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.     

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.     

Effects of long-term cigarette smoke exposure on pulmonary vascular remodeling and TGF-β1 expression in rat pulmonary vessels

AIM: To observe the effects of long-term cigarette smoke exposure on pulmonary vascular remodeling and the protein expression of transforming growth factor-β1(TGF-β1) in the rats, and to explore the mechanism.METHODS: SD rats(n=36) were randomly divided into control group, 2-week smoke exposure(S-2W) group and 12-week smoke exposure(S-12W) groups. HE staining and α-smooth muscle actin staining were performed to observe the pulmonary vascular remodeling.The protein expression of proliferating cell nuclear antigen(PCNA) and TGF-β1 in the pulmonary arteries was determined by the method of immunohistochemistry. The mRNA expression of TGF-β1 in the pulmonary arteries was evaluated by RT-qPCR.RESULTS: Compared with control group, ratio of pulmonary vessel wall thickness to vessel diameter(WT%) and percentage of muscularized vessels were significantly increased in S-2W group and S-12W group(P<0.01). Significant increases in the protein expression of PCNA and TGF-β1 in smoke exposure groups were observed compared with control group. There was significant difference between 2 model groups(P<0.01). Compared with control group, the mRNA expression of TGF-β1 in pulmonary artery walls obviously increased in smoke exposure groups. There was significantly difference between S-2W and S-12W groups(P<0.05). The mRNA expression of TGF-β1 was positively correlated with pulmonary vascular muscularization, WT% and the protein expression of PCNA. CONCLUSION: Long-term cigarette smoke exposure results in pulmonary artery remodeling in rats. The possible mechanism is that cigarette smoking exposure induces the over-expression of TGF-β1 at mRNA level in pulmonary vessels and promotes the proliferation of pulmonary vascular smooth muscle cells in rats.     

Effect of alpha-lipoic acid on expression of miR-21/Smad7 and fibrosis in kidney of diabetic rats

AIM: To investigate the protective effect of alpha-lipoic acid (ALA) on the kidney of the rats with diabetes mellitus (DM), and to discuss the mechanism. METHODS: The DM rats were divided into normal control (NC) group, DM group and ALA group. After treated with ALA for 6 weeks, the rats were sacrificed to detect the relevant biochemical parameters, and the pathological changes of the kidney tissues were observed by HE staining and Masson staining. The protein levels of transforming growth factor-β1 (TGF-1), p-Smad2/3, Smad7, collagen I and collagen Ⅲ were determined by Western blot. In addition, the expression of microRNA-21 (miR-21) was detected by RT-qPCR. RESULTS: Compared with NC group, the kidney weight/body weight, blood glucose (BG), total cholesterol, triglyceride and 24-h urine protein were remarkably increased in DM group (P<0.05). The pathological observation of the kidney tissues showed fibrosis changes in DM group. The level of Smad7 was reduced in DM group, while the levels of TGF-β1, p-Smad2/3, collagen I, collagen Ⅲ and miR-21 in the kidney tissues were increased (P<0.05). After treatment with ALA for 6 weeks, all the relevant biochemical parameters were reduced except BG, and the renal fibrosis lesions were obviously alleviated. Compared with DM group, the levels of TGF-1, p-Smad2/3, collagen I, collagen Ⅲ and miR-21 in the kidney tissues were reduced in ALA group, while the level of Smad7 was increased (P<0.05).CONCLUSION: ALA may prevent the development of renal fibrosis in rats through restraining the expression of TGF-β1 and miR-21, increasing the levels of Smad7 protein, and reducing the deposition of extra cellular matrix.     

Effects of bradykinin on proliferation of porcine pulmonary artery smooth muscle cells induced by TGF-β1

AIM: To investigate the effects of bradykinin (BK) on the proliferation of pulmonary artery smooth muscle cells (PASMCs) induced by transforming growth factor beta 1 (TGF-β1) and its possible mechanisms. METHODS: Primary porcine PASMCs were isolated, cultured and identified, and the cells at passages 2~6 were used in this study. The viability of PASMCs was determined by Cell Counting Kit-8 assay. The protein expression of phosphatidylinositol 3-kinase (PI3K), phosphorylated Akt (p-Akt) and phosphorylated extracellular signal-regulated kinase 1/2 (p-ERK1/2) was detected by Western blotting. RESULTS: TGF-β1 promoted the proliferation of PASMCs in a dose-dependent manner (P<005). BK significantly inhibited the proliferation of PASMCs induced by TGF-β1 (P<005), and attenuated the elevated expression of PI3K, p-Akt and p-ERK1/2 proteins (P<005). HOE-140, a BK type 2 receptor (B2R) inhibitor, reversed the effects of BK (P<005). CONCLUSION: BK inhibits TGF-β1-induced proliferation of PASMCs, which may be associated with inactivation of PI3K/Akt and ERK1/2 signaling pathways.     

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.     

RTN1A induces renal tubular epithelial cells to secrete VEGF and IL-8 and promotes diabetic nephropathy renal fibrosis via ERK signaling pathway

AIM:To investigate the effects of reticulon 1A (RTN1A) on the secretion of vascular endothelial growth facter (VEGF) and interleukin-8 (IL-8) in renal tubular epithelial cells, and on the diabetic nephropathy (DN) renal fibrosis, and to explore the underlying mechanism. METHODS:The mouse model of DN was established, and the blood glucose, kidney index, urine microalbumin (UMA) and creatinine clearance (CCr) were measured. The protein levels of RTN1A, p-ERK, ERK, VEGF, IL-8 and renal fibrosis markers α-smooth muscle actin (α-SMA) and fibronectin (FN) were determined by Western blot. Human renal tubular epithelial cell line HK-2 was treated with high glucose, and the ERK signaling proteins, fibrosis markers and secretion of cytokines were detected by Western blot and ELISA. The cells were treated with high glucose combined with RTN1A silencing or ERK inhibitor PD98059 for 24 h, and the ERK signaling proteins, fibrosis markers and secretion of cytokines were also detected by Western blot and ELISA. RESULTS:The blood glucose, kidney index, UMA and CCr in the DN mice were significantly higher than those in control group (P<0.05), suggesting that DN model was successfully constructed. The protein levels of RTN1A and its downstream protein p-ERK, the cytokines VEGF and IL-8, and the fibrosis markers α-SMA and FN were significantly increased in the DN model mice (P<0.05). The protein levels of RTN1A, p-ERK, VEGF, IL-8, α-SMA and FN were also significantly increased in the HK-2 cells after treated with high glucose for 24 h, while these proteins were significantly decreased after silencing of RTN1A expression. CONCLUSION:RTN1A may be associated with the occurrence and development of DN. Silencing of RTN1A expression inhibits DN renal inflammation and fibrosis through ERK signaling. RTN1A may be an effective therapeutic target.     

TAK1 regulates fibrosis of renal tubular epithelial cells by regulating p38 MAPK signaling pathway

AIM:To investigate the effect of transforming growth factor-β (TGF-β) activated kinase 1(TAK1) on renal tubular epithelial fibrosis. METHODS:The renal tubular epithelial cell line HK-2 was used as the research object. After induced by TGF-β1, real-time PCR and Western blot were used to detect the expression of TAK1 in the HK-2 cells. TAK1 shRNA lentivirus was used to infect HK-2 cells, real-time PCR and Western blot were used to determine the interference effect on TAK1 expression in the HK-2 cells with TGF-β1 stimulation. Under the condition of treating with p38 MAPK activator anisomycin, the levels of type I collagen and type Ⅲ collagen in the supernatant, and the protein levels of α-smooth muscle actin (α-SMA), connective tissue growth factor (CTGF) and p-p38MAPK^{Thr 180/Tyr 182} in the HK-2 cells with TAK1 knock-down were determined by ELISA and Western blot, respectively. RESULTS:TGF-β1 significantly increased the expression of TAK1 in the HK-2 cells(P<0.05). TAK1 shRNA significantly decreased the expression of TAK1 in the HK-2 cells with TGF-β1 stimulation. Type I collagen and type Ⅲ collagen secreted by the HK-2 cells after treatment with TGF-β1 were increased, the protein levels of α-SMA, CTGF and p-p38MAPK^{Thr 180/Tyr 182} were also increased(P<0.05). Knock-down of TAK1 expression significantly inhibited the secretion of type I and type Ⅲ collagen, reduced the protein levels of α-SMA, CTGF and p-p38MAPK^{Thr 180/Tyr 182} in the TGF-β1-induced HK-2 cells(P<0.05). Treatment with p38 MAPK activator reversed the inhibitory effect of TAK1 knock-down on the secretion of type I and type Ⅲ collagens, and the protein levels of α-SMA, CTGF and p-p38 MAPK^{Thr 180/Tyr 182} in the HK-2 cells(P<0.05). CONCLUSION:Knock-down of TAK1 expression attenuates the TGF-β1 induced fibrosis of renal tubular epithelial cells by inhibiting p38 MAPK signaling pathway.     

Effect of salvianolic acid B on high glucose-induced phenotypic transition and extracellular matrix secretion in human glomerular mesangial cells

AIM:To investigate the effect of salvianolic acid B (Sal B) on high glucose-induced phenotypic transition and extracellular matrix (ECM) secretion in human glomerular mesangial cells (HGMCs) and the underlying mechanisms. METHODS:HGMCs were randomly divided into control group, high glucose group and high glucose plus high dose, medium dose and low dose of Sal B groups. The HGMCs except those in control group were exposed to high glucose (33.3 mmol/L) for 72 h, while those in Sal B groups were co-incubated with indicated concentrations of Sal B. The protein levels of α-smooth muscle actin (α-SMA), transforming growth factor-β₁ (TGF-β₁) and phosphorylated Smad2 and p38 mitogen-activated protein kinase (MAPK) were determined by Western blot. The secretion levels of collagen type I (Col I), collagen type Ⅲ (Col Ⅲ), fibronectin (FN) and laminin (LN) were measured by ELISA. RESULTS:Exposure to high glucose markedly increased the protein expression of α-SMA, TGF-β₁, Col I, Col Ⅲ, FN and LN in the HGMCs (P<0.01). The phosphorylation levels of Smad2 and p38 MAPK were also significantly increased (P<0.01). Co-incubation with Sal B evidently decreased the protein expression of α-SMA, TGF-β₁, Col I, Col Ⅲ, FN and LN in the HGMCs induced by high glucose (P<0.05 or P<0.01). The phosphorylated levels of Smad2 and p38 MAPK were also reduced noticeably (P<0.05 or P<0.01). CONCLUSION:Sal B significantly suppresses high glucose-induced phenotypic transition and ECM secretion in the HGMCs, which might be attributed, at least partly, to inhibition of TGF-β₁/Smad signaling pathway and p38 MAPK activation.     

Effects of curcumin analogue L6H4 on myocardial tissue of type 2 diabetic rats

AIM: To explore the effects of curcumin analogue L6H4 on the myocardial tissue of type 2 diabetic rats and its mechanism. METHODS: Male Sprague-Dawley rats were randomly divided into normal control (NC) group, high-fat (HF) group, high-fat treatment (FT) group, diabetes mellitus (DM) group and diabetes treatment (DT) group.The rats in the latter 4 groups were fed high-fat diet for 4 weeks, then the rats in DM groups and DT groups were intraperitoneally injected with streptozotocin (STZ) to induce type 2 diabetes, while the rats in FT group and DT group were given L6H4. The blood glucose and lipid levels were detected by biochemical method, and serum adiponectin (APN) levels were detected by ELISA. The serum insulin levels were measured by radioimmunoassay and homeostasis model assessment of insulin resistance (HOMA-IR) were calculated. The morphological changes of myocardium were observed by Masson staining and electron microscopy. The protein expression of adiponectin receptor 1 (AdipoR1) and transforming growth factor β1(TGF-β1) in myocardial tissue were determined by immunohistochemistry. The protein expression of adipoR1 was also detected by Western blot for verification. RESULTS: Compared with NC group, the blood glucose, lipids, insulin, HOMA-IR and TGF-β1 were increased in HF and DM group, but they were decreased after treated with L6H4. Compared with NC group, the concentration of serum APN were decreased and the expression of AdipoR1 in the myocardium were weakened in HF group and DM group, and they increased after treated with L6H4. The myocardial fibrosis was obvious in HF group and DM group, the mitochondria in cardiomyocytes expanded, and the cristae disordered, partial disappeared. These lesions were significantly reduced after L6H4 treatment. CONCLUSION: L6H4 exerts a protective effect on the heart in type 2 diabetic rats. The increased concentration of serum APN, the enhanced expression of AdipoR1, and the expression of TGF-β1 inhibited by APN may be involved in the mechanism of protection.     

Expression of FBXW7 in diabetic cardiomyopathy

AIM:To investigate the expression of F-box/WD repeat-containing protein 7 (FBXW7) in the myocardium of type 1 diabetic rats and to clarify its role in the development of diabetic cardiomyopathy. METHODS:All rats were randomly divided into non-diabetic (ND) group, 4-week diabetes mellitus (DM) model group, 8-week DM mo-del group and 12-week DM model group. The DM model was prepared by intraperitoneal injection of streptozotocin (60 mg/kg). The change of myocardial pathological structure was investigated by HE staining. The FBXW7 expression level in the myocardium was determined by Western blot and immunohistochemistry methods. RESULTS:DM induced cardiomyocyte degeneration and necrosis as shown by cardiac histological analysis. Both Western blot and immunohistochemistry results showed that the expression level of FBXW7 was significantly increased in 4-week, 8-week and 12-week DM groups compared with control group (P<0.01). However, The FBXW7 expression level in 12-week DM group was decreased compared with 4-week and 8-week DM groups. CONCLUSION:With the development of diabetes, the expression of FBXW7 in the myocardium of the rats with diabetic cardiomyopathy shows a tendency to increase first and then decrease, suggesting that it plays some roles in the development of diabetic cardiomyopathy.     

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.     

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.     

Effects of TRPC1 on TGF-β1-induced epithelial-mesenchymal transition of human bronchial epithelial cells

AIM: To investigate the role of canonical transient receptor potential channel 1 (TRPC1) in the epithelial-mesenchymal transition (EMT) of human bronchial epithelial (HBE) cells induced by transforming growth factor-β1 (TGF-β1). METHODS: EMT of 16HBE cells induced by TGF-β1 were identified by microscopy, immunofluorescence and Western blotting. Immunofluorescence, real-time PCR and Western blotting were applied to detect the mRNA and the protein expression of TRPC1 in the 16HBE cells. The influence of SKF96365 (a TRPC1 blocker) and siRNA-mediated silencing of TRPC1 on the EMT of the 16HBE cells were detected by microscopy and Western blotting. RESULTS: Treatment with TGF-β1 induced significant morphological changes of the 16HBE cells. Exposure to TGF-β1 decreased the expression of E-cadherin protein (P<0.01) and increased the expression of α-SMA protein (P<0.05) in the 16HBE cells. Immunofluorescence observation indicated that TRPC1 expression in the 16HBE cells was positive. The expression of TRPC1 at mRNA and protein levels was significantly increased in the 16HBE cells after stimulation with TGF-β1 (P<0.05). The morphological changes of the 16HBE cells induced by TGF-β1 were inhibited by SKF96365 and TRPC1 silencing compared with TGF-β1 group. The protein expression of E-cadherin and α-SMA induced by TGF-β1 were inhibited by SKF96365 and TRPC1 silencing compared with TGF-β1 group (P<0.05). CONCLUSION: TGF-β1 induces EMT with the mechanism of up-regulating TRPC1 in human bronchial epithelial cells.     

Influence of paricalcitol on renal tubulointerstitial fibrosis in diabetic nephropathy

AIM: To investigate the effect of paricalcitol (P) on renal tubulointerstitial fibrosis and the underlying mechanisms in diabetic nephropathy (DN).METHODS: DN rat model was induced by a single intraperitoneal injection of streptozotocin after fasting. The animals were randomly divided into 2 groups:the DN rats in paricalcitol-intervened group (group P) were injected intraperitoneally with paricalcitol dissolved in propylene glycol after the day when the model was induced successfully at a dose of 0.4 μg/kg (3 times a week); the DN rats in DN group (group D) were given isopyknic propylene glycol. Normal control group (group C) was also set up. The samples of blood, urine and renal tissue were collected after intervention of paricalcitol for 12 weeks. The biochemical indexes were measured. The renal tissues were used for pathologic observation and determining the expression of transforming growth factor-β1 (TGF-β1), Wnt-4, β-catenin and Klotho by immunohistochemistry and Western blotting. In addition, the correlation among the above indexes was analyzed.RESULTS: (1) Scr, BUN and 24 h urine protein increased significantly in group D compared with group C, while decreased in group P compared with group D (P<0.05). (2) The area of renal tubulointerstitial fibrosis increased in group D compared with group C, while decreased in group P compared with group D (P<0.05). (3) The expression of Klotho decreased, while the expression of TGF-β1, Wnt-4 and β-catenin increased in group D compared with group C (P<0.05). Compared with group D, the expression of Klotho increased, while the expression of TGF-β1, Wnt-4 and β-catenin decreased in group P (P<0.05). (4) The expression of Klotho was negatively correlated with the fibrosis area, TGF-β1, Wnt-4 and β-catenin (P<0.05).CONCLUSION: Paricalcitol inhibits renal tubulointerstitial fibrosis in DN by promoting the expression of renal Klotho, and inhibiting Wnt/β-catenin signaling pathway activation and TGF-β1 synthesis.     

Pycnogenol suppresses TGF-β1-induced hepatic stellate cell activation via ERK-mediated autophagy inhibition

AIM: To explore the effect of Pycnogenol on transforming growth factor-β1 (TGF-β1)-induced hepatic stellate cell activation. METHODS: Cultured LX-2 cells were treated with 5 μg/L TGF-β1 and different concentrations (0, 10, 25 and 50 mg/L) of Pycnogenol. The viability of the LX-2 cells under the conditions with or without autophagy inhibitor 3-MA and ERK inhibitor PD98059 was determined by MTT assay. The protein levels of α-SMA, ColⅠ, TIMP-1, LC3-Ⅱ/Ⅰ, beclin 1, p-ERK1/2 and ERK1/2 were detected by Western blot. RESULTS: Compared with control group, 5 μg/L TGF-β1 treatment elevated the cell viability, and increased the protein levels of α-SMA, ColⅠ, TIMP-1, LC3-Ⅱ/Ⅰ, beclin 1, p-ERK1/2, and ERK1/2 in the LX-2 cells (P<0.05). However, these effects were reversed by Pycnogenol pretreatment in a dose-dependent manner and the inhibitory effect of 50 mg/L Pycnogenol was the most significant in the LX-2 cells (P<0.05). Furthermore, compared with TGF-β1 group, pretreatment with 50 mg/L Pycnogenol, 5 mmol/L 3-MA or 20 μmol/L PD98059 downregulated TGF-β1-induced cell viability and the protein levels of α-SMA and LC3-Ⅱ/Ⅰ in the LX-2 cells (P<0.05). CONCLUSION: Pycnogenol suppresses TGF-β1-induced hepatic stellate cell activation via p-ERK and autophagy inhibition.     

Expression of OPN and macrophage colony stimulating factor in diabetic rats and intervention of mycophenolate mofetil

AIM: To explore the effect of mycophenolate mofetil (MMF) on expression of osteopotin (OPN) and macrophage colony stimulating factor (M-CSF) in diabetic rats with renal tubulo-interstitial injury. METHODS: Diabetes was induced in uninephrectomized male Wistar rats by peritoneal injection of streptozotocin (65 mg/kg). The rats were randomly divided into three groups: control group (NC), diabetic group (DM) and MMF treated group ［DM+MMF, treatment of MMF (15 mg·kg^-1·d^-1) by gavage from the next day of the induction for 8 weeks］. Serum biochemistry, 24 h urinary protein and the ratio of left kidney weight/body weight were determined after 8 weeks. The renal tubulo-interstitial morphological change was observed, immunohistochemical method was used to analyze the expression of OPN, M-CSF and CD68. The mRNA of OPN in renal tissue was amplified by quantitative real-time PCR. RESULTS: Compared with control group, serum glucose level, 24 h urinary protein and the ratio of left kidney weight/body weight were significantly increased (P<0.01), and the relative area of interstitial fibrosis was also significantly enlarged in DM group (P<0.01).Compared with NC group, the expressions of OPN, M-CSF, CD68 protein and OPN mRNA were significantly upregulated in DM group (P<0.01). After intervention with MMF, the upregulations of the above-mentioned parameters, except blood glucose and serum creatinine, were all significantly inhibited (P<0.05 or P<0.01). CONCLUSION: The expressions of OPN, M-CSF and CD68 in renal tubulointerstitial decrease in diabetic rats treated with MMF. MMF also inhibits the level of OPN mRNA, reduces proteinuria and prevents renal injury. MMF plays an apparently protective role in renal tubulointerstitial injury, probably associated with inhibiting chemokine and proliferation on macrophages.     

Protectin D1 inhibits renal fibrosis in early-stage diabetic nephropathy mice by inhibiting renal inflammation and podocyte epithelial-mesenchymal transition

AIM:To study the effect of protectin D1 (PD1) as a potent anti-inflammatory lipid mediator on diabetic nephropathy (DN). METHODS:PD1 (0.08 mg·kg-1·d-1) was intraperitoneally injected into the mice for 8 weeks after diabetes was induced by injection of streptozotocin. The 24-h urinary protein and albumin levels, body weight, renal weight, kidney-to-body weight ratio, creatinine clearance, glomerular mesangial matrix accumulation, renal cortical macrophage accumulation, and glomerular expression of fibronectin (FN), α-smooth muscle actin (α-SMA), zonula occludens-1 (ZO-1) and P-cadherin were detected. Thfe effect of PD1 on inhibiting epithelial-mesenchymal transition (EMT) in the podocytes induced by transforming growth factor β1 (TGF-β1), and the effect of PD1 on inhibiting the inflammatory effect of macrophages induced by high glucose were determined. RESULTS:PD1 markedly suppressed diabetes-induced elevation of 24-h urinary protein and albumin levels, body weight, renal weight, kidney-to-body weight ratio, creatinine clearance, glomerular mesangial matrix accumulation, and glomerular expression of FN and α-SMA. PD1 also suppressed diabetes-induced increase in the number of renal cortical macrophages in the mice with DN. Analysis by Western blotting and immunohistochemistry revealed that PD1 suppressed diabetes-induced elevation of mesenchymal/fibrotic markers FN and α-SMA, and increased podocyte-related epithelial markers ZO-1 and P-cadherin in the glomeruli of the mice with DN. PD1 repressed high glucose-induced generation of tumor necrosis factor α and interleukin 1β by macrophages, and inhibited TGF-β1-induced increases in fibroblast-specific protein 1 and α-SMA, and inhibited TGF-β1-induced decreases in epithelial markers P-cadherin and ZO-1 in podocytes in vitro. CONCLUSION: PD1 inhibits renal fibrosis in the early stage of DN, and its mechanisms may be related to its anti-inflammatory and anti-EMT effects on podocytes.