Therapeutic effects of losartan and astragalus membranaceus on diabetic nephropathy

AIM: To observe the protective effects of losartan and astragalus membranace on the kidney of diabetic rats, and to study their possible mechanisms. METHODS: The diabetic rats were induced by a single intraperitoneal injection of streptozotocin. At the end of 12th week,changes in urinary albumin excretion, urinary β₂-MG excretion, Ccr,NO,ET-1 levels in blood, urinary and renal tissue were observed. Serum and urinary TGF-β₁ concentration,average volume of glomeruler,average thickness of glomerular basement membrane were also measured. RESULTS: In the treated diabetic rats, urinary albumin excretion, urinary β₂-MG excretion, Ccr, urinary and renal tissue NO, urinary TGF-β₁, average volume of glomeruler, average thickness of glomerular basement membrane decreased obviously as compared with diabetic untreated rats. These effects were enhanced when losartan was combined with astragalus membranace. CONCLUSION: Losartan or astragalus membranace reversed the injury of renal structure and function in STZ-induced diabetic rats. The protective effects were enhanced when losartan was combined with astragalus membranace. The decrease in NO,ET,TGF-β₁ concentration in renal tissue may be one of mechanisms for this action.     

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 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.     

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.     

Effect of erlotinib on renal injury in rats with STZ-induced diabetic nephropathy

AIM: To investigate the effect of epidermal growth factor receptor (EGFR) inhibitor erlotinib on kidney injury in diabetic nephropathy (DN) rat and the underlying mechanism. METHODS: The rat model of DN was induced by intraperitoneal injection of streptozotocin (STZ) at dose of 55 mg/kg. One week after STZ injection, the rats with blood glucose level exceeding 16.7 mmol/L were identified as diabetic. Diabetic rats were randomly divided into 2 groups:STZ group and STZ+erlotinib group. In addition, the normal rats were used as control group. The rats in STZ+erlotinib group were treated with erlotinib at 100 mg·kg^-1·d^-1 for 4 weeks(5th~8th week). The fasting blood glucose (FBG), serum creatinine (SCr) and 24 h urine protein were measured. The pathological changes of the kidney were observed by HE staining and Masson staining. The protein levels of EGFR, p-EGFR, transforming growth factor β1 (TGFβ1), Smad2/3, p-Smad2/3, collagen Ⅳ (ColⅣ) and fibronectin in the kidney tissues were determined by Western blot. The reactive oxygen species (ROS) level and malondialdehyde (MDA) content in the renal tissues were futher analyzed. RESULTS: Compared with control group, the levels of FBG, 24 h urine protein and Scr were significantly increased in STZ group (P<0.01). Compared with STZ group, the levels of FBG, 24 h urine protein and SCr in STZ+erlotinib group were markedly decreased (P<0.05). In additon, the glomerular structure was restored to normal, the proliferative degree of mesangial cells markedly attenuated, and the epithelial cells were in alignment in STZ+erlotinib group. Moreover, erlotinib significantly inhibited the protein levels of p-EGFR, TGFβ1, p-Smad2/3, ColⅣ and fibronectin in the kidney tissues of STZ rats. In addition, erlotinib also significantly inhibited the levels of ROS and MDA in the kidney tissues of STZ rats. CONCLUSION: Erlotinib ameliorates STZ-induced diabetic nephropathy possibly through inhibiting the activation of EGFR/TGFβ1-Smad2/3 signaling pathway in association with suppression of fibrosis and oxidative stress.     

Estradiol stimulated proliferation and differentiation of prostatic stromal cells through regulation of BPH-1 paracrine

AIM: To characterize the effect of estradiol on proliferation, differentiation and extracellular matrix (ECM) accumulation in stromal cells through regulation of BPH-1 paracrine. METHODS: BPH-1 cells were stimulated with different concentrations of estradiol. Conditioned media (CM) were harvested and their effects on stromal cell cultures were tested. Cell proliferation was determined by MTT assay. mRNA of smoothelin, fibronectin, collagen Ⅳ and transforming growth factor β₁(TGF-β₁) were analyzed by real-time RT-PCR. Western blotting was used to determine smooth muscle myosin heavy chain (SMMHC). ELISA and radioimmunoassay were respectively used to measure fibronectin, TGF-β₁ and collagen Ⅳ protein expressions.RESULTS: Estrodiol stimulated the expression and secretion of TGF-β₁ in BPH-1 cells. The proliferation of stromal cells increased when they were cultured with CM harvested from estrogen treated BPH-1 cells. The mRNA levels of collagen Ⅳ and smoothelin increased in stromal cells treated with CM from BPH-1 cells. The results of radioimmunoassay also showed that the collagen Ⅳ protein level up-regulated in the supernatants and cell extracts of CM-treated stromal cells. A neutralizing antibody to TGF-β₁ inhibited the stimulation of collagen Ⅳ and SMMHC by BPH-1 CM. The expression of fibronectin was only marginally changed in stromal cells cultured in the presence of BPH-1 CM. CONCLUSION: The BPH-1 cells increase ECM accumulation and differentiation of stromal cells through TGF-β₁. Estradiol stimulate differentiation of stromal cells by induction of TGF-β₁ expression. Estradiol stimulate proliferation by influencing the factors secreted from prostatic epithelial cells.     

Effects of IGFBPrP1 and thioacetamide on liver tissues in mice

AIM: To investigate the effects of insulin-like growth factor binding protein related protein 1(IGFBPrP1) and thioacetamide (TAA) on the liver tissues, and to identify the role of IGFBPrP1 in liver fibrosis. METHODS: Thirty-two male C57BL/6 wild-type mice were randomly divided into 4 groups (n=8 in each group): control group, recombinant murine IGFBPrP1(rmIGFBPrP1) 4 weeks group, TAA 2 weeks group and TAA 4 weeks group. The methods of hematoxylin-eosin (HE) staining, picric acid-Sirius red staining, immunohistochemistry and Western blotting were performed. RESULTS: The extensive fatty degeneration of liver cells in rmIGFBPrP1 4 weeks group was observed. The collagen deposition was found in TAA 2 weeks group. In TAA 4 weeks group, the degree of hepatic fibrosis was more serious than that in TAA 2 weeks group. The expression levels of IGFBPrP1, transforming growth factor beta 1(TGF-β₁), Smad3, p-Smad2/3, collagen Ⅲ, collagenⅠand fibronectin (FN) in liver tissues were higher in rmIGFBPrP1 4 weeks group, TAA 2 weeks group and TAA 4 weeks group than those in control group. No significant difference of the expression levels of IGFBPrP1, collagen I and FN between rmIGFBPrP1 4 weeks group and TAA 2 weeks group was observed. CONCLUSION: IGFBPrP1 plays an important role in the process of thioacetamide-induced liver fibrosis. Meanwhile, IGFBPrP1 induces excessive deposition of extracellular matrix through TGF-β₁/Smad3 pathway.     

Diagnostic value of serum miR-21 for diabetic nephropathy

AIM:To evaluate the diagnostic value of serum miR-21 as a molecular biomarker in the patients with diabetic nephropathy (DN). METHODS:Twenty-five patients with DN, 50 patients with type II diabetes ［divided into DMA group and DMB group base on urinary microalbumin (UmAlb), 25 in each group］, 25 patients with diabetic nephropathy-induced uremia (UM) and 25 normal controls (NC) were included in the study. Total RNA was extracted from the serum samples for determining the miR-21 level using real-time PCR. The relationship between miR-21 level and clinical parameters of the patients with diabetic nephropathy was analyzed. RESULTS:The relative expression level of serum miR-21 was significantly lower in DN group than that in DMA group, DMB group and NC group, and was significantly higher than that in UM group (all P<001). The serum miR-21 level showed significant negative correlation with cystatin C, UmAlb, UmAlb/urinary creatinine in DN patients (P<0.01,P<0.05,P<0.05). For discriminating the DN patients from NC, DMA, DMB, DMA＋NC, DMB＋NC and DMA＋DMB, the areas under the receiver operating characteristic curve (AUC-ROC) for serum miR-21 were 0848 (95% CI: 0737~0959), 0896 (95% CI: 0812~0980), 0782 (95% CI: 0641~0922), 0838 (95% CI: 0743~0933), 0796 (95% CI: 0675~0917) and 0808 (95% CI: 0704~0911), and the sensitivity and specificity were 800% and 720%, 720% and 880%, 720% and 840%, 760% and 770%, 760% and 820%, 700% and 860%, respectively. For discriminating the DN patients from DMA＋DMB＋NC, the AUC-ROC was 0845 (95% CI: 0752~0939), and the sensitivity and specificity were 760% and 773%, respectively. CONCLUSION: Serum miR-21 can serve as a molecular diagnostic marker for diagnosis of diabetic nephropathy.     

Relationship between the changes of angiotensin Ⅱ and vascular endothelial growth factor in plasma and the occurrence of diabetic nephropathy

AIM:To measure the concentrations of angiotensinⅡ(ATⅡ) and vascular endothelial growth factor(VEGF) in plasma of diabetics and investigate the relationship between ATⅡ and VEGF and the role of VEGF in occurrence and development of diabetic nephropathy(DN). METHODS:98 type Ⅱ diabetics were divided into two groups: 43 were in diabetic nephropathy group and 55 in non-diabetic nephropathy (NDN) group, and 25 healty persons were in control group. We measured the levels of ATⅡ, VEGF, glycosylated hemoglobinC (GHbAlc) in plasma and urinary microalbumin(UmALB), respectively.RESULTS:The levels of ATⅡ, VEGF, HbAlc and UmALB in DN group and NDN group were increased significantly compared with control group(P＜0.01); The concentrations of ATⅡ, VEGF and UmALB in DN group were obviously higher than that in NDN group(P＜0.01), but there was no significant difference of GHbAlc between DN and NDN; There was a positive correlation between ATⅡ and VEGF(r=0.465,P＜0.05), and VEGF was positively correlated with UmALB(r=0.540,P＜0.01). CONCLUSION:The levels of ATⅡand VEGF increased significantly in plasma of diabetics, and ATⅡ and VEGF may involve in the occurrence and development of diabetic nephropathy.     

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.     

Effect of irbesartan on nephrin expression in the podocyte of early diabetic nephropathy rats

AIM: To investigate the expression of the nephrin in podocyte of the diabetic nephropathy(DN) rats and the mechanism of irbesartan-induced renal protection.METHODS: The DN model was established by a single injection of streptozotocin(STZ),and DN rats were randomly divided into 2 groups: model group and irbesartan treatment group.In addition,the normal rats served as a normal control group. All the rats were received daily gavage respectively for 8 weeks. The urinary protein quality in 24 hours,body weight(BW),kidney weight (KW),KW/BW,glucemia,urea nitrogen,creatinine,total cholesterol, triacylglycerol were detected with correlative methods and the pathological changes of kidney were also detected with optic microscope and transmission electron microscope.The expression of nephrin in podocyte were detected by immunohistochemistry. RESULTS: In DN rats, irbesartan reduced the urinary protein quality in 24 hours (P<0.01) and alleviated the damage of kidney. Meanwhile，the expression of nephrin was declined remarkably in podocytes in irbesartan treatment group compared with model group（P<0.05）.CONCLUSION: Irbesartan might protect kidney against STZ-induced injury via decreasing the expression of nephrin in podocytes.     

Study on relationship between expression of PKCα in glomeruli and development of nephropathy in diabetic rats

AIM:To observe the dynamic changes of expression of PKCα, TGF-β₁ and α-SMA in glomeruli of diabetic rats induced by the alloxon and to invesitigate their roles in the diabetic nephropathy(DN).METHODS:Rats were randomly divided into four groups: normal control group (group A), diabetic group of one week (group B), diabetic group of one month (group C), diabetic group of two months (group D). Immunohistochemistry and Western blotting were used to detect the expression of PKCα, TGF-β₁ and α-SMA in renal tissue of all groups. Blood glucose, triglycerides, cholesterol, creatinine and urine protein were analysed by chemical methods. The morphological changes of renal tissue were checked through microscopy.RESULTS:The expression of PKCα and TGF-β₁ in renal tissue of diabetic groups were increased comparing with those of nomal control group(P<0.05). The mesangial cells expressed α-SMA in two months group. Chronologically the expression of PKCα, TGF-β₁ and α-SMA were positively correlative with each other and the impairment of kidney was also observed.CONCLUSIONS: During the DN process the expression of PKCα increased. PKCα raised GFR and the permeability of glomerular filtration membrane which enhanced urinary albumin excretion. PKCα also increased expression of TGF-β and therefore to induce the expression of α-SMA. The appearance of α-SMA was a marker of the phenotypic transform of renal cells.