Matrix Accumulation In Mesangial Cells Research Articles

KCNQ1OT1/miR-18b/HMGA2 axis regulates high glucose-induced proliferation, oxidative stress, and extracellular matrix accumulation in mesangial cells.

The dysregulated long noncoding RNAs (lncRNAs) are associated with the pathogenesis of diabetic nephropathy (DN). LncRNA potassium voltage-gated channel subfamily Q member 1 overlapping transcript 1 (KCNQ1OT1) plays an important role in diabetes, but the role and mechanism of KCNQ1OT1 in DN are largely unknown. Serum samples were collected from 30 DN patients and normal volunteers. High glucose (HG)-challenged human mesangial cells (HMCs) were used as a cell model of DN. KCNQ1OT1, microRNA-18b (miR-18b), and high mobility group protein A2 (HMGA2) abundances were examined via quantitative reverse transcription polymerase chain reaction or western blot. Cell proliferation was assessed via 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide. Oxidative stress was assessed via the levels of reactive oxygen species (ROS), malondialdehyde (MDA), superoxide dismutase (SOD), and SOD2. Extracellular matrix (ECM) accumulation was investigated by the levels of fibronectin (FN), collagen I (Col I), and Col IV. The relationship between miR-18b and KCNQ1OT1 or HMGA2 was determined via dual-luciferase reporter analysis, RNA immunoprecipitation, and pull-down. KCNQ1OT1 expression was increased and miR-18b expression was decreased in DN patients and HG-challenged HMCs. miR-18b was targeted via KCNQ1OT1. Knockdown of KCNQ1OT1 weakened HG-caused proliferation, oxidative stress, and ECM accumulation of HMCs by increasing miR-18b. HMGA2 was targeted via miR-18b. miR-18b alleviated HG-induced cell proliferation, oxidative stress, and ECM accumulation by decreasing HMGA2. Silence of KCNQ1OT1 reduced HMGA2 expression via miR-18b. KCNQ1OT1 knockdown attenuated HG-induced proliferation, oxidative stress, and ECM accumulation of HMCs by regulating miR-18b/HMGA2 axis.

Circ-AKT3 inhibits the accumulation of extracellular matrix of mesangial cells in diabetic nephropathy via modulating miR-296-3p/E-cadherin signals.

Diabetic nephropathy is a leading cause of end‐stage renal disease globally. The vital role of circular RNAs (circRNAs) has been reported in diabetic nephropathy progression, but the molecular mechanism linking diabetic nephropathy to circRNAs remains elusive. In this study, we investigated the significant function of circ‐AKT3/miR‐296‐3p/E‐cadherin regulatory network on the extracellular matrix accumulation in mesangial cells in diabetic nephropathy. The expression of circ‐AKT3 and fibrosis‐associated proteins, including fibronectin, collagen type I and collagen type IV, was assessed via RT‐PCR and Western blot analysis in diabetic nephropathy animal model and mouse mesangial SV40‐MES13 cells. Luciferase reporter assays were used to investigate interactions among E‐cadherin, circ‐AKT3 and miR‐296‐3p in mouse mesangial SV40‐MES13 cells. Cell apoptosis was evaluated via flow cytometry. The level of circ‐AKT3 was significantly lower in diabetic nephropathy mice model group and mouse mesangial SV40‐MES13 cells treated with high‐concentration (25 mmol/L) glucose. In addition, circ‐AKT3 overexpression inhibited the level of fibrosis‐associated protein, such as fibronectin, collagen type I and collagen type IV. Circ‐AKT3 overexpression also inhibited the apoptosis of mouse mesangial SV40‐MES13 cells treated with high glucose. Luciferase reporter assay and bioinformatics tools identified that circ‐AKT3 could act as a sponge of miR‐296‐3p and E‐cadherin was the miR‐296‐3p direct target. Moreover, circ‐AKT3/miR‐296‐3p/E‐cadherin modulated the extracellular matrix of mouse mesangial cells in high‐concentration (25 mmol/L) glucose, inhibiting the synthesis of related extracellular matrix protein. In conclusion, circ‐AKT3 inhibited the extracellular matrix accumulation in diabetic nephropathy mesangial cells through modulating miR‐296‐3p/E‐cadherin signals, which might offer novel potential opportunities for clinical diagnosis targets and therapeutic biomarkers for diabetic nephropathy.

β-Caryophyllene inhibits high glucose-induced oxidative stress, inflammation and extracellular matrix accumulation in mesangial cells

β-Caryophyllene (BCP) is a bicyclic sesquiterpene compound that has anti-diabetic activity. However, the effect of BCP on diabetic nephropathy (DN) remains unclear. Here, we aimed to evaluate the potential role of BCP in high glucose (HG)-induced glomerular mesangial cells (MCs). MCs were maintained under HG condition to simulate DN in vitro. Our results showed that BCP inhibited HG-induced cell proliferation, ROS production and NADPH oxidase (NOX) 2/4 expression. BCP exhibited anti-inflammatory activity with decreased levels of TNF-α, IL-1β, IL-6 in HG-induced MCs. Moreover, BCP treatment suppressed the HG-induced secretion of fibronectin (FN) and collagen IV (Col IV) in MCs. Furthermore, BCP suppressed the NF-κB activation and enhanced the Nrf2 activation in HG-induced MCs. However, inhibition of Nrf2 attenuated the protective effects of BCP on HG-induced MCs, while inhibition of NF-κB enhanced the nephro-protective effects of BCP on MCs. In conclusion, these findings demonstrated that BCP executed protective effects on HG-induced MCs via regulating NF-κB and Nrf2 signaling pathways.

Knockdown of CTRP6 inhibits high glucose-induced oxidative stress, inflammation and extracellular matrix accumulation in mesangial cells through regulating the Akt/NF-κB pathway.

C1qTNF-related protein 6 (CTRP6) is a member of the CTRP family and exerts a key role in the progression of diabetes mellitus. However, the role of CTRP6 in diabetic nephropathy remains unknown. The present study was designed to examine the roles of CTRP6 in diabetic nephropathy and explore the potential molecular mechanisms. Our results showed that the expression level of CTRP6 was significantly increased in high glucose (HG)-stimulated glomerular mesangial cells (MCs). The following loss/gain-of-function assays demonstrated that CTRP6 knockdown significantly inhibited HG-induced reactive oxygen species (ROS) production in MCs. CTRP6 knockdown caused significant decreases in tumournecrosisfactor-α (TNF-α), interleukin (IL)-1β and IL-6 production levels in HG-induced MCs. Moreover, knockdown of CTRP6 inhibited HG-stimulated extracellular matrix (ECM) accumulation in MCs characterized by decreased expression and production levels of fibronectin (FN) and collagen IV (Col IV). Furthermore, CTRP6 knockdown suppressed HG-induced the activation of Akt/NF-κB pathway in MCs, while overexpression of CTRP6 exhibited the opposite effects. Treatment with LY294002, an inhibitor of Akt, reversed the induction effects of CTRP6 overexpression on ROS production, inflammation and ECM accumulation in MCs. In conclusion, these findings demonstrated that CTRP6 knockdown inhibits HG-induced ROS production, inflammation and ECM accumulation in MCs, which were mediated by the inactivation of the Akt/NF-κB pathway. The roles of CTRP6 in diabetic nephropathy provided evidence for its therapeutic potential for the treatment of diabetic nephropathy.

Berberine attenuates high glucose-induced proliferation and extracellular matrix accumulation in mesangial cells: Involvement of suppression of cell cycle progression and NF-κB/AP-1 pathways

Berberine has been shown to have renoprotective effects on diabetes through attenuating TGF-β1 and fibronectin (FN) expression. However, how berberine regulates TGF-β1 and FN is not fully clear. Here we investigated whether berberine inhibited TGF-β1 and FN expression in high glucose-cultured mesangial cells. Berberine significantly inhibited mesangial cell proliferation and hypertrophy by increasing the cell population in G1-phase and reducing that in S-phase. In addition, berberine reversed high glucose-induced down-regulation of cyclin-dependent kinase inhibitor p21Waf1/Cip1 and p27Kip1. Berberine inhibited p65 translocation to the nucleus and c-jun phosphorylation induced by high glucose. Furthermore, berberine attenuated high glucose-induced expression of TGF-β1 and FN. Using a luciferase reporter assay, we found that high glucose-induced transcription activity of NF-κB and AP-1 was blocked by berberine. Electrophoretic mobility shift assay showed that high glucose increased that NF-κB and AP-1 DNA binding activity. These data indicate that berberine inhibited mesangial cell proliferation and hypertrophy by modulating cell cycle progress. In addition, berberine suppressed high glucose-induced TGF-β1 and FN expression by blocking NF-κB/AP-1 pathways.

Acortatarin A inhibits high glucose-induced extracellular matrix production in mesangial cells

Background Diabetic nephropathy (DN) is the leading cause of end-stage renal disease. Various treatment regimens and combinations of therapies provide only partial renoprotection. Therefore new approaches are needed to retard the progression of DN. The aim of the present study was to evaluate the role of a novel spiroalkaloid from Acorus tatarinowii named acortatarin A (AcorA) in inhibiting high glucose-induced extracellular matrix accumulation in mesangial cells (MCs). Methods The cytotoxity of AcorA on MCs was examined by 3-(4,5-dimethylthiazol-2-yl)-2.5-diphenyltetrazolium bromide (MTT) assay. The expression of fibronectin and collagen IV was examined by real time PCR and western blotting. The expression of p22phox and p47phox was detected by western blot. The interaction between p22phox and p47phox was examined by co-immunoprecipitation. The phosphorylation of p47phox was examined by immunoprecipitation. The phosphorylation of protein kinase C (PKC) α, PKCβ, phospholiase C gamma (PLCγ1), and the p85 subunit of PI3K was determined by Western blotting. Results AcorA significantly inhibited high glucose-induced activation of NADPH oxidase, a ROS-generating enzyme, by increasing phosphorylation of p47phox and enhancing interaction between p22phox and p47phox. Preincubation of AcorA with MCs inhibited high glucose-induced collagen IV and fibronectin production in a dose-dependent manner. Moreover, AcorA attenuated high glucose enhanced phosphorylation of PKCα, PKCβ, PLCγ1, and the p85 subunit of PI3K. Conclusion AcorA inhibits high glucose-induced extracellular matrix production via blocking NADPH oxidase activation.

Dickkopf-1 Promotes Hyperglycemia–Induced Accumulation of Mesangial Matrix and Renal Dysfunction

Wnt/beta-catenin signaling mediates renal fibrosis in several model systems including diabetic nephropathy. Dickkopf-1 (DKK-1) is an endogenous inhibitor of Wnt/beta-catenin signaling, but whether DKK-1 modulates diabetic nephropathy is unknown. Here, we studied whether DKK-1 participates in high glucose (HG)-induced expression of profibrotic factors and renal damage. In vitro, HG increased expression of DKK1, receptor Kremen-2, TGF-beta1, and fibronectin in mesangial cells. Loss and gain of DKK1 function modulated HG-mediated c-Jun, TGF-beta1, and fibronectin expression. DKK1 mediated HG-induced phosphorylation of Ser45-beta-catenin and reduction of nuclear beta-catenin levels, but not phosphorylation of ERK kinase. Wnt3a protein and the beta-catenin (Delta45) mutation increased nuclear beta-catenin but abrogated HG-induced DKK1 and fibronectin expression. Exogenous DKK1 antisense oligonucleotide attenuated the increase in both serum DKK1 and urinary protein excretion in streptozotocin-induced diabetic rats. Knocking down DKK1 inhibited mesangial expression of TGF-beta1 and fibronectin and reduced both the glomerular volume and deposition of mesangial matrix in diabetic kidneys. Taken together, DKK1 mediates HG-induced destabilization of beta-catenin and matrix accumulation in mesangial cells. Knocking down DKK1 prevents diabetes-induced renal dysfunction and microstructure deterioration, suggesting that inhibition of DKK1offers therapeutic potential for diabetic nephropathy.

NAD(P)H OXIDASE-DEPENDENT REACTIVE OXYGEN SPECIES GENERATION MEDIATES AMADORI-INDUCED MATRIX ACCUMULATION IN RENAL CELLS.

Diabetic nephropathy (DN) is the leading cause of chronic kidney disease in the United States. Renal hypertrophy and extracellular matrix (ECM) accumulation are prominent features of DN. Chronic hyperglycemia leads to the accumulation of nonenzymatic glycation proteins known as Amadori and advanced glycation end products. Glycated proteins represent a major mechanism by which hyperglycemia leads to diabetic renal disease. Oxidative stress has emerged as a critical pathogenic factor in the development of diabetic complications, including DN. Research in our laboratory finds novel NAD(P)H oxidases, highly expressed in the kidney, to be major sources of reactive oxygen species (ROS) in diabetes. The purpose of the study is to investigate the mechanisms by which Amadori alter the redox state to mediate matrix expansion in glomerular mesangial cells. Methods NADPH-dependent superoxide generation was measured using lucigenin-enhanced chemiluminescence and intracellular ROS production was measured using the peroxide-sensetive fluorescent probe 2′,7′-dichlorofluorescin diacetate (DCF). To study ECM production, the expression of fibronectin (FN) and collagen IV expression was assessed by Western blot analysis and immunofluorescence confocal microscopy. Summary Amadori products and control albumin were prepared by incubating human serum albumin (HSA) in the presence or absence of glucose for 1 week. In mesangial cells, Amadori-HSA stimulates NADPH-dependent superoxide generation compared with control-HSA measured by lucigenin-enhanced chemiluminescence in a dose- and time-dependent manner. In addition, we show that exposure of mesangial cells to Amadori-HSA resulted in an increase in DCF fluorescence. Amadori-HSA induces FN and collagen IV expression in mesangial cells as detected by Western blot analysis. Pretreatment of the cells with diphenyleneoidonium (DPI), an inhibitor of NAD(P)H oxidase, markedly reduces Amadori-HSA-induced FN expression as assessed by immunofluorescence confocal microscopy. Conclusion NAD(P)H-oxidase isoenzyme(s) are activated by Amadori, leading to matrix accumulation in mesangial cells. This may represent an important mechanism by which Amadori products contribute to the pathogenesis of DN.

Elevated glucose induction of thrombospondin-1 up-regulates fibronectin synthesis in proximal renal tubular epithelial cells through TGF-β1 dependent and TGF-β1 independent pathways

TGF-beta1 bioactivation, consequent to the interaction of latent TGF-beta1 with thrombospondin-1 (TSP-1), correlates with matrix accumulation in mesangial cells. Tubulointerstitial damage predicts poor renal survival. There is little data on TGF-beta1 bioactivation and matrix synthesis in human proximal renal tubular epithelial cells under the influence of high glucose concentrations. This study thus investigates the role of TSP-1 in mediating elevated glucose-induction of TGF-beta1 bioactivation and fibronectin (FN) synthesis in human proximal tubular epithelial cells. Human proximal renal tubular epithelial cells (HK-2 cells) were incubated with 5, 10, 20 or 30 mM D-glucose for up to 3 weeks either in the presence or absence of TSP-1 blocking peptide. In separate studies HK-2 cells were incubated with exogenous TSP-1 (0-10 ng/ml) or TGF-beta1 (0-10 ng/ml) for 24 h. Cell proliferation was assessed by [(3)H]-thymidine incorporation. TGF-beta1 transcript, secretion and bioactivity were investigated by quantitative real-time PCR, ELISA and the MLEC bioassay respectively. TSP-1 and FN synthesis were assessed by quantitative real-time PCR, ELISAs and Western blot analysis. Elevated glucose concentrations increased TSP-1 synthesis, which was associated with reduced cell proliferation, increased TGF-beta1 bioactivity, and stimulation of FN synthesis. The inclusion of TSP-1 blocking peptide to cells stimulated with elevated glucose concentration abrogated activation of TGF-beta1 and induction of FN secretion. Exogenous TSP-1 increased bioactive TGF-beta1 in HK-2 cells to initiate FN accumulation. Of interest is our observation that TSP-1 also increased matrix synthesis through a mechanism independent of TGF-beta1. TGF-beta1 in turn modulated TSP-1 synthesis, indicative of an autocrine loop between TSP-1 and TGF-beta1. TSP-1 plays an important role in the induction of matrix synthesis by high glucose concentrations in human proximal renal tubular epithelial cells, through TGF-beta1 dependent and TGF-beta1 independent pathways. Pharmacological intervention targeting increased TSP-1 expression may interrupt the pathogenesis of diabetic nephropathy.

Matrix accumulation in mesangial cells exposed to cyclosporine A requires a permissive genetic background.

Chronic nephrotoxicity is an important adverse effect of cyclosporine A (CsA) therapy. Tubulo-interstitial lesions and arteriolopathy are common histologic findings. Glomerular lesions are also described, but they are of variable severity. The aim of our study is to determine whether CsA has a direct effect on mesangial cells and whether the cellular response depends on the genetic background. We studied mesangial cells isolated from mice susceptible (ROP/Le-+Es1(b)+Es1(a), ROP) and resistant to glomerulosclerosis (B6SJLF1, C57). We previously showed that sclerosis-prone and sclerosis-resistant phenotypes are maintained in vitro. We examined whether CsA exposure directly affected extracellular matrix turnover in mesangial cells and whether the response is determined by the genetic background. Extracellular matrix synthesis and degradation were studied by proline incorporation, ELISA, reverse transcription-polymerase chain reaction, zymography, and reverse zymography. We chose a CsA dose that induced neither cytotoxicity nor apoptosis (1 microg/ml). At the dose of 1 microg/ml total collagen accumulation was increased in ROP but not in C57 cells. Matrix metalloproteinase (MMP)-2 activity and mRNA levels were selectively decreased in ROP cells. CsA exposure did not affect tissue inhibitors of MMP (TIMP)-1 and -2 activity or TGF-beta1 mRNA expression and protein synthesis in either cell line. CsA increases total collagen accumulation in mesangial cells from sclerosis-prone mice by decreasing MMP-2 activity, but does not affect cells from sclerosis-resistant mice. Thus, CsA directly affects mesangial cells, but only those with a permissive genetic background for glomerulosclerosis.