Role of caveolae in high glucose-induced extracellular matrix production in rat mesangial cells

AIM：To investigate the role of caveolae in high glucose (HG)-induced extracellular matrix (ECM) production in rat mesangial cells (MCs). METHODS：Synchronized rat MCs were divided into normal glucose group, HG group, HG+methyl-β-cyclodextrin (β-MCD) group and HG+β-MCD+cholesterol (Chol) group. Western blotting was used to detect the protein expression of caveolin-1 (Cav-1), phosphorylated caveolin-1 (p-Cav-1-Y14) and collagen type 1 (Col I). The mRNA expression of Cav-1 was determined by real-time PCR. ELISA was used to measure the level of fibronectin (FN) in the supernatant. RESULTS：High glucose significantly increased the expression of FN and Col I. In HG 12, 24 and 48 h groups, the mRNA and protein levels of Cav-1 were not significantly different from those in HG 0 h group, whereas the level of p-Cav-1-Y14 was significantly increased. β-MCD significantly attenuated HG-induced elevation of p-Cav-1-Y14 and FN production, but had no effect on HG-induced Col I expression. All these responses to β-MCD were abolished by Chol. CONCLUSION：High glucose significantly increases the production of Col I and FN in rat MCs. FN production induced by high glucose is mediated by p-Cav-1-Y14.     

Activation of smad signaling and collagenⅠsynthesis in NRK52E cells induced by advanced glycation end products

AIM:To investigate the effects of advanced glycation end products on activation of Smad signaling pathway and collagenⅠ synthesis in proximal tubular epithelial cells. METHODS:Advanced glycation end products (AGE-BSA) were prepared by incubation of bovine serum albumin (BSA) with D-glucose. Normal rat proximal tubular epithelial (NRK52E) cells were cultured in RPMI-1640 medium with AGE-BSA. Phosphorylation and nuclear translocation of Smad2/3 were examined by immunocytochemistry. Levels of TGF-β1 in supernatant of cell culture were measured by enzyme-linked immunosorbent assay (ELISA). Expression of TGF-β1, Smad2, Smad3 and Smad7 mRNA were detected by RT-PCR. Expression of α-SMA , E-cadherin and collagenⅠproteins were detected by Western blotting.RESULTS:AGE-BSA induced Smad2/3 phosphorylation and nuclear translocation, two peaks occured at 30 min (68% vs 16%, P<0.05) and 24 h (76% vs 16%, P<0.05) compared to 0 min. The level of TGF-β1 markedly increased in supernatant of cell culture by induced AGE-BSA at 24 h and 48 h. The expression of TGF-β1 mRNA markedly increased at 24 h, and associated with high expression of Smad2, Smad3 and Smad7 mRNA at 48 h. AGE-BSA up-regulated significantly the expression of α-SMA and collagenⅠproteins, down-regulated the expression of E-cadherin protein. CONCLUSION:AGEs induces activation of Smad signaling, as well as transdifferentiation and collagenⅠ synthesis in proximal tubular epithelial cells.     

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.     

Effect of sitagliptin on autopaghy in mesangial cells induced by AGEs

AIM: To investigate the effect of sitagliptin on the autopaghy and the expression of extracellular matrix in mesangial cells induced by advanced glycation end products (AGEs). METHODS: The cells were divided into 5 groups:control group, AGE group, and sitagliptin (5, 10 and 20 μmol/L) groups. After 48 h, the cell viability was measured by MTT assay, and the content of collagen (Col) Ⅳ in the supernatant of the cell culture was detected by ELISA. The protein levels of beclin-1, adenosine monophosphate-activated protein kinase (AMPK), p-AMPK, p70S6K and p-p70S6K were determined by Western blot. RESULTS: Compared with control group, the viability and the expression of Col IV induced by AGEs in the cultured mesangial cells were significantly increased (P<0.01). Sitagliptin decreased the viability and the expression of Col IV induced by AGEs in the mesangial cells in a dose-dependent manner. AGEs significantly inhibited the protein levels of beclin-1 and p-AMPK, but significantly increased the protein level of p-p70S6K. Compared with AGE group, sitagliptin significantly reversed the above results in a dose-dependent manner. CONCLUSION: Autophagy may mediate the protective effect of sitagliptin on mesangial cells induced by AGEs.     

Effect of advanced glycosylation end products on function of human adipose tissue-derived stem cells in vitro

AIM：To explore the effect of advanced glycosylation end products (AGEs) on the function of human adipose-derived stem cells (hADSCs) in promoting wound healing. METHODS：hADSCs were isolated by conventional method in vitro and divided into control bovine serum albumin (BSA) group, low-dose AGE-BSA group and high-dose AGE-BSA group. The proliferation and migration of hADSCs with different treatments were determined by WST-8 assay and Transwell assay, respectively. In addition, the expression of vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), and insulin-like growth factor-1(IGF-1) at mRNA and protein levels was determined by real-time PCR and ELISA analysis. RESULTS：Compared with control group, the proliferation and migration abilities were significantly inhibited in the hADSCs of AGE-BSA group. The mRNA expression of VEGF, HGF and IGF-1 in AGE-BSA group was obviously lower than that in control group. The contents of VEGF, HGF and IGF-1 in hADSCs-conditioned me-dium in AGE-BSA group were also obviously lower than those in control group. CONCLUSION：AGEs alter the intrinsic properties of hADSCs and impair their functions in promoting wound healing, thus affecting the therapeutic potential of hADSCs in the treatment of diabetic ulcers.     

Expression of SGK isoforms in human proximal tubular epithelial cells under the condition of high glucose concentration

AIM: To investigate the effect of high glucose concentration on serum and glucocorticoid induced protein kinase (SGK) mRNA and protein expressions in human proximal tubular epithelial cells (HKC) and the possible role of SGK in the production of extracellular matrix (ECM) of HKC under the condition of high glucose. METHODS: HKC was divided into 3 groups: control glucose group (CG group, 5.5 mmol/L D-glucose); high glucose group (HG group, 25 mmol/L D-glucose) and osmotic control group (MG group, 19.5 mmol/L mannitol and 5.5 mmol/L D-glucose). The expressions of SGK mRNA and protein were assessed by semi-quantitative RT-PCR and Western blotting respectively. The level of secretary and cytoplasmic fibronectin (FN) were detected by enzyme-linked immunoabsordent assay (ELISA) and indirect-immunofluorescence. RESULTS: HKC expressed SGK1, SGK2 and SGK3 at mRNA and protein levels. Their mRNA level were up-regulated since 2 hours after cells exposed to D-glucose and this up-regulation persisted to the end of 8th hour, and SGK1 protein level elevated simultaneously. On the other hand, the increased FN secretion by high glucose was in a time-dependent manner and its improved secretion threshold was just followed by the high expression of SGK1. CONCLUSIONS: In response to high glucose, the expression of SGK1, SGK2 and SGK3 in human proximal tubular epithelial cells were up-regulated which was accompanied with FN accumulation. The high expression of SGK may mediate overproduction of ECM in proximal tubular epithelial cells and contribute to the diabetic nephropathy.     

Effect of probucol and simvastatin on reactive oxygen species production and HO-1 expression induced by advanced glycation end products in rat renal microvascular endothelial cells

AIM: To investigate the effect of probucol and simvastatin on the production of reactive oxygen species (ROS) and heme oxygenase-1 (HO-1) expression induced by advanced glycation end products (AGEs) in rat renal microvascular endothelial cells (RMECs). METHODS: RMECs isolated and cultured from rat kidney were divided into 4 groups: normal control group, AGEs group, probucol group and simvastatin group. The levels of ROS were determined by the molecular probes of DCFH-DA. The expression of HO-1 at mRNA and protein levels was detected by RT-PCR and Western blotting, respectively. RESULTS: (1) AGEs up-regulated ROS production and HO-1 expression in RMECs. (2) Probucol up-regulated HO-1 expression in RMECs, and inhibited the increasing level of ROS and expression of HO-1 in RMECs induced by AGEs. (3) Simvastatin also inhibited the increasing level of ROS in RMECs induced by AGEs, but it had no effect on HO-1 expression in RMECs with or without AGEs.CONCLUSION: Protective effect of probucol on the dysfunction of RMECs induced by AGEs may be related with its effect on the expression of HO-1 at mRNA and protein levels. Simvastatin also plays roles in antioxidation and renal protection, but is ineffective in the modulation of HO-1 expression.     

Effect of berberine on invasion and migration of PG cells from a high metastatic human giant lung carcinoma cell line

AIM:To study the effect of berberine(Ber) on invasion and migration of PG cells from a high metastatic human giant lung carcinoma cell line and to explore its mechanism. METHODS:Agarose drop method was used to detect PG migration; transwell cabin with FN in lower chamber was adopted to detect PG chemotaxis. PG adhesion to FN and martrigel was detected by MTT; PG invasive ability was determined by transwell cabin covered with martrigel. Expression of MMP2/TIMP2 protein and mRNA were detected by quantitative immunocytochemical method and RT-PCR respectively. RESULTS:After PG was treated by Ber（10 mg/L） for 24 h: 1) migration distance of Ber-treated PG cells was markedly shorter than that of control cells (P<0.01) and the number of passed membrane cells towards FN was much fewer than that of control cells (P<0.01); 2) PG adhesion to FN and martrigel was inhibited remarkably by Ber compared with control PG; 3) the migration of PG cells through the martrigel-coated transwell was significantly inhibited by the addition of Ber; 4) MMP2 expression was reduced significantly(P<0.01), while the TIMP2 expression showed up-regulating tendency, but had no differences compared with control group(P>0.05). The MMP2/TIMP2 ratio was decreased; 5) the MMP2 mRNA/TIMP2 mRNA ratio was decreased by Ber.CONCLUSION:Inhibition of cell migration, adhesion to ECM and invasion into ECM of tumor cells and regulation of homeostasis between MMPs and TIMPs to maintain ECM integrity may be the basic mechanism of inhibitive effect of Ber on invasion and metastasis of tumors.     

Effect of insulin on the SGK1 expression and extracellular matrix synthesis in human mesangial cells cultured in high glucose

AIM:To study the effect of insulin on the serum and glucocorticoid-inducible kinase 1 (SGK1) expression and extracellular matrix synthesis in human glomerular mesangial cells (HMC) cultured in high glucose. METHODS:The HMCs were cultured in the presence of 5.5 or 25 mmol/L glucose with or without 100 nmol/L insulin (i.e. NG, HG, NI and HI groups). 4 h latter, expressions of SGK1, insulin receptor substrate-1 (IRS1) and IRS2 in corresponding groups were detected by immunofluorescence or examined by Western blotting. The phosphorylation of IRS1 and IRS2 was measured by immunoprecipitation. 24 h latter, connective tissue growth factor(CTGF) and fibronectin (FN) were also examined by RT-PCR and ELISA, respectively. RESULTS:Compared with NG, the SGK1 protein expression in HG, NI and HI groups was significantly higher (P<0.01). High glucose mainly caused IRS2 protein and its phosphorylation level increase (P<0.01). When treated with 100 nmol/L insulin, IRS1 protein and its phosphorylation in HI group apparently elevated while slight inhibition of IRS2 protein expression and its phosphorylation were observed (HI vs HG, P<0.05). High glucose enhanced the expression of CTGF and FN, and insulin strengthened this effect.CONCLUSION:Insulin and high glucose up-regulate the expression of SGK1 in mesangial cells through different target molecular pathways and ultimately enhance ECM synthesis. The effect of insulin is highly associated with IRS1 signaling cascades.     

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.     

Inhibition of plasminogen activator inhibitor-1 by small interfering RNA attenuates pulmonary fibrosis in rats

AIM: To examine the effects of silencing of plasminogen activator inhibitor-1 (PAI-1) expression by small interfering RNA (siRNA) on bleomycin (BLM)-induced rat pulmonary fibrosis. METHODS: Total 72 Wistar rats were divided into 4 groups: control, BLM, BLM+non-specific siRNA (BLM+N), and BLM+ PAI-1 siRNA (BLM+P). Pulmonary fibrosis was induced by intratracheal injection of BLM (5 mg/kg), whereas equal volume of normal saline was used in control group. After the administration of BLM or normal saline, the rats were treated with tracheal injection of PAI-1-siRNA (7.5 nmol/0.2 mL per rat) in BLM+P group, non-specific siRNA (7.5 nmol/0.2 mL per rat) in BLM+N group, and 0.2 mL normal saline in BLM group and control group, twice a week, 8 times in 28 d. On day 7, 14, and 28, the rats (n=6 at each time point) were sacrificed. The bronchoalveolar lavage fluid (BALF) from the left lung was harvested to examine the activity of PAI-1. The mRNA expression of collagen type Ⅲ, α-smooth muscle actin (α-SMA) and tissue inhibitor of metalloproteinase-1 (TIMP-1) in the middle lobe of the right lung was detected by RT-PCR. RESULTS: PAI-1 activity and the expression of collagen type Ⅲ, α-SMA and TIMP-1 were increased in BLM group on day 7, 14 and 28. Intratracheal injection of PAI-1 siRNA twice a week continuously reduced PAI-1 activity in the BALF (P<0.05),and decreased the expression of collagen type Ⅲ, α-SMA and TIMP-1 in the fibrotic lung tissues on day 7, 14 and 28. Statistical differences in the expression of collagen type Ⅲ, α-SMA and TIMP-1 between BLM+P group and BLM group at the same time point were observed. CONCLUSION: Intratracheal injection of PAI-1 siRNA twice a week continuously inhibits the expression of PAI-1. PAI-1 siRNA ameliorates BLM-induced pulmonary fibrosis by down-regulation of TIMP-1 expression.     

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.     

Chronic metabolic acidosis markedly induces proliferation of mesangial cells in rats

AIM: To study the effects of chronic metabolic acidosis on glomerulus, mesangial cells and the production of extracellular matrix. METHODS: Chronic metabolic acidosis was induced by addition of 0.28 mol/L NH4Cl to drinking water for 3, 7, or 14 days in male Wistar rats (n=10). Light microscope combined with computer software (Motic Images Advanced 3.2) was used to determine the effect of chronic acid loading on renal morphologic changes. The expressions of proliferation cell nuclear antigen (PCNA) and p27 in glomeruli were detected by Western blotting or immunohistochemistry. Fibronectin (FN) mRNA was detected by real-time PCR. The proliferation of mesangial cells in vitro was determined by ［3H］-TdR incorporation. The concentration of FN in cultured supernatant was detected by ELISA. RESULTS: On day 1, 3, 7 and 14, the arterial pH and plasma ［HCO3-］ in experimental rats were significantly decreased. There was a significantly increased in the kidney weight and the ratio of kidney to body weigh in experimental rats on day 3, 7 and 14. The glomerular area and cell numbers also increased significantly. Immunoblotting demonstrated decreased p27 expression and increased PCNA expression in isolated glomeruli, and the expression of PCNA increased in a time-dependent manner following the time of chronic metabolic acidosis. Immunohistochemistry showed increased positive PCNA expression mainly localized to mesangial cells. The expression of FN mRNA was significantly elevated in experimental rats on day 7 and 14. In vitro, acid loading induced mesangial cell proliferation and synthesis of FN. CONCLUSION: These results suggest that chronic metabolic acidosis induces mesangial cell proliferation, and its mechanism may be associated with the downregulation of cell cycle kinase inhibitor p27.     

Autophagy plays a protective role in advanced glycation end product-induced apoptosis of vascular endothelial cells

AIM: To investigate the effect of advanced glycation end products (AGEs) on autophagy in human umbilical endothelial cells (HUVECs) and to identify the role of autophagy in advanced glycation end product-induced cell apoptosis. METHODS: HUVECs were cultured and treated with AGEs or bovine serum albumin. The protein expression was detected by Western blotting. Autophagosomes were observed under electron microscope. The cell apoptotic rate was determined by flow cytometry. The cell viability was quantified by MTT assay. RESULTS: After treated with AGEs, the level of autophagy-associated protein LC3-Ⅱ in HUVECs was up-regulated, and the number of autophagosomes was increased. Compared with control group, the apoptotic rate of HUVECs increased and the viability of HUVECs was decreased in AGEs treatment group. Furthermore, pretreating the cells with an autophagy inhibitor 3-methyladenine aggravated these effects. The levels of phospho-protein kinase B(Akt) and phospho-mammalian target of rapamycin(mTOR) in HUVECs were also decreased by treatment with AGEs. Pretreatment with Akt activator insulin-like growth factor 1 (IGF-1) increased Akt phosphorylation and suppressed the AGE-induced LC3-Ⅱ expression. CONCLUSION: AGEs induce autophagy in HUVECs through PI3K/Akt/mTOR signal pathway. Autophagy plays a protective role in AGE-induced apoptosis in HUVECs.     

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.     

The role of adrenomedullin in diabetic nephropathy

AIM: To investigate the role of adrenomedullin (AM) in diabetic nephropathy. METHODS: We observed the changes in the expression and secretion of AM, TGF-β₁ in the cultured human mesangial cells under high glucose condition and the contents of the laminin and type IV collagen in the supernatants. The effect of intervention with AM was also observed. RESULTS: High glucose condition resulted in increase in the expression and secretion of AM、 TGF-β₁、 laminin and type IV collagen. AM reversed the influence of high glucose on the cultured human mesangial cells. CONCLUSION: These results showed that high glucose condition is one of stimulating factors of AM and the renal protective action of AM may be associated with suppression of TGF-β₁ and reducing excessive accumulat ion of laminin and type IV collagen.