Mesangial Cell Apoptosis Research Articles

YM155 enhances ABT-737-mediated apoptosis through Mcl-1 downregulation in Mcl-1-overexpressed cancer cells.

ABT-737 is a BH3 mimetic inhibitor of Bcl-xL, Bcl-2, and Bcl-w, and it has been reported for anti-cancer effects in various types of cancer cells. However, ABT-737 fails to induce apoptosis in cancer cell with high levels of Mcl-1 expression. The pharmacological survivin inhibitor YM155 has been reported to induce downregulation of Mcl-1 expression. Therefore, we investigated the effect of YM155 to sensitize resistance against ABT-737 in Mcl-1-overexpressed human renal carcinoma Caki cells. We found that ABT-737 alone and YM155 alone did not induce apoptosis, but YM155 markedly sensitized ABT-737-mediated apoptosis in Mcl-1-overexpressed Caki cells, human glioma cells (U251MG), and human lung carcinoma cells (A549). In contrast, combined treatment with ABT-737 and YM155 did not increase apoptosis in normal mouse kidney cells (TCMK-1) and human mesangial cells (MC). YM155 induced lysosome-dependent downregulation of Mcl-1 expression in Mcl-1-overexpressed Caki cells. In addition, combined treatment with ABT-737 and YM155 induced loss of mitochondrial membrane potential and inhibited interaction of Bcl-xL and Bax. Taken together, our results suggested that YM155 effectively improves sensitivity to ABT-737 through downregulation of Mcl-1 expression.

Effect of norcantharidin on the proliferation, apoptosis, and cell cycle of human mesangial cells

Aims: Norcantharidin (NCTD) regulates immune system function and reduces proteinuria. We sought to investigate the effect of NCTD on proliferation, apoptosis and cell cycle of cultured human mesangial cells (HMC) in vitro.Methods: HMC cells were divided into a normal control group, and various concentrations of NCTD group (2.5, 5, 10, 20, or 40 μg/mL). Cell proliferation was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, apoptosis was detected by Annexin V/propidium iodide (PI) assays, and morphological analysis was performed by Hoechest 33258 staining. Finally, cell cycle was analyzed by flow cytometry.Results: NCTD dose and time dependently inhibits HMC proliferation significantly (p < .05). Apoptosis dose and time dependently increased after NCTD treatment. Cell-cycle analysis revealed that the number of cells in the G2 phase increased significantly, whereas the fraction of cells in the S phase decreased, especially 24 h after 5 μg/ml NCTD treatment.Conclusion: NCTD inhibits HMC cell proliferation, induces apoptosis, and affects the cell cycle.

Ochratoxin A induces ER stress and apoptosis in mesangial cells via a NADPH oxidase-derived reactive oxygen species-mediated calpain activation pathway.

Ochratoxin A (OTA) contaminated food increases reactive oxygen species (ROS) production in glomerulus and causes glomerulopathy. The molecular mechanisms still remain uncertain. In this study, we used mouse and rat glomerular mesangial cells and delineate the signaling pathway behind the OTA-triggered cell apoptosis. OTA dose-dependently induced expression of ER stress markers including phospho-PERK, phospho-eIF2α, GRP78, GRP94, and CHOP. Apoptosis events including cleavage of caspase-12, caspase-7, and PARP are also observed. OTA activated oxidative stress and increased NADPH oxidase activity. NADPH oxidase inhibitor, apocynin, significantly attenuated OTA-induced cell apoptosis. Moreover, OTA markedly increased the calpain activity which significantly inhibited by apocynin. Transfection of calpain-siRNA effectively inhibited the OTA-increased ER stress-related protein expression. These findings suggest that OTA activated NADPH oxidase and calpain, induced ER stress and ROS production, and caused glomerular mesangial cells apoptosis which leads to glomerulopathy.

Hsa‑miR‑371‑5p inhibits human mesangial cell proliferation and promotes apoptosis in lupus nephritis by directly targeting hypoxia‑inducible factor 1α.

MicroRNAs (miRNAs/miR) have emerged as a novel class of gene expression modulators in kidney disease. Lupus nephritis (LN) is the predominant cause of morbidity and mortality in patients with systemic lupus erythematosus (SLE). Hsa‑miR‑371‑5p has previously been reported to be dysregulated in LN using a miRNA microarray analysis. The present study aimed to determine the function and molecular mechanisms of hsa‑miR‑371‑5p in human mesangial cells of LN. Quantitative polymerase chain reaction (qPCR) was used to detect hsa‑miR‑371‑5p expression in LN tissues. Furthermore, the MTT assay and flow cytometric analyses were performed to analyze the effects of hsa‑miR‑371‑5p on mesangial cell proliferation and apoptosis. Bioinformatics analysis, luciferase reporter assay, qPCR and western blotting were also conducted to predict and confirm the target gene of hsa‑miR‑371‑5p in mesangial cells. The results demonstrated that hsa‑miR‑371‑5p expression was markedly downregulated in LN renal tissues compared with in normal kidney tissues. Restoration of hsa‑miR‑371‑5p expression using synthetic hsa‑miR‑371‑5p mimics was able to significantly inhibit mesangial cell proliferation and induce apoptosis. In addition, mechanistic exploration demonstrated that hypoxia‑inducible factor1α (HIF‑1α) was a direct target gene of hsa‑miR‑371‑5p in mesangial cells. In conclusion, these results suggested that hsa‑miR‑371‑5p is downregulated in LN, and overexpression of hsa‑miR‑371‑5p may inhibit mesangial cell proliferation and promote apoptosis by directly targeting HIF‑1α.

Inhibition of autophagy increased AGE/ROS-mediated apoptosis in mesangial cells

The aim of our study was to investigate the role of autophagy, a homeostatic process involved in the lysosomal degradation of damaged cell organelles and proteins, in regulating the survival of mesangial cells treated with advanced glycation end products (AGEs). In the present study, AGEs induced mitochondrial depolarization and led to mitochondrial-dependent apoptosis in mesangial cells, as shown by the loss of the mitochondrial membrane potential; increased Bax processing; increased Caspase-9, Caspase-3 and PARP cleavage; and decreased Bcl-2 expression. Meanwhile, AGEs also triggered autophagy flux in mesangial cells, as confirmed by the presence of autophagic vesicles, the conversion of LC3II/LC3I and the increase/decrease in Beclin-1/p62 expression. Interestingly, this study reported apparent apoptosis and autophagy that were dependent on reactive oxygen species (ROS) production. Scavenging ROS with N-acetyl-l-cysteine could prevent the appearance of the autophagic features and reverse AGE-induced apoptosis. Moreover, AGE-triggered mitophagy, which was confirmed by the colocalization of autophagosomes and mitochondria and Parkin translocation to mitochondria, played a potential role in reducing ROS production in mesangial cells. Additionally, inhibition of autophagy significantly enhanced AGE-induced cell apoptosis. Taken together, our data suggest that ROS were the mediators of AGE-induced mesangial cell apoptosis and that autophagy was likely to be the mechanism that was triggered to repair the ROS-induced damage in the AGE-treated cells and thereby promote cell survival. This study provides new insights into the molecular mechanism of autophagy involved in AGE-induced apoptosis in mesangial cells.

Involvement of Endoplasmic Reticulum Stress, Autophagy, and Apoptosis in Advanced Glycation End Products-Induced Glomerular Mesangial Cell Injury.

Advanced glycation end-products (AGEs)-induced mesangial cell death is one of major causes of glomerulus dysfunction in diabetic nephropathy. Both endoplasmic reticulum (ER) stress and autophagy are adaptive responses in cells under environmental stress and participate in the renal diseases. The role of ER stress and autophagy in AGEs-induced mesangial cell death is still unclear. Here, we investigated the effect and mechanism of AGEs on glomerular mesangial cells. AGEs dose-dependently decreased mesangial cell viability and induced cell apoptosis. AGEs also induced ER stress signals in a time- and dose-dependent manner. Inhibition of ER stress with 4-phenylbutyric acid effectively inhibited the activation of eIF2α and CHOP signals and reversed AGEs-induced cell apoptosis. AGEs also activated LC-3 cleavage, increased Atg5 expression, and decreased p62 expression, which indicated the autophagy induction in mesangial cells. Inhibition of autophagy by Atg5 siRNAs transfection aggravated AGEs-induced mesangial cell apoptosis. Moreover, ER stress inhibition by 4-phenylbutyric acid significantly reversed AGEs-induced autophagy, but autophagy inhibition did not influence the AGEs-induced ER stress-related signals activation. These results suggest that AGEs induce mesangial cell apoptosis via an ER stress-triggered signaling pathway. Atg5-dependent autophagy plays a protective role. These findings may offer a new strategy against AGEs toxicity in the kidney.

Inhibitory effects of Shenkang injection and its main component emodin on the proliferation of high glucose‑induced renal mesangial cells through cell cycle regulation and induction of apoptosis.

Increased mesangial cell proliferation is a major pathological feature of early-stage diabetic nephropathy (DN). The present study investigated the effects of the Traditional Chinese Medicine Shenkang injection (SKI) and its main component emodin (EM) on high glucose‑cultured mesangial cells. The proliferation rate, cell cycle distribution, apoptosis and morphology of rat renal mesangial cells (RMCs) cultured in the presence of various concentrations of glucose (5.6 or 25mM), SKI (25, 50 or 100mg/l) or EM (10, 20 or 40µM) were assessed at time‑points of 12, 24 or 48h. High‑glucose treatment promoted the proliferation of RMCs, which was significantly inhibited by SKI and EM, while these drugs had no effect on RMCs under normal glucose conditions, as indicated by an MTT assay. Furthermore, flow cytometric analysis revealed that SKI and EM inhibited the cell cycle progression of RMCs and induced apoptosis. Transmission electron microscopy revealed morphological characteristics of apoptosis and western blot analysis demonstrated the upregulation of B‑cell lymphoma2‑associated X protein (bax) and activation of caspases in RMCs following treatment with SKI or EM under high‑glucose conditions. In conclusion, SKI and its major active component EM were shown to inhibit high‑glucose‑induced proliferation of RMCs via inducing cell cycle arrest at G1 phase as well as cellular apoptosis via upregulation of pro‑apoptotic mediators bax and caspase activation, and may therefore be suitable for the treatment of DN.

TRPC6 channel activation promotes neonatal glomerular mesangial cell apoptosis via calcineurin/NFAT and FasL/Fas signaling pathways.

Glomerular mesangial cell (GMC) proliferation and death are involved in the pathogenesis of glomerular disorders. The mechanisms that control GMC survival are poorly understood, but may include signal transduction pathways that are modulated by changes in intracellular Ca2+ ([Ca2+]i) concentration. In this study, we investigated whether activation of the canonical transient receptor potential (TRPC) 6 channels and successive [Ca2+]i elevation alter neonatal GMC survival. Hyperforin (HF)-induced TRPC6 channel activation increased [Ca2+]i concentration, inhibited proliferation, and triggered apoptotic cell death in primary neonatal pig GMCs. HF-induced neonatal GMC apoptosis was not associated with oxidative stress. However, HF-induced TRPC6 channel activation stimulated nuclear translocation of the nuclear factor of activated T-cells, cytoplasmic 1 (NFATc1). HF also increased cell death surface receptor Fas ligand (FasL) level and caspase-8 activity in the cells; effects mitigated by [Ca2+]i chelator BAPTA, calcineurin/NFAT inhibitor VIVIT, and TRPC6 channel knockdown. Accordingly, HF-induced neonatal GMC apoptosis was attenuated by BAPTA, VIVIT, Fas blocking antibody, and a caspase-3/7 inhibitor. These findings suggest that TRPC6 channel-dependent [Ca2+]i elevation and the ensuing induction of the calcineurin/NFAT, FasL/Fas, and caspase signaling cascades promote neonatal pig GMC apoptosis.

Endometrial gene expression of women with recurrent pregnancy losses and infertility

Tumor necrosis factor-like weak inducer of apoptosis (TWEAK) is a cytokine of the TNF superfamily that activates the Fn14 receptor. TWEAK may regulate cell proliferation, cell death, cell differentiation, and inflammation. TWEAK and Fn14 are constitutively present in the kidney. Sources of TWEAK and Fn14 include intrinsic renal cells and infiltrating leukocytes. Basal Fn14 expression is low, but Fn14 is greatly upregulated during kidney injury. TWEAK contributes to kidney inflammation promoting chemokine secretion by renal cells through canonical and non-canonical NFκB activation. TWEAK also promotes tubular cell proliferation. However, TWEAK induces mesangial and tubular cell apoptosis under proinflammatory conditions. These data indicate that TWEAK is a multifunctional cytokine in the kidney, the actions of which are modulated by the cell microenvironment. Confirmation of the role of TWEAK in kidney injury came from functional studies in experimental animal models. The TWEAK/Fn14 pathway contributed to cell death and interstitial inflammation during acute kidney injury, to glomerular injury in lupus nephritis, to hyperlipidemia-associated kidney injury, and to tubular cell hyperplasia following unilateral nephrectomy. Circulating soluble TWEAK (sTWEAK) levels are a potential biomarker of adverse outcomes in chronic kidney disease and urinary sTWEAK is a potential biomarker of lupus nephritis activity. The available evidence suggests that TWEAK may provide diagnostic information and be a therapeutic target in renal injury. Its role in human kidney disease should be further explored.

Sublytic C5b-9 triggers glomerular mesangial cell apoptosis in rat Thy-1 nephritis via Gadd45 activation mediated by Egr-1 and p300-dependent ATF3 acetylation.

The apoptosis of glomerular mesangial cells (GMCs) is considered to be an important contributor to the initiation and development of rat Thy-1 nephritis (Thy-1N) and is accompanied by sublytic C5b-9 deposition. However, the mechanism by which sublytic C5b-9 triggers GMC apoptosis has not been elucidated. In this study, functional and histological examinations were performed on GMCs treated with sublytic C5b-9 (in vitro) and renal tissues of Thy-1N rats (in vivo). The in vitro studies found that sublytic C5b-9 could trigger GMC apoptosis through upregulating Egr-1, ATF3, and Gadd45 expression. Egr-1-mediated post-transcriptional modulation of ATF3, Egr-1/ATF3-enhanced Gadd45 promoter activity, and p300-mediated ATF3 acetylation were all involved in GMC apoptosis. More importantly, the effective binding elements for Egr-1 and ATF3 to Gadd45β/γ promoters and the ATF3 acetylation site were identified. In vivo, silencing renal p300, Egr-1, ATF3, and Gadd45β/γ significantly decreased GMC apoptosis, secondary GMC proliferation, and urinary protein secretion in Thy-1N rats. Together, these findings implicate that sublytic C5b-9-induced activation of Egr-1/p300-ATF3/Gadd45 axis plays a critical role in GMC apoptosis in Thy-1N rats.

Effects of p53 on aldosterone-induced mesangial cell apoptosis in vivo and in vitro.

Aldosterone (ALD) is a well-known hormone, which may initiate renal injury by inducing mesangial cell (MC) injury in chronic kidney disease (CKD); however, the molecular mechanism remains unknown. The aim of the present study was to investigate the effects of p53 on ALD-induced MC apoptosis and elucidate the underlying molecular mechanism. For the in vivo studies, rats were randomly assigned to receive normal saline or ALD for 4 weeks. The ratio of MC apoptosis was analysed by terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) assay. In addition, the expression level and localisation of p53, a well-known cell apoptosis-associated key protein, were detected by immunofluorescence. For the in vitro studies, rat MCs were incubated in medium containing either buffer (control) or ALD (10−6 M) for 24 h. The cell apoptosis ratio was assessed by flow cytometry, and the expression level of p53 was assessed by reverse transcription quantitative polymerase chain reaction and western blotting. In order to confirm the role of p53 in ALD-regulated cell apoptosis, a rescue experiment was performed using targeted small interfering (si)RNA to downregulate the expression of p53. The ALD-treated rats exhibited greater numbers of TUNEL-positive MCs and higher expression levels of p53 when compared with the control group. Furthermore, the ratio of MC apoptosis and the p53 expression level were significantly increased following ALD exposure, compared with the control group. Additionally, in the rescue experiment, the effects of ALD on MC were blocked by downregulating the expression level of p53 in MCs. The present study hypothesized that ALD may directly contribute to the occurrence of MC apoptosis via p53, which may participate in ALD-induced renal injury.

Asymmetric dimethylarginine (ADMA) treatment induces apoptosis in cultured rat mesangial cells via endoplasmic reticulum stress activation

Asymmetric dimethylarginine (ADMA), a high risk factor for endothelial dysfunction and cardiovascular disease (CVD), has been reported to promote cellular dysfunction via endoplasmic reticulum (ER) stress activation in various cells. Additionally, increased serum ADMA levels have been observed in incipient kidney diseases. Previously, we reported that activated ER stress is associated with mesangial cell apoptosis, observed mainly in overt nephropathy or chronic kidney disease (CKD). However, the effect of ADMA on mesangial cell apoptosis is unknown. Thus, we investigated the effects of ADMA on mesangial cell apoptosis and ER stress signaling. ADMA treatment increased caspase-3 activity and activated three branches of ER stress signaling (PERK, IRE1, and ATF6) that induce mesangial cell apoptosis. Pharmacological inhibitors of ER stress (inhibitors of PERK, IRE1, and S1P) attenuated ADMA-induced cleavage of caspase-3 and induced a decrease in the mitochondrial membrane potential. Furthermore, these inhibitors diminished the number of apoptotic cells induced by ADMA treatment. Taken together, our results indicated that ADMA treatment induces mesangial cell apoptosis via ER stress signaling. These results suggest that ADMA-induced mesangial cell apoptosis could contribute to the progression of overt nephropathy and CKD.

CagA promotes proliferation and secretion of extracellular matrix by inhibiting signaling pathway of apoptosis in rat glomerular mesangial cells

Cytotoxin-associated antigen A (CagA), a major virulence factor of Helicobacter pylori (Hp), is associated with the pathogenesis of peptic ulcer and gastric cancer. Recent researches demonstrated that Hp exists in palatine tonsil in all studied IgA nephropathy (IgAN) patients, most of which were CagA-positive, suggesting that CagA may be a causative pathogenic factor of IgAN. However, the underlying molecular mechanisms and signaling pathway are still largely unclear. In the present study, CCK8 assay, enzyme-linked immunosorbent assay, and immunohistochemistry were performed to investigate the effect of CagA on cell proliferation and extracellular matrix secretion in rat glomerular mesangial cells. RT-PCR and western blotting were used to reveal the potential signaling pathway. Rat glomerular mesangial cells were treated with recombinant CagA protein for 72 h, in a dose- and time-dependent manner. We found that CagA promoted cell proliferation and extracellular matrix secretion by inhibiting signaling pathway of apoptosis. Taken together, these findings suggested that CagA induced cellular injury in glomerular mesangium by proliferation and secretion of extracellular matrix, and may play an important role in pathogenesis of IgAN.

Role of the ER stress in prostaglandin E2/E-prostanoid 2 receptor involved TGF-β1-induced mice mesangial cell injury.

This study aims to investigate the relationship between prostaglandin E2 E-prostanoid 2 receptor (EP2) and Endoplasmic reticulum (ER) stress in transforming growth factor-β1 (TGF-β1)-induced mouse glomerular mesangial cells (MCs) injury. We cultured primary WT, EP2(-/-) MCs (EP2 deleted), and adenovirus-EP2-infected WT MCs (EP2 overexpressed). PCR, Western blot, flow cytometry, and immunohistochemical technique were used in in vitro and in vivo experiments. We found that TGF-β1-induced PGE2 synthesis decreased in EP2-deleted MCs and increased in EP2-overexpressed MCs. EP2 deficiency in these MCs augmented the coupling of TGF-β1 to ER stress-associated proteins [chaperone glucose-regulated protein 78 (GRP78), transient receptor potential channel 1 (TRPC1), and transient receptor potential channel 4 (TRPC4)], and upregulation of EP2 showed no significant change of GRP78, but augmented the expression of TRPC1, while TRPC4 expression was downregulated in comparison to normal MCs. In addition, EP2 deficiency in MCs augmented TGF-β1-induced fibronectin (FN), cyclooxygenase-2 (COX2), and CyclinD1 expression. Silencing of EP2 also strengthened TGF-β1-induced extracellular-signal-regulated kinase 1/2 (ERK1/2) phosphorylation. Flow Cytometry showed that silencing of EP2 significantly promoted the apoptosis of MCs. In contrast, EP2 overexpression reversed the effects of EP2 deficiency. 8weeks after 5/6 nephrectomy (Nx), blood urea nitrogen and creatinine concentrations were significantly increased in EP2(-/-) 5/6Nx mice as compared to those of WT 5/6Nx mice. The pathological changes in kidney of EP2(-/-) mice were markedly aggravated compared with WT mice. Immunohistochemical analysis showed significant augment of TRPC4 and ORP150 in the kidney of EP2(-/-) mice compared with WT mice. Considering all the findings, it is suggested that increased expression of EP2 may prevent TGF-β1-induced MCs damage through ER stress regulatory pathway.

Extracellular matrix‐induced Hic‐5 expression in glomerular mesangial cells leads to a prosclerotic phenotype independent of TGF‐β

Chronic fibroproliferative diseases account for approximately 45% of all deaths in the developed world. In the kidney, glomerulosclerosis is the underlying pathology in approximately half of patients with renal failure receiving dialysis. Mesangial cell expression of the LIM protein hydrogen peroxide-induced clone-5 (Hic-5) is important in its pathogenesis. Hic-5 expression increases following mesangial cell attachment to collagen I, associated with increased collagen I expression and increased susceptibility to apoptosis both in vitro and in experimental glomerulosclerosis. TGF-β has an established role in many fibrotic diseases, including glomerulosclerosis, where it increases collagen I deposition in vivo and promotes mesangial cell apoptosis in vitro. In other cell types, TGF-β induces Hic-5 expression. We investigated whether Hic-5-induced changes in mesangial cell phenotype were TGF-β-dependent. Adding exogenous TGF-β to mesangial cell cultures failed to increase Hic-5 expression; blocking TGF-β signaling did not reduce Hic-5 expression. However, inducing Hic-5 expression in mesangial cells by adhesion to collagen I led to TGF-β expression, which was abolished by small interfering RNA (siRNA) Hic-5 knockdown. Mesangial cells expressing Hic-5 showed altered latent TGF-β-binding protein expression and Smad signaling, with enhanced susceptibility to TGF-β-induced apoptosis. Mesangial cell attachment to collagen I led to increased Hic-5 expression within 2-4 h and increased procollagen I transcription within 12 h, whereas adding TGF-β to siRNA Hic-5 knockdown mesangial cells increased procollagen I transcription to a lesser degree after 48 h. Mesangial cell Hic-5 expression was associated with increased α-smooth muscle actin and plasminogen activator inhibitor-1 expression. Taken together, these data indicate that there is a prosclerotic feedback loop in mesangial cells dependent on matrix-derived signals in which Hic-5 is a pivotal signaling protein. This feedback loop is TGF-β-independent. The role of TGF-β-dependent and -independent sclerotic pathways merit further investigation.

Modulation of cyclins and p53 in mesangial cell proliferation and apoptosis during Habu nephritis.

BackgroundMesangial cell (MC) proliferation and apoptosis are the main pathological changes observed in mesangial proliferative nephritis. In this study, we explored the role of cyclins and p53 in modulating MC proliferation and apoptosis in a mouse model of Habu nephritis.MethodsThe Habu nephritis group was prepared by injection of Habu toxin. Mesangiolysis and mesangial expansion were determined by periodic acid-Schiff (PAS) reagent staining. Immunohistochemical analysis of PCNA and KI67, and TUNEL staining were used to detect cell proliferation and apoptosis, respectively. Expression levels of cyclins and p53 were examined by Western blotting.ResultsPAS staining showed that mesangial dissolution appeared on days 1 and 3, and mesangial proliferation with extracellular matrix accumulation was apparent by days 7 and 14. Both PCNA and KI67 immunohistochemical analysis showed that MC proliferation began on day 3, peaked on day 3 and 7, and recovered by day 14. TUNEL staining results showed that MC apoptosis began to increase on day 1, continued to rise on day 7, and peaked on day 14. Western blot analysis showed that cyclin D1 was upregulated on day 1, cyclins A2 and E were upregulated on days 3 and 7, and p53 was upregulated on days 3, 7 and 14. There was no change in the expression levels of Bax or p21.ConclusionWe explored the tendency for MC proliferation and apoptosis during the process of Habu nephritis and found that cyclins and p53 may modulate the disease pathology. This will help us determine the molecular pathogenesis of MC proliferation and provide new targets for disease intervention.

Metformin ameliorates lipotoxicity-induced mesangial cell apoptosis partly via upregulation of glucagon like peptide-1 receptor (GLP-1R)

Glucagon like peptide-1 receptor (GLP-1R), known to be expressed in pancreatic beta cells, is also expressed in glomerular mesangial cells and its agonist has protective effects in diabetic nephropathy. However, its regulatory mechanisms by lipotoxicity in glomerular mesangial cells are not understood. We found that palmitate-mediated lipotoxicity increased apoptosis and decreased GLP-1R expression in a rat mesangial cell line. Silencing GLP-1R expression also increased mesangial cell apoptosis. Interestingly, metformin, one of the biguanide drugs that has anti-diabetic effects, attenuated lipotoxicity-induced mesangial cell apoptosis and restored GLP-1R expression. Moreover, this treatment alleviated GLP-1R knockdown-induced mesangial cell apoptosis. To further evaluate in vivo, diabetic obese db/db mice were administered metformin. Glomerular GLP-1R expression was diminished in db/db mice, as compared with db/m control mice. However, this decrease significantly recovered on metformin administration. Together, these data provide novel evidence that lipotoxicity decreases the mesangial GLP-1R expression in intact cells and in vivo. The decrease induced mesangial cell apoptosis. Furthermore, we provided the evidence that metformin treatment has a renal protective effect partly via increased mesangial GLP-1R expression. Our data suggested that regulation of GLP-1R expression could be a promising approach to treat diabetic nephropathy and the novel mechanism of metformin mediated GLP-1R regulation.

CHOP mediates XBP1S-induced renal mesangial cell necrosis following high glucose treatment

High glucose (HG)-induced apoptosis in mesangial cells (MCs) is a critical determinant during the pathogenesis of diabetic nephropathy. The signaling cascade inducing MCs apoptosis by HG involves overproduction of reactive oxygen species. Our previous studies have demonstrated that HG-induced oxidative stress is mediated by suppression of spliced/active X-box binding protein 1 (XBP1S), suggesting the importance of XBP1S in HG-induced MCs apoptosis. CHOP, an endoplasmic reticulum stress-associated proapoptotic signal, is involved in downstream of XBP1S. In the present study, we explored the effect of XBP1S in modulating HG-induced apoptosis in renal MCs and then identified the role of CHOP in these processes. Apoptosis and necrosis were quantified by flow cytometry; protein levels of XBP1S, caspase3, Bax, Bcl2, BNIP3, and CHOP were analyzed by Western blotting. The cellular localization of XBP1S was determined by immunofluorescence histochemistry. The binding of XBP1 to CHOP promoter was determined by chromatin immunoprecipitation assays. In addition, adenoviruses harboring XBP1S gene (Ad-XBP1S) were used to overexpress XBP1S, whereas the knockdown of CHOP was achieved by small interference RNA. HG suppressed nuclear distribution of XBP1S and induced apoptosis and necrosis in MCs. Ad-XBP1S infection enhanced the nuclear translocation of XBP1S and reduced MCs apoptosis and necrosis. XBP1S bound to the promoter region of CHOP and upregulated CHOP expression. Conversely, CHOP expression was reduced upon HG exposure and knockdown of CHOP increased necrosis but not apoptosis in MCs. These results suggest that XBP1S protected MCs from HG-induced apoptosis and necrosis, and CHOP participates in XBP1S-regulated necrosis but not apoptosis.

TRPC6 Channel‐Mediated Neonatal Glomerular Mesangial Cell Apoptosis Involves Activation of Calcineurin/Nuclear Factor of Activated T‐Cell Signaling

Alterations in glomerular mesangial cell (GMC) survival are involved in the pathogenesis of progressive glomerulopathies. However, the mechanisms that underlie GMC survival are not fully resolved. In this study, we examined whether activation of the canonical transient receptor potential (TRPC) 6 channels modulates GMC survival. Hyperforin (HF)-induced TRPC6 channel activation elevated intracellular Ca2+ ([Ca2+]i) concentration, inhibited proliferation, and promoted apoptosis in primary GMCs derived from newborn pig kidneys. Dichlorodihydrofluorescein diacetate assay revealed that HF-induced GMC apoptosis is not associated with oxidative stress. However, HF-induced GMC apoptosis was attenuated by BAPTA-AM, a [Ca2+]i chelator, Ac-DEVD-CHO, a caspase-3/7 inhibitor, and siRNA-mediated TRPC6 channel knockdown. HF also stimulated nuclear translocation of NFATC1 in the cells in a [Ca2+]i-dependent manner. Furthermore, HF-induced apoptosis was abrogated in GMCs pretreated with calcineurin/NFAT signaling inhibitors FK506 and 11R-VIVIT. These findings suggest that an elevation in [Ca2+]i elicited by TRPC6 channel activation promotes neonatal GMC apoptosis by activating the calcineurin/NFAT signaling pathway.

CXCL10 expression induced by Mxi1 inactivation induces mesangial cell apoptosis in mouse Habu nephritis

MAX interactor 1 (Mxi1) proteins are c-myc antagonists that primarily exert their biological functions by inhibiting Myc-dependent gene transcription. In this study, Mxi1−/− mice were used to generate a model of mesangial proliferative glomerulonephritis for the first time. In the present study, we demonstrated that Mxi1−/− mice exhibited a more typical and severe pathological phenotype, which was displayed primarily as a noticeable dissolution phenotype with a higher proportion of apoptotic cells and higher chemokine CXCL10 expression during the early days of modeling, compared with wild-type mice. Additionally, we determined that IRF3-mediated TLR4 signaling was likely involved in regulating CXCL10 expression, which might participate in the mesangial dissolution process. We also found increases in CXCL10 expression, caspase 3 activation, and the proportion of apoptotic cells when Mxi1 expression was inhibited in mouse mesangial cells. Furthermore, the proportion of apoptotic cells decreased after inhibiting CXCL10 expression. Therefore, we concluded that the mesangial cell apoptosis observed in this mesangial proliferative glomerulonephritis model was related to CXCL10 expression induced by Mxi1 inactivation. This finding provides a new theoretical basis for the mechanism of mesangial proliferative glomerulonephritis progression and reveals potential intervention targets for the early treatment of this disease.