Gene expression of metalloproteinase and its inhibitor in mesangial cells exposed to high glucose.

To clarify the roles of metalloproteinases and their inhibitor (TIMP) in diabetic glomerulopathy, we studied the effect of a high glucose concentration on the gene expression of metalloproteinase transin and TIMP as well as collagen type IV and laminin in cultured rat mesangial cells (MCs). In the high glucose group, collagen type IV, laminin, and TIMP mRNA levels were all elevated in a concentration-dependent manner, whereas transin expression was suppressed. Osmotic control of high glucose with mannitol selectively stimulated TIMP expression. We hypothesize that high glucose decreases matrix-degrading activity as well as increases matrix productivity in MCs.     

Sphingosine kinase-1 pathway mediates high glucose-induced fibronectin expression in glomerular mesangial cells

Diabetic nephropathy is characterized by accumulation of glomerular extracellular matrix proteins, such as fibronectin (FN). Here, we investigated whether sphingosine kinase (SphK)1 pathway is responsible for the elevated FN expression in diabetic nephropathy. The SphK1 pathway and FN expression were examined in streptozotocin-induced diabetic rat kidney and glomerular mesangial cells (GMC) exposed to high glucose (HG). FN up-regulation was concomitant with activation of the SphK1 pathway as reflected in an increase in the expression and activity of SphK1 and sphingosine 1-phosphate (S1P) production in both diabetic kidney and HG-treated GMC. Overexpression of wild-type SphK1 (SphK(WT)) significantly induced FN expression, whereas treatment with a SphK inhibitor, N,N-dimethylsphingosine, or transfection of SphK1 small interference RNA or dominant-negative SphK1 (SphK(G82D)) abolished HG-induced FN expression. Furthermore, addition of exogenous S1P significantly induced FN expression in GMC with an induction of activator protein 1 (AP-1) activity. Inhibition of AP-1 activity by curcumin attenuated the S1P-induced FN expression. Finally, by inhibiting SphK1 activity, both N,N-dimethylsphingosine and SphK(G82D) markedly attenuated the HG-induced AP-1 activity. Taken together, these results demonstrated that the SphK1 pathway plays a critical role in matrix accumulation in GMC under diabetic condition, suggesting that the SphK1 pathway could be a potential therapeutic target for diabetic nephropathy.     

HMGB1 regulates ferroptosis through Nrf2 pathway in mesangial cells in response to high glucose

Ferroptosis, a novel type of programmed cell death, is involved in inflammation and oxidation of various human diseases, including diabetic kidney disease. The present study explored the role of high-mobility group box-1 (HMGB1) on the regulation of ferroptosis in mesangial cells in response to high glucose. Compared with healthy control, levels of serum ferritin, lactate dehydrogenase (LDH), reactive oxygen species (ROS), malonaldehyde (MDA), and HMGB1 were significantly elevated in diabetic nephropathy (DN) patients, accompanied with deregulated ferroptosis-related molecules, including long-chain acyl-CoA synthetase 4 (ACSL4), prostaglandin-endoperoxide synthase 2 (PTGS2), NADPH oxidase 1 (NOX1), and glutathione peroxidase 4 (GPX4). In vitro assay revealed that erastin and high glucose both induced ferroptosis in mesangial cells. Suppression of HMGB1 restored cellular proliferation, prevented ROS and LDH generation, decreased ACSL4, PTGS2, and NOX1, and increased GPX4 levels in mesangial cells. Furthermore, nuclear factor E2-related factor 2 (Nrf2) was decreased in DN patients and high glucose-mediated translocation of HMGB1 in mesangial cells. Knockdown of HMGB1 suppressed high glucose-induced activation of TLR4/NF-κB axis and promoted Nrf2 expression as well as its downstream targets including HO-1, NQO-1, GCLC, and GCLM. Collectively, these findings suggest that HMGB1 regulates glucose-induced ferroptosis via Nrf2 pathway in mesangial cells.     

Hyperosmolarity induced by high glucose promotes senescence in human glomerular mesangial cells

Hyperglycemia is involved in the diabetic complication of different organs and can elevate serum osmolarity. Here, we tested whether hyperosmolarity promoted by high glucose levels induces cellular senescence in renal cells. We treated Wistar rats with streptozotocin to induce diabetes or with consecutive daily injections of mannitol to increase serum osmolarity and analyzed p53 and p16 genes in renal cortex by immunohistochemistry. Both diabetic and mannitol treated rats showed a significant increase in serum osmolarity, without significant signs of renal dysfunction, but associated with increased staining for p53 and p16 in the renal cortex. An increase in p53 and p16 expression was also found in renal cortex slices and glomeruli isolated from healthy rats, which were later treated with 30 mM glucose or mannitol. Intracellular mechanisms involved were analyzed in cultured human glomerular mesangial cells treated with 30 mM glucose or mannitol. After treatments, cells showed increased p53, p21 and p16 expression and elevated senescence-associated β-galactosidase activity. Senescence was prevented when myo-inositol was added before treatment. High glucose or mannitol induced constitutive activation of Ras and ERK pathways which, in turn, were activated by oxidative stress. In summary, hyperosmolarity induced renal senescence, particularly in glomerular mesangial cells, increasing oxidative stress, which constitutively activated Ras-ERK 1/2 pathway. Cellular senescence could contribute to the organ dysfunction associated with diabetes.     

RhoA/rho kinase signaling reduces connexin43 expression in high glucose-treated glomerular mesangial cells with zonula occludens-1 involvement

RhoA/Rho kinase (ROCK) signaling has been suggested to be involved in diabetic nephropathy (DN) pathogenesis. Altered expression of connexin43 (Cx43) has been found in kidneys of diabetic animals. Both of them have been found to regulate nuclear factor kappa-B (NF-κB) activation in high glucose-treated glomerular mesangial cells (GMCs). The aim of this study was to investigate the relationship between RhoA/ROCK signaling and Cx43 in the DN pathogenesis. We found that upregulation of Cx43 expression inhibited NF-κB p65 nuclear translocation induced by RhoA/ROCK signaling in GMCs. Inhibition of RhoA/ROCK signaling attenuated the high glucose-induced decrease in Cx43. F-actin accumulation and an enhanced interaction between zonula occludens-1 (ZO-1) and Cx43 were observed in high glucose-treated GMCs. ZO-1 depletion or disruption of F-actin formation also inhibited the reduction in Cx43 protein levels induced by high glucose. In conclusion, activated RhoA/ROCK signaling induces Cx43 degradation in GMCs cultured in high glucose, depending on F-actin regulation. Increased F-actin induced by RhoA/ROCK signaling promotes the association between ZO-1 and Cx43, which possibly triggered Cx43 endocytosis, a mechanism of NF-κB activation in high glucose-treated GMCs.     

Inhibition kinetics and regulation of sphingosine kinase 1 expression in prostate cancer cells: functional differences between sphingosine kinase 1a and 1b

Sphingosine kinase 1 catalyses the formation of the bioactive lipid, sphingosine 1-phosphate and is a target for anti-cancer agents. We demonstrate here that 2-(p-hydroxyanilino)-4-(p-chlorophenyl)thiazole (SKi, also referred to as SKI-II), FTY720 (Fingolimod), and (S)-FTY720 vinylphosphonate inhibit sphingosine kinase 1 activity with distinct kinetics, indicating that these compounds exhibit different binding modalities with sphingosine kinase 1. Thus, SKi is a mixed inhibitor of sphingosine and ATP binding, whereas FTY720 is competitive with sphingosine and uncompetitive with ATP, and (S)-FTY720 vinylphosphonate is uncompetitive with sphingosine and is a mixed inhibitor with respect to ATP. A novel 'see-saw' model is proposed for the binding of inhibitor to catalytic and allosteric sites, the latter dependent on substrate binding, that provides an explanation for the different inhibitor kinetics. In addition, we demonstrate that the expression level and properties unique to an N-terminal 86 amino-acid isoform variant of sphingosine kinase 1 (SK1b) in prostate cancer cells reduce its sensitivity to SKi-induced proteasomal degradation in comparison to SK1a, i.e. these two N-terminal variants of sphingosine kinase 1 (SK1a and SK1b) have different properties. The reduced sensitivity of SK1b to proteasomal degradation in response to SKi is translated into specific changes in ceramide and S1P levels that leads to apoptosis of androgen-sensitive but not androgen-independent LNCaP prostate cancer cells. Therefore, our proposed 'see-saw' model might be usefully employed in the design of sphingosine kinase inhibitors to promote apoptosis of chemotherapeutic resistant cancer cells.     

Berberine reduces fibronectin and collagen accumulation in rat glomerular mesangial cells cultured under high glucose condition

Diabetic nephropathy, one of the microvascular complications of diabetes mellitus, is a leading cause of end-stage renal disease. Berberine is one of the main constituents of Coptidis Rhizoma and Cortex Phellodendri. In this study, we investigated the effects of berberine on fibronectin and collagen production, and explored the role of p38MAPK signaling pathway in rat glomerular mesangial cells cultured under high glucose condition. Six groups were divided according to the different experimental conditions: (1) Normal glucose group (NG); (2) Mannitol group (Mannitol); (3) High glucose group (HG); (4) SB203580 treatment group (HG + SB203580); (5) Berberine low dosage group (HG + BBR 30 μM); (6) Berberine high dosage group (HG + BBR 90 μM). Cell proliferation and collagen synthesis were measured by MTT and ³H-proline incorporation assay, respectively. The phospho-p38MAPK, phospho-cAMP response element binding protein (CREB) and fibronectin were detected by western blot analysis. Fibronectin protein expression and collagen synthesis were significantly increased in HG-treated group compared with normal glucose group (P < 0.05). In SB203580 treatment group and two groups of berberine, protein expression of fibronectin and collagen synthesis were obviously decreased compared with HG-treated group (P < 0.05). Berberine significantly decreased protein expression of fibronectin compared with SB203580 treatment group (P < 0.05). Berberine at high dosage significantly decreased collagen synthesis compared with SB203580 treatment group (P < 0.05). Both SB203580 and berberine significantly decreased phospho-p38MAPK and phospho-CREB level compared with HG-treated group (P < 0.05). These results indicated that berberine might inhibit fibronectin and collagen synthesis partly via p38MAPK signal pathway in rat glomerular mesangial cells exposed to high glucose.     

Sphingosine kinase 1 and 2 regulate the capacity of mesangial cells to resist apoptotic stimuli in an opposing manner

Abstract Sphingosine kinases (SKs) are key enzymes regulating the production of sphingosine-1-phosphate (S1P), which determines important cell responses including cell growth and death. Here we show that renal mesangial cells isolated from wild-type, SK-1(-/-), and SK-2(-/-) mice show a differential response to apoptotic stimuli. Wild-type mesangial cells responded to staurosporine with increased DNA fragmentation and caspase-3 processing, which was enhanced in SK-1(-/-) cells. In contrast, SK-2(-/-) cells were highly resistant to staurosporine-induced apoptosis. Furthermore, the basal phosphorylation and activity of the anti-apoptotic protein kinase B (PKB) and of its substrate Bad were decreased in SK-1(-/-) but not in SK-2(-/-) cells. Upon staurosporine treatment, phosphorylation of PKB and Bad decreased in wild-type and SK-1(-/-) cells, but remained high in SK-2(-/-) cells. In addition, the anti-apoptotic Bcl-X(L) was significantly upregulated in SK-2(-/-) cells, which may further contribute to the protective state of these cells. In summary, our data show that SK-1 and SK-2 have opposite effects on the capacity of mesangial cells to resist apoptotic stimuli. This is due to differential modulation of the PKB/Bad pathway and of Bcl-X(L) expression. Thus, subtype-selective targeting of SKs will be critical when considering these enzymes as therapeutic targets for the treatment of inflammation or cancer.     

高糖对原代培养人肾小球系膜细胞表达MMP-9、TIMP-1的影响

目的了解高糖对原代培养的人肾小球系膜细胞表达MMP-9、TIMP-1和MMP-9/TIMP-1的影响,进一步探讨糖尿病肾病的发病机制。方法取自愿水囊引产的胎儿肾,解剖取肾皮质剪碎,应用肾皮质组织块法合优生选择法对人肾小球系膜细胞进行原代培养。ELISA方法检测MMP-9、TIMP-1的表达变化。结果与正常组相比较,高糖组MMP-9在24h、48h、72h均显著降低（P〈0.01）,TIMP-1在24h、72h可显著升高（P〈0.05）,48h表达降低;MMP-9/TIMP-1的比值在24h、48h、72h均显著降低（P〈0.01）。结论高糖能够降低MMP-9/TIMP-1,与肾小球系膜细胞细胞外基质降解能力降低密切相关。     

Plasmin reduces fibronectin deposition by mesangial cells in a protease-activated receptor-1 independent manner

BackgroundProtease-activated receptor-1 (PAR-1) potentiates diabetic nephropathy (DN) as evident from reduced kidney injury in diabetic PAR-1 deficient mice. Although thrombin is the prototypical PAR-1 agonist, anticoagulant treatment does not limit DN in experimental animal models suggesting that thrombin is not the endogenous PAR-1 agonist driving DN.ObjectivesTo identify the endogenous PAR-1 agonist potentiating diabetes-induced nephropathy.MethodsUnbiased protease expression profiling in glomeruli from human kidneys with DN was performed using publically available microarray data. The identified prime candidate PAR-1 agonist was subsequently analysed for PAR-1-dependent induction of fibrosis in vitro.ResultsOf the 553 proteases expressed in the human genome, 247 qualified as potential PAR-1 agonists of which 71 were significantly expressed above background in diabetic glomeruli. The recently identified PAR-1 agonist plasmin(ogen), together with its physiological activator tissue plasminogen activator, were among the highest expressed proteases. Plasmin did however not induce mesangial proliferation and/or fibronectin deposition in vitro. In a PAR-1 independent manner, plasmin even reduced fibronectin deposition.ConclusionExpression profiling identified plasmin as potential endogenous PAR-1 agonist driving DN. Instead of inducing fibronectin expression, plasmin however reduced mesangial fibronectin deposition in vitro. Therefore we conclude that plasmin may not be the endogenous PAR-1 agonist potentiating DN.     

Role of aldose reductase in the high glucose induced expression of fibronectin in human mesangial cells

Aldose reductase (AR) has emerged as a key contributor to the diabetic nephropathy (DN), however, the mechanisms by which AR increases DN remain poorly understood. Here, we report that exposure to high glucose (HG) stimulates fibronectin (FN) from human mesangial cells in culture. Our results show that exposure to HG and overexpression AR increase the expression of FN. This increase was prevented by the AR inhibitors sorbinil and zopolrestat. Treatment with HG and transfected with plasmid PcDNA3.0-AR, resulted in phosphorylation and activation of ERK, JNK and AKT signaling pathway, and increase the expression of FN. Treatment with inhibitor of JNK and AKT signaling pathway decreased the expression of FN. These results show that inhibition of AR may be useful to prevented extracellular matrix (ECM) deposition in diabetic nephropathy, which is regulated by JNK and AKT.