changes and play an important protective role in diabetic kidneys.     

Effects of atorvastatin on iopromide-induced apoptosis of renal tubular epithelial cells in diabetic rats

AIM：To investigate the protective effect of atorvastatin (ATO) against contrast medium (CM)-induced apoptosis of renal tubular epithelial cells in diabetic rats. METHODS：Streptozocin-induced diabetic Wistar rats were fed for 8 weeks and then randomly divided into 5 groups: diabetes mellitus (DM) group, DM with iopromide (a kind of CM) treatment group (DM+CM group), and groups of DM rats treated with ATO at 5 mg·kg-1·d-1 (ATO1 group), 10 mg·kg-1·d-1 (ATO2 group) and 30 mg·kg-1·d-1 (ATO3 group) before iopromide injection. Healthy Wistar rats served as normal controls (N group). Urine creatinine (UCr) and 24-hour urinary albumin (24 h-UAlb) were determined 24 h after iopromide injection. Serum creatinine (SCr) and blood urea nitrogen (BUN) were detected 48 h after iopromide injection, and then creatinine clearance (CCr) and 24-hour urinary albumin excretion rate (24 h-UAER) were calculated. The rats were sacrificed and both kidneys were removed 48 h after iopromide injection. For the left kidney, the morphology by HE staining, the renal tubular apoptosis by TUNEL and the expression of Bax and Bcl-2 by immunohistochemistry were detected. For the right kidney, the expression of Bax and Bcl-2 was measured by Western blotting. RESULTS：Compared with N and DM groups, the levels of SCr, BUN and 24 h-UAER, as well as the expression of Bax in the renal medulla were higher, the levels of Ccr and Bcl-2 expression in the renal medulla were lower and TUNEL-positive cells were more in DM+CM group. Compared with DM+CM group, ATO attenuated these changes, especially in ATO3 group. CONCLUSION: Iopromide could cause renal tubular apoptosis. Early application of ATO could dose-dependently attenuate the development of contrast-induced acute kidney injury, partly due to suppression of iopromide-induced renal tubular apoptosis.     

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.     

Effect of rosiglitazone on expressions of insulin receptor substrate-1 and glucose transporter 4 in skeletal muscle of type 2 diabetic rats with hyperlipemia

AIM: To investigate the effect of rosiglitazone on the expressions of insulin receptor substrate-1 (IRS-1) and glucose transporter 4 (GLUT4) in skeletal muscles of type 2 diabetic rats with hyperlipemia, and to explore the different pharmacological mechanism. METHODS: The model of type 2 diabetic rats with hyperlipemia was established by injecting low dosage of streptozotocin (STZ) and feeding with high fat diet. Then the diabetic rats were divided into two groups: untreated diabetic group and rosiglitazone-intervened diabetic group. The course of treatment lasted for 4 weeks. The expressions of IRS-1 and the GLUT4 proteins in the cell membrane of isolated rats skeletal muscles were detected by Western blotting. RESULTS: The fasting blood glucose, insulin and triglyceride contents in rosiglitazone-intervened diabetic group were lower than those in untreated diabetic group, but they were still higher than those in control group. The result of Western blotting showed that the expression of GLUT4 protein in rosiglitazone-intervened diabetic group was increased compared with untreated diabetic group, but its level was still lower than that in control group. The protein expression and tyrosine phosphorylation of IRS-1 in rosiglitazone-intervened diabetic group were significantly higher than those in untreated diabetic group and their levels were lower than those in control group. CONCLUSION: The effect of rosiglitazone on GLUT4 protein may link to its ability to induce the protein expression and tyrosine phosphorylation of IRS-1 in skeletal muscles in type 2 diabetic rats.     

Protective effects of luteolin on STZ-induced diabetic kidneys

AIM: To explore the protective effects of luteolin on the diabetic kidneys. METHODS: Male Sprague-Dawley rats were randomly divided into 5 groups: normal control group, diabetic model group and the groups of diabetic rats treated with luteolin at a low dose, a middle dose and a high dose. The diabetic model was induced by a single intraperitoneal injection of streptozotocin (STZ,65 mg/kg). Blood glucose, urine protein, the activity of superoxide dismutase and catalase in serum and kidney, and the content of malonaldehyde(MDA) in kidney were analyzed by biochemical methods. Western blotting was used to detect the protein expression of transforming growth factor-β₁(TGF-β₁) and plasminogen activator inhibitor-1(PAI-1) in the renal cortex. The morphological changes of the renal tissues were observed under microscope. RESULTS: Compared with diabetic model group, luteolin significantly reduced the level of blood glucose (P<0.01), the content of urine protein (P<0.01) and MDA (P<0.01) in the kidneys, and increased the activity of superoxide dismutase and catalase (P<0.01) in serum and kidneys in the diabetic rats. The protein levels of TGF-β₁ and PAI-1 in the renal cortex were dramatically decreased as the rats were treated with luteolin. CONCLUSION: Luteolin may exert an important protective effect on diabetic kidneys by relieving oxidative stress and inhibiting the protein expression of TGF-β₁ and PAI-1 in the renal tissues of STZ-induced diabetic rats.     

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.