Aim: Aberrantly glycosylated IgA1 is a key factor in the pathogenesis of IgA nephropathy (IgAN). In this study we investigated the effects of aggregated IgA1 derived from... More
Aim: Aberrantly glycosylated IgA1 is a key factor in the pathogenesis of IgA nephropathy (IgAN). In this study we investigated the effects of aggregated IgA1 derived from IgAN patients (P-aIgA1) on human renal mesangial cells (HMCs) and the anti-proliferative and antifibrotic effects of histone deacetylase (HDAC) inhibitors in vitro. Methods: Three types of IgA1 were prepared, ie, N-IgA1 (IgA1 from healthy volunteers), P-IgA1 (IgA1 from IgAN patients), and P-aIgA1 (aggregated IgA1 from IgAN patients). The isolated IgA1 was heated for thermal polymerization. The proliferation of human renal mesangial cells (HMCs) were assessed using MTT assay. The expression levels of relevant proteins were examined using immunoblotting assays or immunohistochemistry. Results: P-aIgA1 (25–250 μg/mL) dose-dependently promoted the proliferation of HMCs, and markedly increased the protein levels of type I histone deacetylase (HDAC1, HDAC2 and HDAC8) in the cells. Both P-IgA1 and N-IgA1 were much weaker in stimulating cell proliferation and HDAC expression. P-aIgA1 (50 μg/mL) markedly increased the protein levels of Col1a1 and PAI-1, as well as pSmad2/3 and pStat3 in the cells. Pretreatment with the HDAC inhibitor trichostatin A (TSA, 250 nmol/L) or valproic acid (VPA, 400 μg/mL) partially reversed P-aIgA1-induced cell proliferation and extracellular matrix synthesis in HMCs. Conclusion: P-aIgA1 produces pro-proliferative and profibrotic actions in HMCs via upregulating the expression of HDACs, and subsequently activating TGF-β/Smad2/3 and Jak2/Stat3 signaling pathways. Both VPA and TSA attenuate P-aIgA1-induced cell proliferation and fibrosis in HMCs. Keywords: IgA nephropathy; human renal mesangial cells; HDAC; trichostatin A; valproic acid; extracellular matrix synthesis; Smad2/3; Stat3; antifibrosis Less
Background. miR-192, miR-194, and miR-215 are enriched in the kidney and play roles in the pathogenesis of diabetic nephropathy (DN). Extracellular vesicles (EVs) can be ... More
Background. miR-192, miR-194, and miR-215 are enriched in the kidney and play roles in the pathogenesis of diabetic nephropathy (DN). Extracellular vesicles (EVs) can be detected in body fluids and may serve as disease biomarkers. Methods. Eighty type 2 diabetes patients with normoalbuminuria (n = 30), microalbuminuria (n = 30), or macroalbuminuria (n = 20), as well as 10 healthy controls, were enrolled in this study. Real-time PCR was used to evaluate urinary EV miRNAs expression. Results. The miR-192 levels were significantly higher than the miR-194 and miR-215 levels in urine EVs and all three miRNAs were significantly increased in the microalbuminuric group compared with the normoalbuminuric and control subjects but were decreased in the macroalbuminuric group. In patients with normoalbuminuria and microalbuminuria, miR-192 was positively correlated with albuminuria (r = 0.357, P = 0.005) levels and transforming growth factor- (TGF-) β1 (r = 0.356, P = 0.005) expression. Receiver operating characteristic (ROC) curve analysis revealed that miR-192 was better than miR-194 and miR-215 in discriminating the normoalbuminuric group from the microalbuminuric group. Exposure of human renal tubular epithelial cells to high glucose increased the expression of both miRNAs in cellular supernatant EVs, indicating a potential source. Conclusion. These results suggest the potential use of urinary EV miR-192 as a biomarker of the early stage of DN. Less
Oxidative damage to mitochondrial DNA (mtDNA) has been linked to the pathogenicity of diabetic nephropathy. We tested the hypothesis that mtDNA copy number may be increas... More
Oxidative damage to mitochondrial DNA (mtDNA) has been linked to the pathogenicity of diabetic nephropathy. We tested the hypothesis that mtDNA copy number may be increased in human mesangial cells in response to high glucose-induced reactive oxygen species (ROS) to compensate for damaged mtDNA. The effect of manganese superoxide dismutase mimetic (MnTBAP) on glucose-induced mtDNA copy number was also examined. The copy number of mtDNA was determined by real-time PCR in human mesangial cells cultured in 5 mM glucose, 25 mM glucose, and mannitol (osmotic control), as well as in cells cultured in 25 mM glucose in the presence and absence of 200 μ M MnTBAP. Intracellular ROS was assessed by confocal microscopy and flow cytometry in human mesangial cells. The copy number of mtDNA was significantly increased when human mesangial cells were incubated with 25 mM glucose compared to 5 mM glucose and mannitol. In addition, 25 mM glucose rapidly generated ROS in the cells, which was not detected in 5 mM glucose. Furthermore, mtDNA copy number was significantly decreased and maintained to normal following treatment of cells with 25 mM glucose and MnTBAP compared to 25 mM glucose alone. Inclusion of MnTBAP during 25 mM glucose incubation inhibited mitochondrial superoxide in human mesangial cells. Increased mtDNA copy number in human mesangial cells by high glucose could contribute to increased mitochondrial superoxide, and prevention of mtDNA copy number could have potential in retarding the development of diabetic nephropathy. Less
Diabetic nephropathy (DN) is one of the major diabetic complications and the leading cause of end-stage renal disease. Abnormal angiogenesis results in new vessels that a... More
Diabetic nephropathy (DN) is one of the major diabetic complications and the leading cause of end-stage renal disease. Abnormal angiogenesis results in new vessels that are often immature and play a pathological role in DN, contributing to renal fibrosis and disrupting glomerular failure. Purple corn has been utilized as a daily food and exerts disease-preventive activities. This study was designed to investigate whether anthocyanin-rich purple corn extract (PCE) prevented glomerular angiogenesis under hyperglycemic conditions. Human endothelial cells were cultured in conditioned media of mesangial cells exposed to 33 mM high glucose (HG-HRMC-CM). PCE decreased endothelial expression of vascular endothelial growth factor (VEGF) and hypoxia inducible factor (HIF)-1α induced by HG-HRMC-CM. Additionally, PCE attenuated the induction of the endothelial marker of platelet endothelial cell adhesion molecule (PECAM)-1 and integrin β3 enhanced in HG-HRMC-CM. Endothelial tube formation promoted by HG-HRMC-CM was disrupted in the presence of PCE. In the in vivo study employing db/db mice treated with 10 mg/kg PCE for 8 weeks, PCE alleviated glomerular angiogenesis of diabetic kidneys by attenuating the induction of VEGF and HIF-1α. Oral administration of PCE retarded the endothelial proliferation in db/db mouse kidneys, evidenced by its inhibition of the induction of vascular endothelium-cadherin, PECAM-1 and Ki-67. PCE diminished the mesangial and endothelial induction of angiopoietin (Angpt) proteins under hypeglycemic conditions. The induction and activation of VEGF receptor 2 (VEGFR2) were dampened by treating PCE to db/db mice. These results demonstrate that PCE antagonized glomerular angiogenesis due to chronic hyperglycemia and diabetes through disturbing the Angpt-Tie-2 ligand-receptor system linked to renal VEGFR2 signaling pathway. Therefore, PCE may be a potent therapeutic agent targeting abnormal angiogenesis in DN leading to kidney failure. Less
Development of diabetic nephropathy with fibrosis is associated with hypereglycemia-linked inflammation. Increased levels of proinflammatory factors have been found in di... More
Development of diabetic nephropathy with fibrosis is associated with hypereglycemia-linked inflammation. Increased levels of proinflammatory factors have been found in diabetic patients with nephropathy. The present study was to test the hypothesis that isoangustone A, a novel compound present in licorice, can inhibit renal fibrosis and inflammation inflamed by high glucose (HG) in human mesangial cells through disturbing transforming growth factor β (TGF-β) and nuclear facor κB (NF-κB) pathways. Serum-starved mesangial cells were cultured in 33 mmol/L glucose media. Cells were treated with 1-20 μmol/L isoangustone A isolated from Glycyrrhiza uralensis licorice for three days. Exposure of cells to HG elevated connective tissue growth factor and collagen production, which was dose-dependently reversed by isoangustone A. Isoangustone A boosted HG-plummeted membrane type matrix metalloproteinase (MMP)-1 expression and diminished HG-elevated tissue inhibitor of MMP-2 expression. HG activated mesangial TGF-β1-SMAD-responsive signaling, which was repealed by ≥10 μmol/L isoangustone A. Furthermore, HG upregulated intracellular cell adhesion molecule-1 (ICAM-1) level and monocyte chemoattractant protein-1 (MCP-1) mRNA expression, and such increases were dose-dependently suppressed by isoangustone A most likely through hampering TGF-β signaling pathways. Blockade of NF-κB signaling appeared to be responsible for attenuating HG-triggered induction of ICAM-1 and MCP-1. Our findings provide the first evidence that isoangustone A dampens mesangial sclerosis associated with inflammation in response to HG through hindering TGF-β and NF-κB signaling. Less
Honokiol has been shown to possess a lot of pharmacologic benefits, including antioxidative, antiangiogenic and antineoplastic effects. In the present study, we investiga... More
Honokiol has been shown to possess a lot of pharmacologic benefits, including antioxidative, antiangiogenic and antineoplastic effects. In the present study, we investigated the anti-inflammatory effects of honokiol and the signaling mechanisms involved in lipopolysaccharide (LPS)-induced conditions in human renal mesangial cells (HRMCs). Honokiol did not significantly change HRMC viability when used at a concentration of <20 μmol/l but markedly altered cell viability at concentrations of >40 μmol/l. In this study, LPS treatment led to a marked upregulation of the levels of IL-1β, IL-18, TNF-α, TGF-β1, CCL2, CCL3, and CCL5 in HRMCs. The expression of COX-2, iNOS, and their products PGE(2) and NO also increased. The upregulation of these molecules was significantly abolished by honokiol in a dose-dependent manner. Moreover, honokiol almost completely reversed IL-1β, CCL3, and NO expression at 10 μmol/l, and IL-18, TNF-α, TGF-β1, and COX-2 expression at 20 μmol/l. In addition, phospho-NF-κB p65 at Ser536, phospho-Akt, and phospho-p42/44 were dramatically suppressed by honokiol in LPS-treated HRMCs. These results indicate that honokiol can inhibit the LPS-induced expression of inflammatory cytokines and mediators in HRMCs. The anti-inflammatory mechanisms of honokiol are partly due to the suppression of the phospho-NF-κB p65, phospho-Akt and phospho-p42/44 pathways. Copyright © 2010 Published by Elsevier B.V. Less
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, ... More
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. Less
Diabetic nephropathy characterized as mesangial fibrosis and glomerulosclerosis results in renal failure and end-stage renal diseases. Enhanced expression and secretion o... More
Diabetic nephropathy characterized as mesangial fibrosis and glomerulosclerosis results in renal failure and end-stage renal diseases. Enhanced expression and secretion of connective tissue growth factor (CTGF) play an important role in the expansion of glomerular mesangial matrix mostly composed of type IV collagen. Isoliquiritigenin can prevent various renal injuries via its anti-inflammatory action. However, the effect of isoliquiritigenin on diabetic nephropathy has never been explored. The present study was to investigate whether nontoxic isoliquiritigenin inhibited high glucose (HG)-induced mesangial fibrosis by retarding formation of type IV collagen as well as CTGF in human mesangial cells (HRMC). Serum starved cells were cultured in media containing 5.5 mM glucose plus 27.5 mM mannitol as an osmotic control or 33 mM glucose for 3 days with and without 1-20 microM isoliquiritigenin. Exposure of cells to HG caused marked increases in collagen secretion and CTGF expression, which was dose-dependently reversed by isoliquiritigenin at the transcriptional levels. Additionally, isoliquiritigenin boosted HG-plummeted type matrix metalloproteinase-1 (MT-1 MMP) expression and dampened HG-elevated tissue inhibitor of MMP-2 (TIMP-2) expression, facilitating the degradation of mesangial matrix. Isoliquiritigenin inhibited HG-upregulated CTGF and TIMP-2 expression via disturbing TGF-beta1 signaling in HRMC, as evidenced by TGF-beta receptor I kinase (TGF-beta RI) inhibitor. HG-activated SMAD2 through autocrine TGF-beta signaling was repealed by > or =10 microM isoliquiritigenin. HG induced SMAD4 expression of HRMC and obliterated antagonistic SMAD7, whereas isoliquiritigenin suppressed induction of TGF-beta RII and TGF-beta RI with blunting their downstream SMAD signaling. The results demonstrate that the bioactive isoliquiritigenin in licorice diminished mesangial matrix accumulation in response to ambient HG through retarding TGF-beta1-SMAD signaling transduction. Therefore, isoliquiritigenin may be a potential therapeutic agent for the prevention and treatment of mesangial fibrosis and glomerulosclerosis leading to diabetic nephropathy due to longstanding diabetes mellitus. Less
Aim: The previous studies demonstrated that transforming growth factor-beta(1) (TGF-beta(1)) could upregulate the expression of aldose reductase (AR). The aim of this stu... More
Aim: The previous studies demonstrated that transforming growth factor-beta(1) (TGF-beta(1)) could upregulate the expression of aldose reductase (AR). The aim of this study is to clarify (investigate) the mechanism of TGF-beta(1)-induced AR expression. Methods: Real-time polymerase chain reaction and western blot were used to analyse the AR expression in mRNA and protein levels in human mesangial cells, and reporter assay was used to analyse the function of various sites within 5'-flanking region of AR gene in AR expression. Results: TGF-beta(1) (4 ng/mL) stimulation could upregulate AR expression. The cells pretreated with pharmacological inhibitors, U0126 and PD98059 for blocking extracellular signal-related kinase (ERK) signalling pathway or SP6000125 for blocking c-Jun N-terminal kinase (JNK) signalling pathway, respectively, showed reduced expression of AR after TGF-beta(1) stimulation. Similarly, the cells transiently transfected with pCMVTAM67, which is an expression plasmid for DN-c-Jun showed decreasing AR expression. Reporter assay revealed that the 5'-promoter region of AR consisting of an AP-1 site and two putative antioxidant response elements (ARE) was responsible for TGF-beta(1) stimulation. Mutation of either ARE did not affect the promoter activity in the reporter assay while mutation of AP-1 site caused a significant decrease in the responsiveness to TGF-beta(1). Conclusion: TGF-beta(1) upregulate AR expression in both mRNA and protein levels. The results demonstrated that ERK and JNK are involved in the downstream signalling pathways and transcription factor AP-1 plays an important role in the regulation of TGF-beta(1)-induced AR expression in mesangial cells. Less
Both clinical and experimental reports indicate that aldosterone contributes to the progression of renal failure independent of its hemodynamic effects. In the present st... More
Both clinical and experimental reports indicate that aldosterone contributes to the progression of renal failure independent of its hemodynamic effects. In the present study, we evaluated effect of aldosterone on human mesangial cell (MC) growth. Aldosterone induced apoptotic and mitogenic effects on MCs. Aldosterone promoted MC apoptosis in a dose- and time-dependent manner. Spironolactone, a mineralocorticoid receptor antagonist, inhibited aldosterone-induced MC apoptosis. Similarly, antioxidants and free radical scavengers partially attenuated proapoaptotic effects of aldosterone. Aldosterone also enhanced dephosphorylation of phospho-Bad and accumulation of cytosolic cytochrome c in MCs. In in vivo studies, rats were randomly assigned to receive normal saline, aldosterone, or eplerenone + aldosterone for 28 days. Systolic blood pressure, urinary albumin excretion rate, serum creatinine, and aldosterone were measured. Aldosterone-infused rats developed elevated systolic blood pressure and albuminuria when compared with control rats. Aldosterone-treated rats also showed greater numbers of apoptosed MCs. This proapoptotic effect of aldosterone was inhibited by eplerenone, a selective aldosterone antagonist. These findings suggest that aldosterone, besides its hemodynamic effects, may also directly contribute to the occurrence of MC apoptosis. Keywords: mesangial cells, apoptosis, aldosterone Less
Both increased aldose reductase (AR) activity and oxidative/nitrosative stress have been implicated in the pathogenesis of diabetic nephropathy, but the relation between ... More
Both increased aldose reductase (AR) activity and oxidative/nitrosative stress have been implicated in the pathogenesis of diabetic nephropathy, but the relation between the two factors remains a subject of debate. This study evaluated the effects of AR inhibition on nitrosative stress and poly(ADP-ribose) polymerase (PARP) activation in diabetic rat kidney and high-glucose-exposed human mesangial cells. In animal experiments, control (C) and streptozotocin-diabetic (D) rats were treated with/without the AR inhibitor fidarestat (F, 16 mg kg−1 day−1) for 6 weeks starting from induction of diabetes. Glucose, sorbitol, and fructose concentrations were significantly increased in the renal cortex of D vs C (p < 0.01 for all three comparisons), and sorbitol pathway intermediate, but not glucose, accumulation, was completely prevented in D + F. F at least partially prevented diabetes-induced increase in kidney weight as well as nitrotyrosine (NT, a marker of peroxynitrite-induced injury and nitrosative stress), and poly(ADP-ribose) (a marker of PARP activation) accumulation, assessed by both immunohistochemistry and Western blot analysis, in glomerular and tubular compartments of the renal cortex. In vitro studies revealed the presence of both AR and PARP-1 in human mesangial cells, and none of these two variables were affected by high glucose or F treatment. Nitrosylated and poly(ADP-ribosyl)ated proteins (Western blot analysis) accumulated in cells cultured in 30 mM D-glucose (vs 5.55 mM glucose, p < 0.01), but not in cells cultured in 30 mM L-glucose or 30 mM D-glucose plus 10 μM F. AR inhibition counteracts nitrosative stress and PARP activation in the diabetic renal cortex and high-glucose-exposed human mesangial cells. These findings reveal new beneficial properties of the AR inhibitor F and provide the rationale for detailed studies of F on diabetic nephropathy. Less
Peroxisome proliferator-activated receptor-gamma (PPAR-gamma) is a ligand-dependent transcription factor that plays an important role in the regulation of insulin sensiti... More
Peroxisome proliferator-activated receptor-gamma (PPAR-gamma) is a ligand-dependent transcription factor that plays an important role in the regulation of insulin sensitivity and lipid metabolism. Evidence shows that PPAR-gamma agonists also ameliorate renal fibrotic lesions in both diabetic nephropathy and nondiabetic chronic kidney disease. However, little is known about the mechanism underlying their antifibrotic action. This study demonstrated that PPAR-gamma agonists could exert their actions by inducing antifibrotic hepatocyte growth factor (HGF) expression. Incubation of mesangial cells with natural or synthetic PPAR-gamma agonists 15-deoxy-Delta12,14-prostaglandin J2 (15d-PGJ2) or troglitazone and ciglitazone suppressed TGF-beta1-mediated alpha-smooth muscle actin, fibronectin, and plasminogen activator inhibitor-1 expression. PPAR-gamma agonists also induced HGF mRNA expression and protein secretion. Transfection studies revealed that 15d-PGJ2 stimulated HGF gene promoter activity, which was dependent on the presence of a novel peroxisome proliferator response element. Treatment of mesangial cells with 15d-PGJ2 induced the binding of PPAR-gamma to the peroxisome proliferator response element in the HGF promoter region. PPAR-gamma agonists also activated c-met receptor tyrosine phosphorylation, induced Smad transcriptional co-repressor TG-interacting factor expression, and blocked TGF-beta/Smad-mediated gene transcription in mesangial cells. Furthermore, ablation of c-met receptor through the LoxP-Cre system in mesangial cells abolished the antifibrotic effect of 15d-PGJ2. PPAR-gamma activation also induced HGF expression in renal interstitial fibroblasts and repressed TGF-beta1-mediated myofibroblast activation. Both HGF and 15d-PGJ2 attenuated Smad nuclear translocation in response to TGF-beta1 stimulation in renal fibroblasts. Together, these findings suggest that HGF may act as a downstream effector that mediates the antifibrotic action of PPAR-gamma agonists. Less
Retinoic acid (RA), an active metabolite of vitamin A, plays a critical role in the regulation of cell proliferation, survival, and differentiation. RA action is primaril... More
Retinoic acid (RA), an active metabolite of vitamin A, plays a critical role in the regulation of cell proliferation, survival, and differentiation. RA action is primarily mediated through its receptors, ligand-dependent transcription factors of the steroid/thyroid/vitamin D nuclear receptor superfamily. Recent studies indicate that administration of RA mitigates progressive kidney disease, underscoring its renoprotective potential. In this study, we investigated the effects of 9-cis-RA on glomerular mesangial cell activation induced by transforming growth factor (TGF)-β1 using an in vitro cell culture system. In human mesangial cells 9-cis-RA suppressed TGF-β1-induced α-smooth muscle actin, fibronectin, and plasminogen activator inhibitor-1 expression, but it did not significantly affect cell proliferation and survival. Interestingly, 9-cis-RA induced hepatocyte growth factor (HGF) mRNA expression and protein secretion, stimulated HGF promoter activity, and activated c-met receptor phosphorylation. Similar to HGF, 9-cis-RA induced expression of the Smad transcriptional co-repressor TGIF in mesangial cells. Overexpression of exogenous TGIF by transfection or 9-cis-RA treatment suppressed trans-activation of the TGF-β-responsive promoter. Moreover, conditional ablation of the c-met receptor completely abolished the anti-fibrotic effect of 9-cis-RA and abrogated TGIF induction. Collectively, these results indicate that 9-cis-RA possesses anti-fibrotic ability by antagonizing TGF-β1 in mesangial cells and that 9-cis-RA activity is likely mediated through a mechanism dependent on HGF/c-met receptor signaling. Less
Mesangial cell activation is a predominant pathologic feature of diabetic nephropathy that precedes the accumulation of extracellular matrix leading to glomerulosclerosis... More
Mesangial cell activation is a predominant pathologic feature of diabetic nephropathy that precedes the accumulation of extracellular matrix leading to glomerulosclerosis. For understanding the potential mechanism by which hepatocyte growth factor (HGF) ameliorates diabetic nephropathy, the effects of HGF on mesangial cell activation induced by TGF-beta1 were investigated. Western blot analysis and immunostaining revealed that HGF suppressed alpha-smooth muscle actin expression induced by TGF-beta1 in cultured rat and human mesangial cells. HGF also inhibited TGF-beta1-mediated fibronectin and type I collagen expression. Such action of HGF was dependent on the activation of extracellular signal-regulated kinase-1 and -2 but not on Akt and p38 mitogen-activated protein kinase. HGF did not affect TGF-beta1-mediated Smad2 phosphorylation and its nuclear translocation. However, it rapidly upregulated Smad transcriptional corepressor TG-interacting factor (TGIF) abundance in mesangial cells, which was primarily mediated by stabilizing its protein from degradation. Ectopic expression of TGIF markedly suppressed Smad-mediated activation of TGF-beta1-responsive promoter activity and completely blocked TGF-beta1-induced alpha-smooth muscle actin expression. In vivo, TGIF expression was dramatically downregulated in the glomeruli of diabetic kidneys, and delivery of exogenous HGF induced TGIF expression. These results suggest that HGF specifically antagonizes the profibrotic action of TGF-beta1 in mesangial cells by stabilizing Smad transcriptional corepressor TGIF. Less
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