Mouse Mesangial Cells Research Articles

Betaine alleviates high glucose‑induced mesangial cell proliferation by inhibiting cell proliferation and extracellular matrix deposition via the AKT/ERK1/2/p38 MAPK pathway

Diabetic nephropathy (DN) is a major cause of chronic renal failure in diabetic patients worldwide. Betaine, a zwitterionic quaternary ammonium salt compound, is involved in numerous biological processes. The present study aimed to investigate the effects of betaine on mouse mesangial cells (MMCs) cultured under high glucose (HG) conditions and its underlying mechanisms. MMCs were treated with betaine under HG conditions. Cell proliferation and the cell cycle distribution were investigated with an MTT assay and flow cytometry, respectively. Western blotting and reverse transcription-quantitative polymerase chain reaction analyses were applied to respectively determine protein and mRNA expression levels. The results suggested that betaine decreased cell proliferation in a dose-dependent manner, while G1-phase arrest was significantly induced in MMCs. Compared with the control group, the expression levels of p21 and p27 decreased under HG conditions, but were reversed by betaine. Furthermore, the expression levels of fibronectin and type IV collagen were significantly decreased in cells treated with betaine compared with the HG group. Additionally, betaine decreased the phosphorylation of Akt, extracellular-signal-regulated kinase (Erk)1/2 and p38 mitogen-activated protein kinase (MAPK), but was enhanced under HG conditions. Overall, the results of the present study indicated that betaine serves a protective role in HG-induced MMCs by inhibiting cell proliferation and extracellular matrix deposition via regulating regulation of the Akt/Erk1/2/p38 MAPK signaling pathway.

Hydrogen sulphide mitigates homocysteine-induced apoptosis and matrix remodelling in mesangial cells through Akt/FOXO1 signalling cascade

Cellular damage and accumulation of extracellular matrix (ECM) protein in the glomerulo-interstitial space are the signatures of chronic kidney disease (CKD). Hyperhomocysteinemia (HHcy), a high level of homocysteine (Hcy) is associated with CKD and further contributes to kidney damage. Despite a large number of studies, the signalling mechanism of Hcy-mediated cellular damage and ECM remodelling in kidney remains inconclusive. Hcy metabolizes to produce hydrogen sulphide (H2S), and a number of studies have shown that H2S mitigates the adverse effect of HHcy in a variety of diseases involving several signalling molecules, including forkhead box O (FOXO) protein. FOXO is a group of transcription factor that includes FOXO1, which plays important roles in cell growth and proliferation. On the other hand, a cell survival factor, Akt regulates FOXO under normal condition. However, the involvement of Akt/FOXO1 pathway in Hcy-induced mesangial cell damage remains elusive, and whether H2S plays any protective roles has yet to be clearly defined. We treated mouse mesangial cells with or without H2S donor, GYY4137 and FOXO1 inhibitor, AS1842856 in HHcy condition and determined the involvement of Akt/FOXO1 signalling cascades. Our results indicated that Hcy inactivated Akt and activated FOXO1 by dephosphorylating both the signalling molecules and induced FOXO1 nuclear translocation followed by activation of the FOXO1 transcription factor. These led to the induction of cellular apoptosis and synthesis of excessive ECM protein, in part, due to increased ROS production, loss of mitochondrial membrane potential (ΔΨm), reduction in intracellular ATP concentration, increased MMP-2, -9, -14 mRNA and protein expression, and Col I, IV and fibronectin protein expression. Interestingly, GYY4137 or AS1842856 treatment prevented these changes by modulating Akt/FOXO1 axis in HHcy. We conclude that GYY4137 and/or AS1842856 mitigates HHcy induced mesangial cell damage and ECM remodelling by regulating Akt/FOXO1 pathway.

Identification of potential therapeutic target genes in mouse mesangial cells associated with diabetic nephropathy using bioinformatics analysis

The aim of the present study was to identify genes under the effect of transforming growth factor-β (TGF-β1), high glucose (HG) and glucosamine (GlcN) in MES-13 mesangial cells and elucidate the molecular mechanisms of diabetic nephropathy (DN). The gene expression datasets GSE2557 and GSE2558 were downloaded from the Gene Expression Omnibus database. Differentially expressed genes (DEGs) were independently screened using the GEO2R online tool. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were performed using the Database for Annotation, Visualization, and Integrated Discovery. The protein-protein interaction (PPI) network was constructed using the Search Tool for the Retrieval of Interacting Genes and Cytoscape software. The hub genes were identified by the NetworkAnalyzer plugin. Overlapping genes were subjected to molecular docking analysis using SystemsDock. A total of 202 upregulated and 158 downregulated DEGs from the HG-treated groups, 138 upregulated and 103 downregulated DEGs from the GlcN-treated groups, and 81 upregulated and 44 downregulated DEGs from the TGF-β1-treated groups were identified. The majority of the DEGs were independently enriched in ‘nucleosome assembly’, ‘chromatin silencing’ and ‘xenobiotic glucuronidation’. In addition, KEGG pathways were significantly enriched in ‘systemic lupus erythematosus’, ‘protein processing in endoplasmic reticulum’ and ‘aldarate metabolism pathway’, and ‘TNF signaling pathway’ intersected in the TGF-β1-treated and HG-treated groups. In total, eight hub genes, Jun, prostaglandin-endoperoxide synthase 2 (Ptgs2), fibronectin 1 (Fn1), cyclin-dependent kinase (Cdk)2, Fos, heat shock protein family A (Hsp70) member 5 (Hspa5), Hsp90b1 and homo sapiens hypoxia upregulated 1 (Hyou1), and three overlapping genes, Ras homolog gene family, member B (RHOB), complement factor H (CFH) and Krüppel-like factor 15 (KLF15), were selected. Valsartan with RHOB, and fosinopril with CFH and KLF15 had preferential binding activity. In conclusion, Jun, Ptgs2, Fn1, Cdk2, Fos, Hspa5, Hsp90b1, Hyou1, RHOB, CFH and KLF15 may be potential therapeutic targets for mesangial cells associated with DN, which may provide insight into DN treatment strategies.

PTPN2 improved renal injury and fibrosis by suppressing STAT-induced inflammation in early diabetic nephropathy.

Diabetic nephropathy (DN) is a chronic inflammatory disease triggered by disordered metabolism. Recent studies suggested that protein tyrosine phosphatase non‐receptor type 2 (PTPN2) could ameliorate metabolic disorders and suppress inflammatory responses. This study investigated PTPN2's role in modulating DN and the possible cellular mechanisms involved. In a mouse model combining hyperglycaemia and hypercholesterolaemia (streptozotocin diabetic, ApoE‐/‐ mice), mice showed severe insulin resistance, renal dysfunction, micro‐inflammation, subsequent extracellular matrix expansion and decreased expression of PTPN2. We found that mice treated with PTPN2 displayed reduced serum creatinine, serum BUN and proteinuria. PTPN2 gene therapy markedly attenuated metabolic disorders and hyperglycaemia. In addition, PTPN2 gene transfer significantly suppressed renal activation of signal transducers and activators of transcription (STAT), STAT‐dependent pro‐inflammatory and pro‐fibrotic genes expression, and influx of lymphocytes in DN, indicating anti‐inflammatory effects of PTPN2 by inhibiting the activation of STAT signalling pathway in vivo. Furthermore, PTPN2 overexpression inhibited the high‐glucose induced phosphorylation of STAT, target genes expression and proliferation in mouse mesangial and tubuloepithelial cells, suggesting that the roles of PTPN2 on STAT activation was independent of glycaemic changes. Our results demonstrated that PTPN2 gene therapy could exert protective effects on DN via ameliorating metabolic disorders and inhibiting renal STAT‐dependent micro‐inflammation, suggesting its potential role for treatment of human DN.

Long noncoding RNA NONHSAG053901 promotes diabetic nephropathy via stimulating Egr‐1/TGF‐β‐mediated renal inflammation

Diabetic nephropathy (DN) is an important factor leading to end-stage kidney disease that affects diabetes mellitus patients globally. Our previous transcriptome sequencing has identified a large group of differentially expressed long noncoding RNA (lncRNA) in early development of DN. On basis of this, we aimed to investigate the function of lncRNA NONHSAG053901 in DN pathogenesis. In this study, we revealed that the expression of NONHSAG053901 was drastically elevated in both DN mouse model and mesangial cells (MCs). It was found that overexpression of NONHSAG053901 remarkably promoted inflammation, fibrosis and proliferation in MCs. Consistently, further investigations suggested that the stimulation of NONHSAG053901 on proinflammatory cytokines via direct binding to early growth response protein 1 (Egr-1). Interaction between Egr-1 and transforming growth factor β (TGF-β) could augment TGF-β function in DN inflammation. Furthermore, the effects of NONHSAG053901 on stimulation of proinflammatory cytokines were abolished by knockdown of Egr-1. These results together suggested that NONHSAG053901 promoted proinflammatory cytokines via stimulating Egr-1/TGF-β mediated renal inflammation.

Amelioration of diabetic nephropathy by SGLT2 inhibitors independent of its glucose-lowering effect: A possible role of SGLT2 in mesangial cells

Several clinical studies have shown the beneficial effects of sodium-glucose cotransporter 2 (SGLT2) inhibitors on diabetic nephropathy. The underlying mechanisms are not fully understood. We found that administration of canagliflozin at a low dose (0.01 mg/kg/day) did not affect either blood glucose levels or glycosuria, but it improved albuminuria and mesangial expansion in db/db mice to a similar extent as at a high dose (3.0 mg/kg/day) that lowered blood glucose levels. This indicated the existence of a tubular SGLT2-independent reno-protective mechanism. Here we focused on the potential role of SGLT2 in mesangial cells (MCs). Western blot analysis revealed the expression of SGLT2 in cultured mouse MCs. Exposure of MCs to high glucose levels for 72 h significantly increased the expression of SGLT2. Canagliflozin or ipragliflozin (both 100 nM) treatment inhibited glucose consumption in the medium under high-glucose conditions but not under normal-glucose conditions. Furthermore, canagliflozin inhibited high-glucose-induced activation of the protein kinase C (PKC)-NAD(P)H oxidase pathway and increases in reactive oxygen species (ROS) production. Thus, the inhibition of mesangial SGLT2 may cause an inhibition of PKC activation and ROS overproduction in diabetic nephropathy, and this may at least in part account for the reno-protective effect of SGLT2 inhibitors.

Lysophosphatidic acid increases mesangial cell proliferation in models of diabetic nephropathy via Rac1/MAPK/KLF5 signaling

Mesangial cell proliferation has been identified as a major factor contributing to glomerulosclerosis, which is a typical symptom of diabetic nephropathy (DN). Lysophosphatidic acid (LPA) levels are increased in the glomerulus of the kidney in diabetic mice. LPA is a critical regulator that induces mesangial cell proliferation; however, its effect and molecular mechanisms remain unknown. The proportion of α-SMA+/PCNA+ cells was increased in the kidney cortex of db/db mice compared with control mice. Treatment with LPA concomitantly increased the proliferation of mouse mesangial cells (SV40 MES13) and the expression of cyclin D1 and CDK4. On the other hand, the expression of p27Kip1 was decreased. The expression of Krüppel-like factor 5 (KLF5) was upregulated in the kidney cortex of db/db mice and LPA-treated SV40 MES13 cells. RNAi-mediated silencing of KLF5 reversed these effects and inhibited the proliferation of LPA-treated cells. Mitogen-activated protein kinases (MAPKs) were activated, and the expression of early growth response 1 (Egr1) was subsequently increased in LPA-treated SV40 MES13 cells and the kidney cortex of db/db mice. Moreover, LPA significantly increased the activity of the Ras-related C3 botulinum toxin substrate (Rac1) GTPase in SV40 MES13 cells, and the dominant-negative form of Rac1 partially inhibited the phosphorylation of p38 and upregulation of Egr1 and KLF5 induced by LPA. LPA-induced hyperproliferation was attenuated by the inhibition of Rac1 activity. Based on these results, the Rac1/MAPK/KLF5 signaling pathway was one of the mechanisms by which LPA induced mesangial cell proliferation in DN models.

Methylglyoxal evokes acute Ca2+ transients in distinct cell types and increases agonist-evoked Ca2+ entry in endothelial cells via CRAC channels

Methylglyoxal (MG) is a by-product of glucose metabolism and its accumulation has been linked to the development of diabetic complications such as retinopathy and nephropathy by affecting multiple signalling pathways. However, its influence on the intracellular Ca2+ homeostasis and particularly Ca2+ entry, which has been reported to be mediated via TRPA1 channels in DRG neurons, has not been studied in much detail in other cell types. In this study, we report the consequences of acute and long-term MG application on intracellular Ca2+ levels in endothelial cells. We showed that acute MG application doesn’t evoke any instantaneous changes in the intracellular Ca2+ concentration in immortalized mouse cardiac endothelial cells (MCECs) and murine microvascular endothelial cells (muMECs). In contrast, an MG-induced rise in intracellular Ca2+ level was observed in primary mouse mesangial cells within 30 s, indicating that the modulation of Ca2+ homeostasis by MG is strictly cell type specific. The formation of the MG-derived advanced glycation end product (AGE) MG-H1 was found to be time and concentration-dependent in MCECs. Likewise, MG pre-incubation for 6 h increased the angiotensin II-evoked Ca2+ entry in MCECs and muMECs which was abrogated by inhibition of Calcium release activated calcium (CRAC) channels with GSK-7975A, but unaffected by an inhibitor specific to TRPA1 channels. Quantitative PCR analysis revealed that MG pre-treatment did not affect expression of the genes encoding the angiotensin receptors AT1R (Agtr 1a & Agtr 1b), Trpa1 nor Orai1, Orai2, Orai3, Stim1, Stim2 and Saraf which operate as constituents or regulators of CRAC channels and store-operated Ca2+ entry (SOCE) in other cell types. Together, our results show that long-term MG stimulation leads to the formation of glycation end products, which facilitates the agonist-evoked Ca2+ entry in endothelial cells, and this could be a new pathway that might lead to MG-evoked vasoregression observed in diabetic vasculopathies.

All-trans retinoic acid suppresses bone morphogenetic protein 4 in mouse diabetic nephropathy through a unique retinoic acid response element.

Diabetic nephropathy (DN) causes mesangial matrix expansion, which results in glomerulosclerosis and renal failure. Collagen IV (COL4) is a major component of the mesangial matrix that is positively regulated by bone morphogenetic protein 4 (BMP4)/suppressor of mothers against decapentaplegic (Smad1) signaling. Because previous studies showed that retinoids treatment had a beneficial effect on kidney disease, we investigated the therapeutic potential of retinoids in DN, focusing especially on the regulatory mechanism of BMP4. Diabetes was induced with streptozotocin in 12-wk-old male Crl:CD1(ICR) mice, and, 1 mo later, we initiated intraperitoneal injection of all-trans retinoic acid (ATRA) three times weekly. Glomerular matrix expansion, which was associated with increased BMP4, phosphorylated Smad1, and COL4 expression, worsened in diabetic mice at 24 wk of age. ATRA administration alleviated DN and downregulated BMP4, phosopho-Smad1, and COL4. In cultured mouse mesangial cells, treatment with ATRA or a retinoic acid receptor-α (RARα) agonist significantly decreased BMP4 and COL4 expression. Genomic analysis suggested two putative retinoic acid response elements (RAREs) for the mouse Bmp4 gene. Chromatin immunoprecipitation analysis and reporter assays indicated a putative RARE of the Bmp4 gene, located 11,488-11,501 bp upstream of exon 1A and bound to RARα and retinoid X receptor (RXR), which suppressed BMP4 expression after ATRA addition. ATRA suppressed BMP4 via binding of a RARα/RXR heterodimer to a unique RARE, alleviating glomerular matrix expansion in diabetic mice. These findings provide a novel regulatory mechanism for treatment of DN.

LncRNA NEAT1 promotes extracellular matrix accumulation and epithelial-to-mesenchymal transition by targeting miR-27b-3p and ZEB1 in diabetic nephropathy.

Diabetic nephropathy (DN) is a kind of microvascular complications of diabetes. Long noncoding RNAs (lnRNAs) can participate in the development of various diseases, including DN. However, the function of lncRNA NEAT1 is unclear. In our present study, we reported that NEAT1 was significantly increased in streptozotocin-induced DN rat models and high-glucose-induced mice mesangial cells. We observed that knockdown of NEAT1 greatly inhibited renal injury of DN rats. Meanwhile, downregulation of NEAT1-modulated extracellular matrix (ECM) proteins (ASK1, fibronectin, and TGF-β1) expression and epithelial-mesenchymal transition (EMT) proteins (E-cadherin and N-cadherin) in vitro. Previously, miR-27b-3p has been reported to be involved in diabetes. Here, miR-27b-3p was decreased in DN rats and high-glucose-induced mice mesangial cells. The direct correlation between NEAT1 and miR-27b-3p was validated using the dual-luciferase reporter assay and RNA immunoprecipitation experiments. In addition, zinc finger E-box binding homeobox 1 (ZEB1), which has been identified in the process of EMT clearly contributes to EMT progression. ZEB1 was predicted as a target of miR-27b-3p and overexpression of miR-27b-3p dramatically repressed ZEB1 expression. Therefore, our data implied the potential role of NEAT1 in the fibrogenesis and EMT in DN via targeting miR-27b-3p and ZEB1.

Long noncoding RNA NEAT1 accelerates the proliferation and fibrosis in diabetic nephropathy through activating Akt/mTOR signaling pathway.

Accumulating evidence has indicated the significant roles of long noncoding RNAs (lncRNAs) in the pathophysiology of diabetic nephropathy (DN). LncRNA nuclear enriched abundant transcript 1 (NEAT1) has been reported to exert a key role in the progression of several diseases including diabetes. However, the role of NEAT1 in the regulation of DP progression remains barely known. Therefore, our study aimed to investigate the role of NEAT1 in a streptozotocin-induced diabetes model (DM) of rats and glucose-induced mouse mesangial cell models. Currently, we found that NEAT1 was greatly upregulated in DM rats and glucose-induced mice mesangial cells, in which a high activation of Akt/mTOR signaling was also observed. Then, it was shown that knockdown of NETA1 was able to reduce renal injury in DM rats obviously. In addition, cell counting kit-8 assay and 5-ethynyl-2'-deoxyuridine assay were carried out and we observed downregulation of NEAT1 significantly inhibited mesangial cell proliferation. Meanwhile, extracellular matrix proteins and messenger RNA (transforming growth factor β1, fibronectin, and collagen IV) expression was dramatically restrained by silencing of NEAT1 in the high glucose-induced mesangial cells. Finally, knockdown of NEAT1 greatly reduced the expression of the phosphorylation of Akt and mammalian target of rapamycin (mTOR) in vitro. These findings revealed that the decrease of NEAT1 repressed the proliferation and fibrosis in DN via activating the Akt/mTOR signaling pathway, which might represent a novel pathological mechanism of DN progression.

Early growth response protein-1 upregulates long noncoding RNA Arid2-IR to promote extracellular matrix production in diabetic kidney disease

Diabetic kidney disease (DKD) has surpassed chronic glomerulonephritis as the leading cause of end-stage renal disease. Previously, we showed that early growth response protein-1 (Egr1) plays a key role in DKD by enhancing mesangial cell proliferation and extracellular matrix (ECM) production. The long noncoding RNA (lncRNA) AT-rich interactive domain 2-IR (Arid2-IR) has been identified as a mothers against decapentaplegic homolog 3 (Smad3)-associated lncRNA in unilateral ureteral obstructive kidney disease. However, the effect of Egr1 on Arid2-IR in the development of DKD is still unknown. In this study, we found that Arid2-IR was increased in mice with high-fat diet and streptozotocin-induced type 2 diabetes and in mouse mesangial cells cultured with high glucose to mimic diabetes. Knockdown of Arid2-IR in mouse mesangial cells reduced the high expression levels of collagen-α1(I) (Col1a1) and α-smooth muscle actin (α-SMA) induced by high glucose. Furthermore, Arid2-IR expression changed the increased expression of Col1a1 and α-SMA caused by overexpression of Egr1. Overall, these data suggest that increased Arid2-IR likely contributes to ECM production in DKD and that Egr1 promotes ECM production in DKD partly by upregulating Arid2-IR. Thus, Arid2-IR may be a new target in the treatment of DKD.

Long noncoding RNA Gm6135 functions as a competitive endogenous RNA to regulate toll-like receptor 4 expression by sponging miR-203-3p in diabetic nephropathy.

We aim to explore the relationship between Gm6135 and diabetic nephropathy. We detected the relative expression levels of Gm6135 and toll-like receptor 4 (TLR4) in diabetic nephropathy mice and high-glucose-cultured mouse mesangial cells SV40-MES-13 by the quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and western blot detection. Cell proliferation and apoptosis were detected after small interfering RNA (siRNA) interference or plasmid overexpression of Gm6135/TLR4, and bioinformatics method was used to predict and screen miR-203 as an intermediate factor. Through dual-luciferase reporter gene, RNA pull-down, qRT-PCR, and western blot, the binding relationship between Gm6135, miR-203-3p, and TLR4 was confirmed. The possibility of the competing endogenous RNA mechanism was demonstrated by cell localization assays and rip assays. Finally, the proliferation of mouse mesangial cells SV40-MES-13 was detected after mimics and inhibitor of microRNA, which were reversed with TLR4 overexpression and siRNA. The results showed that the relative expression levels of Gm6135 and TLR4 in the kidney and high-glucose-cultured mouse mesangial cells of diabetic nephropathy mice increased significantly. Overexpression or downregulation of Gm6135/TLR4 significantly affected the proliferation and apoptosis of mouse mesangial cells. Gm6135 upregulates TLR4 by competitively binding to miR-203-3p.

Deletion of ApoM gene induces apoptosis in mouse kidney via mitochondrial and endoplasmic reticulum stress pathways

Apolipoprotein M (ApoM) is involved in lipid metabolism, and especially is involved in reverse cholesterol transport. However, the relationship between ApoM and apoptosis has been rarely reported. This study aimed to investigate the effect of ApoM on apoptosis using an ApoM gene-deficient mice (ApoM−/−) model and a mouse mesangial cell model with suppressed ApoM gene expression. First, we observed by transmission electron microscopy that mitochondrial damage and endoplasmic reticulum stress were abnormally altered in the kidneys of ApoM−/− mice compared with wild-type mice, showing mitochondrial swelling, vacuolization, myeloid changes, and expansion of the rough endoplasmic reticulum. At the molecular level, the expression of pro-apoptotic related proteins such as AIF, Bax, chop, clever-caspase 3, clever-caspase 7, clever-caspase 9, and clever-caspase 12 increased, and the expression of anti-apoptotic protein Bcl-2 decreased. Secondly, by interfering with the expression of the ApoM gene in mouse mesangial cells, we found that, compared with the control group (NC-si), the cells of the experimental group (siApoM) showed decreased cell viability, nuclear chromatin condensation, nuclear lysis, and an increased proportion of early apoptotic cells. The results in cells at the molecular level were consistent with those at the tissue level. These data indicated that the deletion of the ApoM gene led to upregulation of apoptosis in mouse kidney tissues and mesangial cells through the mitochondrial and endoplasmic reticulum pathways.

Circular RNA circRNA_15698 aggravates the extracellular matrix of diabetic nephropathy mesangial cells via miR-185/TGF-β1.

Circular RNAs (circRNAs) are a novel type of noncoding RNAs that modulate the pathogenesis of multiple diseases. Nevertheless, the role of circRNAs in diabetic nephropathy (DN) pathogenesis is still ambiguous. In the current study, our team aims to investigate the expression profiles of circRNAs in DN and identify the function of circRNA on mesangial cells. CircRNAs microarray analysis revealed dysregulated circRNA in db/db DN mice, and circRNA_15698 was validated to be upregulated in both db/db mice and mouse mesangial cells (SV40-MES13) that were exposed to high glucose (25 mM) using real-time polymerase chain reaction. Loss-of-functional experiments showed that circRNA_15698 knockdown significantly inhibited the expression levels of collagen type I (Col. I), collagen type IV (Col. IV), and fibronectin. Moreover, the cellular localization of circRNA_15698 was mainly in the cytoplasm. Bioinformatics tools and luciferase reporter assay confirmed that circRNA_15698 acted as a 'sponge' of miR-185, and then positively regulated the transforming growth factor-β1 (TGF-β1) protein expression, suggesting a circRNA_15698/miR-185/TGF-β1 pathway. Further validation experiments validated that circRNA_15698/miR-185/TGF-β1 promoted extracellular matrix (ECM)-related protein synthesis. In summary, our study preliminarily investigates the role of circRNAs in mesangial cells and ECM accumulation, providing a novel insight for DN pathogenesis.

Administration of cytokine-induced myeloid-derived suppressor cells ameliorates renal fibrosis in diabetic mice

BackgroundDiabetes is a proinflammatory state. Fibrosis of the renal glomerulus is the most common cause of end-stage renal disease. Glomerulosclerosis is caused by the accumulation of extracellular matrix (ECM) proteins in the mesangial interstitial space. Mesangial cells are unique stromal cells in the renal glomerulus that form the vascular pole of the renal corpuscle along with the mesangial matrix. Myeloid-derived suppressor cells (MDSCs) are heterogeneous immature myeloid cells that rapidly expand to regulate host immunity during inflammation, infection, and cancer. High concentrations of granulocyte–macrophage colony-stimulating factor (GM-CSF) alone or in combination with other molecules represent the most common ex-vivo protocol for differentiating MDSCs from bone marrow or from peripheral blood mononuclear cells. In this study, we analyzed and characterized the functions of MDSCs under the influence of mouse mesangial cells (MMCs) in a hyperglycemic environment and investigated whether cytokine-induced MDSCs ameliorated renal glomerulosclerosis in diabetic mice.MethodsCytokine-induced MDSCs were propagated from bone marrow cells cultured with mouse recombinant GM-CSF, IL-6, and IL-1β. Diabetic mice were induced with streptozotocin (STZ) and maintained at a blood glucose concentration exceeding 350 mg/dl. The ECM of the renal cortex and fibronectin expression of MMCs were analyzed through immunohistochemistry and western blotting. Arginase 1 and inducible NO synthase expressions of MDSCs were evaluated using quantitative reverse-transcriptase PCR. Cytokines released from MMCs were examined using a cytokine array assay.ResultsMDSCs in the diabetic mice were redistributed from the bone marrow into peripheral organs. An increase in fibronectin production was also observed in the renal glomerulus. MMCs in vitro produced more fibronectin and proinflammatory cytokines, such as macrophage inflammatory protein-2, RANTES, and stromal-cell-derived factor-1, under hyperglycemic conditions. The adoptive transfer of cytokine-induced MDSCs into STZ-induced mice normalized the glomerular filtration rate to reduce the kidney to body weight ratio and decrease fibronectin production in the renal glomerulus, ameliorating renal fibrosis. These results demonstrate the anti-inflammatory properties of cytokine-induced MDSCs and offer an alternative immunotherapy protocol for the management of diabetic nephropathy.ConclusionsThe application of cytokine-induced MDSCs provides a promising treatment for renal fibrosis and the prevention of diabetic nephropathy.

Early Growth Response Protein 1 Mediates Long Noncoding RNA Arid2-IR to Promote Extracellular Matrix Production in Diabetic Kidney Disease

Background: In order to investigate the mechanism through which Early growth response protein 1 (Egr1) mediates long noncoding RNA Arid2-IR to promote diabetic kidney disease. Method: In vivo, we induced type 2 diabetes and overexpressed or knocked down Egr1 by rapidly injecting of a large volume of DNA solution through the tail vein. In vitro, we assessed mouse mesangial cells that were stimulated with high glucose conditions and transiently transfected with siRNA and plasmids. Results: Compared with those in the control group, the expression of Egr1 and Arid2-IR in diabetes mice increased with the up-regulation of Col1a1 and α-SMA. Arid2-IR expression changes with Egr1 knockdown and overexpression both in vivo and in vitro. Knockdown Arid2-IR in vitro reduced Col1a1 and α-SMA expression. Overexpressing Egr1 and silencing Arid2-IR reduced Col1a1 and α-SMA expression. Conclusion: Egr1 promoted ECM production by up-regulating Arid2-IR. Thus, Arid2-IR may be a new target for treating diabetic kidney disease. Disclosure Y. Yang: None. F. Hu: None. M. Xue: None. Y. Jia: None. M. Zou: None. Y. Xue: None.

Rho-Kinase Induces CTGF Expression through Actin Dynamics in Mesangial Cells

Introduction: Transforming growth factor (TGF)-β promotes the development of diabetic nephropathy by enhancing excessive production of extracellular matrix such as connective tissue growth factor (CTGF) in mesangial cells. However, the mechanism by which TGF-β induces CTGF expression has not been elucidated. In this study, we aimed to investigate contribution of small GTPase Rho and Rho-kinase to TGF-β-mediated CTGF expression in mesangial cells. Methods and Results: Exposure of mouse mesangial cells (MES-13) to TGF-β induced activation of Rho and Rho-kinase. Y-27632, a specific Rho-kinase inhibitor, reduced TGF-β-mediated CTGF protein expression, indicating that TGF-β induces CTGF expression via Rho-kinase activation. Western blot analysis demonstrated that Y-27632 attenuates TGF-β-mediated phosphorylation of Erk1/2 and JNK/SAPK and nuclear translocation of NF-κB. Rho-kinase has been shown to regulate cytoskeleton reorganization. We therefore investigated whether Rho-kinase is involved in actin dynamics-induced NF-κB activation. Immunostaining studies demonstrated that Y-27632 and Cytochalasin D, an inhibitor of actin polymerization, attenuated TGF-β-mediated nuclear translocation of NF-κB, indicating that Rho-kinase-mediated actin reorganization regulates NF-κB activation. Furthermore, siRNA-mediated knockdown of Rho-kinase isoforms demonstrated ROCK2 but not ROCK1 is involved in these observations. In the present study, we demonstrate for the first time that Rho-kinase, especially ROCK2, mediates TGF-β-induced expression of CTGF via phosphorylation of Erk1/2 and JNK/SAPK and subsequent actin reorganization-mediated nuclear translocation of NF-κB in mesangial cells. Conclusion: Rho-kinase could be an important therapeutic target against diabetic nephropathy. Disclosure Y. Nagai: None. D. Kawanami: None. K. Matoba: None. Y. Takeda: None. T. Akamine: None. S. Ishizawa: None. Y. Kanazawa: None. T. Yokota: None. K. Utsunomiya: None.

Association between serum albumin level and incidence of end-stage renal disease in patients with Immunoglobulin A nephropathy: A possible role of albumin as an antioxidant agent.

Serum albumin is the major intravascular antioxidant. Though oxidative stress plays an important role in the pathophysiology of Immunoglobulin A nephropathy (IgAN), the association between serum albumin and the progression of IgAN is not entirely understood. This retrospective cohort study of 1,352 participants with biopsy-proven IgAN determined the associations between serum albumin level and the incidence of end-stage renal disease (ESRD) using a Cox proportional hazards model. Patients were divided into three groups by tertiles of serum albumin level: Low, Middle, and High group (≤3.9 g/dL, 4.0–4.3 g/dL, ≥4.4 g/dL, respectively). During the median 5.1-year follow-up period, 152 patients (11.2%) developed ESRD. Participants in the Low group had a 1.88-fold increased risk for ESRD compared with those in the High group after adjustment for clinical parameters, including urinary protein excretion, and pathological parameters (Oxford classification). We also experimentally proved the antioxidant capacity of albumin on mesangial cells. The intracellular reactive oxygen species and mitochondrial injury, induced by hydrogen peroxide were significantly attenuated in albumin-pretreated mouse mesangial cells and human kidney cells compared with γ-globulin-pretreated cells. Low serum albumin level is an independent risk factor for ESRD in patients with IgAN. The mechanism could be explained by the antioxidant capacity of serum albumin.

LincRNA 1700020I14Rik alleviates cell proliferation and fibrosis in diabetic nephropathy via miR-34a-5p/Sirt1/HIF-1\u03b1 signaling

Long intergenic noncoding RNAs (lincRNAs) have been gradually identified to be functional in a variety of different mechanisms associating with development and epigenetic regulation of cellular homeostasis. However, the study of lincRNAs in diabetic nephropathy (DN) is still in its infancy. Here, we have found dysexpressed long noncoding RNAs (lncRNAs) in renal tissues of db/db DN mice compared with db/m mice by RNA sequencing. In this study, 5 lincRNAs were confirmed to express in a consistent trend among these DN-related lncRNAs both in vivo and in vitro. Particularly, 1700020I14Rik was the downregulated one. Moreover, our data showed overexpression or knockdown of 1700020I14Rik could regulate cell proliferation and fibrosis in mouse mesangial cells (MCs). Furthermore, 1700020I14Rik was found to interact with miR-34a-5p via both the directly targeting way by bioinformatic investigation and luciferase assay and the Ago2-dependent manner by RIP assay. Results also displayed that overexpression of 1700020I14Rik inhibited cell proliferation and expressions of renal fibrosis markers through miR-34a-5p/Sirt1/HIF-1α pathway in MCs under high glucose condition, while knockdown of 1700020I14Rik could increase cell proliferation and expressions of renal fibrosis markers. In conclusion, these results provide new insights into the regulation between 1700020I14Rik and miR-34a-5p/Sirt1/HIF-1α signaling pathway during the progression of DN.