Mouse Glomerular Mesangial Cells Research Articles

Upregulation of the Urotensin II System in Diabetic Kidney Disease Includes Urotensin II-Related Peptide

The urotensin II (UII) system consists of endogenous ligands UII and its paralog urotensin II-related peptide (URP) and their receptor, UT. Originally isolated from the rat brain, URP is conserved across all vertebrates and shares the same cyclic hexapeptide core sequence with UII. However, UII and URP exhibit different N-terminal amino acid sequences. Beyond the central nervous system, UII and URP are functionally expressed in peripheral tissues. Alterations in tissue expression levels of UII and UT and blood or urinary UII concentrations have been associated with cardiovascular, pulmonary, and kidney dysfunctions, including hypertension, chronic heart failure, pulmonary hypertension, glomerulonephritis, and diabetes, making the UII system a potential therapeutic target for cardiovascular and kidney disease. The skeletal muscle and plasma levels of UII are increased in diabetic mice. Increased kidney tissue expression levels of UII and UT have also been demonstrated in humans and animals with diabetic kidney disease (DKD). Our recent work showed that UT is functionally expressed in cultured mouse glomerular mesangial cells (GMCs). Activation of UT by UII contributes to Ca2+-dependent GMC proliferation and extracellular matrix protein accumulation under high glucose conditions. We have also shown that DKD is attenuated in UT knockout mice. Despite the structural homology between UII and URP, studies have shown that the peptides may exhibit differential central and peripheral expression and pathophysiological function. However, whether URP is dysregulated in DKD remains unclear. Here, we examined whether URP production and kidney expression levels are altered in a hyperglycemic milieu and associated with DKD. Using a streptozotocin (STZ)-induced diabetes model, we examined the kidney tissue, plasma, and urinary levels of UII peptides. We also tested the hypothesis that, like UII, URP stimulates GMC proliferation and matrix protein synthesis. Our data show that protein and mRNA expression levels of both UII and URP were increased in the kidneys of hyperglycemic mice that developed albuminuria. Urinary and plasma UII and URP concentrations were also elevated in the mice. Although both UII and URP stimulated proliferation and type IV collagen production in cultured mouse GMCs by activating UT, the effect of UII was greater. Our data suggests that changes in the UII system in DKD are not limited to UII and UT but include the less-studied URP. GMC proliferation and extracellular matrix accumulation driven by UII and URP may contribute to DKD. NHLBI: R01 HL151735. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Inhibition of ALKBH5 inhibits inflammation and excessive proliferation by promoting TRIM13 m6A modifications in glomerular mesangial cells.

Chronic glomerulonephritis (CGN) refers to the inflammation of glomeruli in the kidneys. Glomerular mesangial cells (GMCs) play a pivotal role in the development of CGN. In the present study, we investigated the impact of ALKBH5, a m6A demethylase, on inflammation and hyperproliferation in mouse glomerular mesangial cells (MMCs) and elucidated the molecular mechanisms contributing to CGN.Western blotting and reverse transcriptase-polymerase chain reaction (RT-qPCR) were employed to evaluate the expression of ALKBH5 and TRIM13. In addition, enzyme-linked immunosorbent assay (ELISA) was used to measure the levels of inflammatory factors (IL-1β, TNF-α, and IL-10) in the lipopolysaccharide (LPS)-induced MMCs supernatant. Methylated RNA immunoprecipitation (MeRIP) was performed to investigate the effect of ALKBH5 on the levels of TRIM13-m6A mRNA. The stability of TRIM13 mRNA was evaluated using an actinomycin D assay.Significantly elevated expression of ALKBH5 was found in LPS-induced MMCs. Interference with ALKBH5 expression inhibited inflammation and excessive proliferation in LPS-induced MMCs. Moreover, interfering with ALKBH5 expression significantly reduced the levels of TRIM13-m6A modification. The overexpression of TRIM13 in MMCs reversed the inflammation and proliferation induced by ALKBH5 interference. In addition, interference with TRIM13 expressioninhibited the activation of the NF-κB pathway and suppressed inflammation and proliferation in MMCs.Inhibiting ALKBH5 hinders inflammation and hyperproliferation by improving TRIM13-m6A modification in glomerular MCs. We believe these findings will further provide insights into the molecular mechanisms and potential therapeutic targets for CGN.

Ursolic acid alleviates lupus nephritis by suppressing SUMO1-mediated stabilization of NLRP3

BackgroundSystemic lupus erythematosus (SLE) is a multi-system autoimmune disease that affects multiple organs and cause a wide range of severe clinical manifestations, including lupus nephritis (LN), which is a major risk factor for morbidity and mortality in individual with SLE. Ursolic acid (UA) is a natural compound with favorable anti-inflammatory properties and has been employed to treat multiple disease, including inflammatory diseases, diabetes, and Parkinson's disease. However, its therapeutic potential on LN and the underlying mechanisms remains unclear. PurposeThis aim of this study was to investigate the impact of UA on LN and its underlying mechanism. MethodsMRL/lpr lupus-prone mouse model was used and UA was administered orally for 8 weeks. Dexamethasone was used as a positive control. After 8 weeks of administration, the spleen-to-body-weight ratio, renal function, urine albumin excretion, cytokines levels, and the deposition of immune complex were measured. The primary mouse glomerular mesangial cells (GMCs) and SV40-MES-13 were stimulated by lipopolysaccharide (LPS), either alone or in combination with nigericin, to establish an in vitro model. The activation of NLRP3 inflammasome were investigated both in vivo and in vitro using qRT-PCR, immunoblotting, and immunofluorescence. ResultsOur results revealed that UA prominently alleviated LN in MRL/lpr lupus-prone mice, leading to a significant reduction in proteinuria production, infiltration of immune cells infiltration, and histopathological damage in the renal tissue. In addition, UA exerted inhibitory effects on the secretion of IL-1β, IL-18, and caspase-1, pyroptosis, and ASC speck formation in primary mouse GMCs and SV40-MES-13 cells. Furthermore, UA facilitated the degradation of NLRP3 by suppressing SUMO1-mediated SUMOylation of NLRP3. ConclusionUA possess a therapeutical effect on LN in MRL/lpr mice by enhancing the degradation of NLRP3 through inhibition of SUMO1-mediated SUMOylation of NLRP3. Our findings provide a basis for proposing UA as a potential candidate for the treatment of LN.

TRF3-IleAAT reduced extracellular matrix synthesis in diabetic kidney disease mice by targeting ZNF281 and inhibiting ferroptosis.

It is well established that the synthesis of extracellular matrix (ECM) in mesangial cells is a major determinant of diabetic kidney disease (DKD). Elucidating the major players in ECM synthesis may be helpful to provide promising candidates for protecting against DKD progression. tRF3-IleAAT is a tRNA-derived fragment (tRF) produced by nucleases at tRNA-specific sites, which is differentially expressed in the sera of patients with diabetes mellitus and DKD. In this study we investigated the potential roles of tRFs in DKD. Db/db mice at 12 weeks were adapted as a DKD model. The mice displayed marked renal dysfunction accompanied by significantly reduced expression of tRF3-IleAAT and increased ferroptosis and ECM synthesis in the kidney tissues. The reduced expression of tRF3-IleAAT was also observed in high glucose-treated mouse glomerular mesangial cells. We administered ferrostatin-1 (1 mg/kg, once every two days, i.p.) to the mice from the age of 12 weeks for 8 weeks, and found that inhibition of the onset of ferroptosis significantly improved renal function, attenuated renal fibrosis and reduced collagen deposition. Overexpression of tRF3-IleAAT by a single injection of AAV carrying tRF3-IleAAT via caudal vein significantly inhibited ferroptosis and ECM synthesis in DKD model mice. Furthermore, we found that the expression of zinc finger protein 281 (ZNF281), a downstream target gene of tRF3-IleAAT, was significantly elevated in DKD models but negatively regulated by tRF3-IleAAT. In high glucose-treated mesangial cells, knockdown of ZNF281 exerted an inhibitory effect on ferroptosis and ECM synthesis. We demonstrated the targeted binding of tRF3-IleAAT to the 3'UTR of ZNF281. In conclusion, tRF3-IleAAT inhibits ferroptosis by targeting ZNF281, resulting in the mitigation of ECM synthesis in DKD models, suggesting that tRF3-IleAAT may be an attractive therapeutic target for DKD.

LINC01232 targeting miR-1250-3p/MSH2 axis attenuates mesangial cell proliferation and fibrosis in diabetic nephropathy.

The significance of long non-coding RNA (ncRNAs) in the initiation and progression of diabetic nephropathy (DN) has attracted much interest. The purpose of this work was to ascertain the role of LINC01232 in cell models and animal models of DN. C57BL/6J mice were administered with streptozotocin (STZ) to develop animal models of DN, and mouse glomerular mesangial cells (MCs) were exposed to high glucose (HG) to establish cell models of DN. Expression levels of LINC01232, miR-1250-3p and MSH2 were identified by quantitative real-time PCR (qPCR) or western blotting. Fibrosis-related proteins were quantified by western blotting. MC proliferative capacity was checked by EdU assay. DN progression and fibrosis level in animal models were assessed by hematoxylin and eosin (HE) and Masson staining. The potential binding sites between miR-1250-3p and LINC01232 or MSH2 were examined by dual-luciferase reporter assay. LINC01232 expression was heightened in kidney tissues of DN patients. Its overexpression in HG-treated MCs alleviated MC proliferation and fibrosis. Overexpression of LINC01232 alleviated the pathological state of glomerular hypertrophy, MC hyperplasia, basement membrane thickening, and fibrosis in the DN models. LINC01232 bound to miR-1250-3p and competed for miR-1250-3p binding sites with MSH2. LINC01232 overexpression decoyed miR-1250-3p to increase MSH2 expression, and MSH2 depletion restored LINC01232 overexpression-inhibited MC proliferation and fibrosis. LINC01232 alleviated the mesangial cell proliferation and fibrosis in the progression of DN by targeting miR-1250-3p/MSH2 pathway.

Construction of chronic glomerulonephritis‑related lncRNA‑mRNA regulatory network and lncRNA‑-miRNA‑mRNA ceRNA network by bioinformatics analysis.

Long non-coding RNAs (lncRNAs) are ncRNA transcripts >200 nucleotides that are important genetic regulators. LncRNAs can directly regulate mRNA through a lncRNA-mRNA regulatory mode and can also regulate mRNA through competitive binding to micro (mi)RNA, which is generally known as the competitive endogenous RNA (ceRNA) network. The present study evaluated the functional roles and regulatory networks of lncRNAs in chronic glomerulonephritis (CGN). The proliferative ability of mouse glomerular mesangial cells (GMCs) induced by different concentrations of lipopolysaccharide (LPS) was assessed using the Cell Counting Kit-8 assay, and RNA sequencing (RNA-seq) was performed to identify differentially expressed lncRNAs in LPS-induced GMCs. Based on the sequencing results, six lncRNAs were selected for validation using reverse transcription-quantitative PCR (RT-qPCR). Furthermore, the lncRNA-mRNA regulatory network and the lncRNA-miRNA-mRNA ceRNA network were constructed to assess the role and mechanism of CGN-related lncRNAs. To elucidate the biological functions of lncRNAs, Gene Ontology (GO) biological process term enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were performed on all mRNAs involved in the lncRNA-mRNA regulatory network and in the ceRNA network. A total of 1,532 differentially expressed lncRNAs, including 594 upregulated lncRNAs and 938 downregulated lncRNAs, were identified using RNA-seq. The results of RT-qPCR validation were consistent with RNA-seq results. An lncRNA-mRNA regulatory network, including 236 lncRNAs and 556 mRNAs, and a ceRNA network, including 6 lncRNAs, 18 miRNAs and 419 mRNAs, were successfully constructed. The GO biological process term enrichment and KEGG pathway enrichment analyses demonstrated that those lncRNAs were often related to inflammatory response and substance metabolism. The present study identified key CGN-related lncRNAs in LPS-induced GMCs, and further demonstrated a global view of the lncRNA-mRNA regulatory network and ceRNA network involved in CGN. These results offered novel insights into the roles of lncRNAs in the pathogenesis of CGN and identified potential diagnostic biomarkers.

Isoliquiritigenin attenuates high glucose-induced proliferation, inflammation, and extracellular matrix deposition in glomerular mesangial cells by suppressing JAK2/STAT3 pathway.

To investigate the effect of isoliquiritigenin (ISL) on high glucose (HG)-induced glomerular mesangial cells (GMCs) proliferation, extracellular matrix (ECM) deposition and inflammation, and the underlying mechanisms. Mouse GMCs (SV40-MES-13) were cultured in HG medium, with or without ISL. The proliferation of GMCs was determined by MTT assay. The production of proinflammatory cytokines was detected by qRT-PCR and ELISA. The expression of connective tissue growth factor (CTGF), TGF-β1, collagen IV, and fibronectin was measured by qRT-PCR and western blot. The phosphorylation of JAK2 and STAT3 was examined by western blot. Next, JAK2 inhibitor AG490 was applied to HG-exposed GMCs. The levels of JAK2/STAT3 phosphorylation and pro-fibrotic markers were analyzed by western blot, and the secretion of TNF-α and IL-1β was evaluated by ELISA. GMCs were treated with HG, HG plus ISL or HG plus ISL, and recombinant IL-6 (rIL-6) which is a JAK2 activator. The levels of JAK2/STAT3 activation, ECM formation, and proinflammatory cytokines secretion were determined by western blot and ELISA, respectively. In mouse GMCs, ISL successfully repressed HG-induced hyperproliferation; production of TNF-α and IL-1β; expression of CTGF, TGF-β1, collagen IV, and fibronectin; and activation of JAK2/STAT3. Similar to ISL, AG490 was able to reverse the inflammation and ECM generation caused by HG. Moreover, rIL-6 impeded the amelioration of ISL on HG-induced adverse effects. Our study demonstrated that ISL displayed preventive effects on HG-exposed GMCs through inhibiting JAK2/STAT3 pathway and provided an insight into the application of ISL for diabetic nephropathy (DN) treatment.

Diphlorethohydroxycarmalol, a phlorotannin contained in brown edible seaweed Ishige okamurae, prevents AGE-related diabetic nephropathy by suppression of AGE-RAGE interaction

Advanced glycation end-products (AGEs) are complex and heterogeneous compounds generated through various steps and are naturally produced during the glycation reaction. Excessively generated AGEs bind to long-lived proteins such as collagen and cause renal dysfunction, leading to AGE-related diabetic nephropathy (DN). In this study, we aimed to confirm the therapeutic potential of marine biological resources by evaluating the role of diphlorethohydroxycarmalol (DPHC), a major phlorotannin compound contained in edible brown algae Ishige okamurae, in the key mechanisms involved in the pathogenesis and progression of AGE-induced DN. The AGE formation inhibitory and AGE-collagen cross-link inhibiting/breaking ability assay were performed to evaluate the anti-glycation effect of DPHC. The AGE-RAGE interaction inhibitory effect was evaluated through molecular docking study. The renoprotective effect of DPHC was confirmed in mouse glomerular mesangial cells. DPHC treatment exhibited anti-glycation ability through suppression of AGE formation and AGE-collagen cross-linking generation, and disruption of cross-links formed between AGE-collagen. In addition, DPHC pre-treatment significantly suppressed reactive oxygen species (ROS) production, intracellular methylglyoxal (MGO)/AGEs accumulation, and activation of apoptosis cascade by MGO. Moreover, DPHC suppressed AGE-receptor for AGE (RAGE) interaction by competing with MG-H1 for the binding site of RAGE. Furthermore, DPHC pre-treatment regulated apoptosis-related protein expression and increased Nrf2, Glo-1, HO-1, CAT, NQO1, and SOD1. These findings prove that the chemical structural characteristics of DPHC have the potential to prevent or manage AGE-induced DN by effectively mediating the key mechanisms responsible for its pathogenesis.

Dieckol, Derived from the Edible Brown Algae Ecklonia cava, Attenuates Methylglyoxal-Associated Diabetic Nephropathy by Suppressing AGE–RAGE Interaction

The formation of advanced glycation end products (AGE) is linked to the pathogenesis of diabetic nephropathy. The aim of this work was to assess the therapeutic potential and underlying mechanism of action of dieckol (DK), isolated from Ecklonia cava, on renal damage induced by methylglyoxal (MGO) in mouse glomerular mesangial cells. The antiglycation properties of DK were evaluated using ELISA. We conducted molecular docking, immunofluorescence analysis, and Western blotting to confirm the mechanism by which DK prevents AGE-related diabetic nephropathy. DK treatment exhibited antiglycation properties through the inhibition of AGE production, inhibition of cross-linking between AGE and collagen, and breaking of its cross-linking. DK pretreatment exhibited protective effects on renal cells by suppressing MGO-induced intracellular reactive oxygen species (ROS) formation, intracellular MGO and AGE accumulation, activation of the apoptosis cascade and apoptosis-related protein expression, activation of receptor for AGE (RAGE) protein expression, and suppression of the glyoxalase system. Furthermore, DK exhibited a stronger binding affinity for RAGE than AGE, which was confirmed as exerting a competitive inhibitory effect on the AGE-RAGE interaction. These results demonstrated that DK is a potential natural AGE inhibitor that can be utilized to prevent and treat AGE-induced diabetic nephropathy.

Transcriptional expression of CXCL10 and STAT1 in lupus nephritis and the intervention effect of triptolide

ObjectiveThis study screened out the key genes associated with the occurrence and development of lupus nephritis (LN) using bioinformatics methods, and then explored the expression of key genes in LN and the inhibitory effect of triptolide.MethodsThe GEO2R online tool in the GEO database was used to perform differential analysis of gene expression in LN tissues and normal kidney tissues. The GO function and KEGG pathway enrichment analysis of differentially expressed genes (DEGs), STRING, and Cytoscape software were used to build a protein–protein interaction network (PPI) to screen out the Hub gene. Mouse glomerular mesangial cells (MMC) were randomly divided into a control group, an interferon-γ (IFN-γ) stimulation group, and a triptolide intervention group. The relative expression of CXCL10 mRNA in each group was detected by real-time fluorescent quantitative PCR (RT-PCR). CXCL10 secretion was detected by enzyme-linked immunosorbent assay (ELISA), and Western blot was used to detect the expression of the JAK/STAT1 signaling pathway–related proteins STAT1 and p-STAT1 in each group.ResultsBioinformatics showed that there were 22 DEGs expression differences in the GEO database. The GO enrichment analysis showed that biological process (BP) such as the type I interferon signaling pathway, innate immune response, IFN-γ-mediated signaling pathway, virus defense response, and immune response were significantly regulated by DEGs. Through the combination of String database analysis and cytoscape software, it was found that STAT1 and CXCL10 are closely related to LN. Experimental results showed that IFN-γ induces the expression of CXCL10 mRNA and protein by activating the JAK/STAT1 signaling pathway, while triptolide inhibits the expression of CXCL10 mRNA and protein by inhibiting the JAK/STAT1 signaling pathway.ConclusionSTAT1 and CXCL10 are the key genes in the occurrence and development of LN. IFN-γ induces the expression of CXCL10 by activating the JAK/STAT1 signaling pathway, while triptolide inhibits the expression of CXCL10 by blocking the JAK/STAT1 signaling pathway. Inhibition of the JAK/STAT1 signaling pathway and CXCL10 expression is expected to become a potential target for the treatment of LN.Key Points• Bioinformatics showed that there were 22 DEGs expression differences in the GEO database.• Through the combination of String database analysis and Cytoscape software, it was found that STAT1 and CXCL10 are closely related to LN.• Experimental results showed that IFN-γ induces the expression of CXCL10 mRNA and protein by activating the JAK/STAT1 signaling pathway, while triptolide inhibits the expression of CXCL10 mRNA and protein by inhibiting the JAK/STAT1 signaling pathway.

Therapeutic Potential of Phlorotannin-Rich Ecklonia cava Extract on Methylglyoxal-Induced Diabetic Nephropathy in In Vitro Model.

Advanced glycation end-products (AGEs) play a vital role in the pathogenesis of diabetic complications. Methylglyoxal (MGO), one of the major precursors of AGEs, is a highly reactive dicarbonyl compound that plays an important role in the pathogenesis of diabetic nephropathy. This study was designed to evaluate the therapeutic potential of phlorotannin-rich Ecklonia cava extract (ECE) on MGO-induced diabetic nephropathy in in vitro models using mouse glomerular mesangial cells. ECE showed anti-glycation activity via breaking of AGEs-collagen cross-links and inhibition of AGEs formation and AGE-collagen cross-linking formation. The renoprotective effects were determined by assessing intracellular reactive oxygen species (ROS) and MGO accumulation, cell apoptosis, and the Nrf-2/ARE signaling pathway. MGO-induced renal damage, intracellular ROS production level, and MGO-protein adduct accumulation were significantly decreased by pretreating ECE. Moreover, ECE pretreatment exhibited preventive properties against MGO-induced dicarbonyl stress via activation of the Nrf2/ARE signaling pathway and reduction of RAGE protein expression in mouse glomerular mesangial cells. Collectively, these results indicated potential anti-glycation properties and prominent preventive effects of ECE against MGO-induced renal damage. Additionally, ECE may be utilized for the management of AGE-related diabetic nephropathy.

Catalpol Alleviates Ang II-Induced Renal Injury Through NF-κB Pathway and TGF-β1/Smads Pathway.

Catalpol is an iridoid glycoside obtained from Rehmannia glutinosa, which in previous studies showed various pharmacological properties, including anti-inflammatory, antioxidant, antidiabetic, antitumor, and dopaminergic neurons protecting effects. Here, we examined the effect of catalpol on renal injury induced by angiotensin II (Ang II) and further to explore its latent molecular mechanisms. We used an in vivo model of Ang II-induced renal injury mice; catalpol (25, 50, and 100 mg/kg) was administered for 28 days. Mouse glomerular mesangial cells (SV40 MES 13), rat kidney interstitial fibroblasts cells (NRK-49F), and human proximal tubular epithelial cells (HK-2) were induced by Ang II (10 µM) in the presence or absence of catalpol (1, 5, and 10 µM) and incubated for 48 hours in vitro. In our study, periodic acid-Schiff and Masson staining of renal tissue showed that catalpol reduced Ang II-induced renal injury in a concentration-dependent manner. The positive expressions of collagen IV and TGF-β1 were observed to decrease sharply after catalpol treatment. In renal tissue, the levels of pro-inflammatory cytokines tumor necrosis factor α and interleukin 6 were evidently decreased after catalpol intervention. Catalpol can relieve Ang II-induced renal injury by inactivating NF-κB and TGF-β1/Smads signaling pathways. Therefore, catalpol may act as a potential drug to treat Ang II-induced renal injury.

Ishige okamurae Ameliorates Methylglyoxal-Induced Nephrotoxicity via Reducing Oxidative Stress, RAGE Protein Expression, and Modulating MAPK, Nrf2/ARE Signaling Pathway in Mouse Glomerular Mesangial Cells.

Advanced glycation end-products (AGEs) such as methylglyoxal (MGO) play a vital role in the pathogenesis of nephropathy, a diabetic complication. In the present study, we evaluated the anti-glycation and renal protective properties of Ishige okamurae extract (IOE) against AGE-induced oxidative stress. HPLC analysis confirmed that bioactive phlorotannins such as diphlorethohydroxycarmalol and ishophloroglucin A are predominantly present in IOE. IOE showed strong anti-glycation activities via inhibition of AGE formation, inhibition of AGE–protein cross-linking, and breaking of AGE–protein cross-links. In addition, in vitro studies using mesangial cells demonstrated that IOE effectively suppressed intracellular reactive oxygen species production, intracellular MGO accumulation, and apoptotic cell death by MGO-induced oxidative stress, in addition to regulating the expression of proteins involved in the receptor for AGEs and nuclear factor erythroid 2-related factor 2 (Nrf2)/antioxidant response elements (ARE) signaling pathways. Therefore, IOE can serve as a natural therapeutic agent for the management of AGE-related nephropathy.

The occurrence of lupus nephritis is regulated by USP7-mediated JMJD3 stabilization

Ubiquitin-specific peptidases7 (USP7) participates in the regulation of various metabolic and immune disorders. However, the role of USP7 in lupus nephritis (LN) remains unknown. The current study set out to elucidate the regulatory role of USP7 in LN together with JMJD3 and NF-κB. SLE MRL/LPR mice and mouse glomerular mesangial cells SV40 MES 13 cells were employed for in vivo or vitro experiments. USP7, JMJD3 and NF-κB expression in MRL/LPR mice were detected, followed by investigation of their functions in the proliferation of mesangial cells and mesangial matrix. Subsequently, the interaction among USP7, JMJD3 and NF-κB was determined by means of ChIP and co-immunoprecipitation assay. The results indicated that USP7, JMJD3, p-NF-κB p65 were all highly-expressed in MRL/LPR mice. USP7 promoted the proliferation of mesangial cells and mesangial matrix, and stabilized the JMJD3 protein via deubiquitination in SV40 MES 13 cells. Meanwhile, silencing of JMJD3 inhibited the promotive effect of USP7 on the proliferation of mesangial cells and mesangial matrix. Furthermore, JMJD3 increased the expression of NF-κB p65 through demethylation, whereas silencing JMJD3 alleviated the proliferation of mesangial cells and mesangial matrix. Lastly, NF-κB p65 was proved to aggravate LN pathogenesis. Altogether, our findings highlighted that USP7 promoted the occurrence of LN by regulating the NF-κB p65 signaling pathway via stabilization of JMJD3.

Quercetin Antagonizes Glucose Fluctuation Induced Renal Injury by Inhibiting Aerobic Glycolysis via HIF-1α/miR-210/ISCU/FeS Pathway.

Background and Objective: Glucose fluctuation (GF) has been reported to induce renal injury and diabetic nephropathy (DN). However, the mechanism still remains ambiguous. Mitochondrial energy metabolism, especially aerobic glycolysis, has been a hotspot of DN research for decades. The activation of HIF-1α/miR210/ISCU/FeS axis has provided a new explanation for aerobic glycolysis. Our previous studies indicated quercetin as a potential therapeutic drug for DN. This study aims to evaluate levels of aerobic glycolysis and repressive effect of quercetin via HIF-1α/miR210/ISCU/FeS axis in a cell model of GF.Methods: The mouse glomerular mesangial cells (MCs) were exposed in high or oscillating glucose with or without quercetin treatment. Cell viability was measured by CCK8 assay. Aerobic glycolysis flux was evaluated by lactate acid, pH activity of PFK. Apoptosis level was confirmed by Annexin V-APC/7-AAD double staining and activity of caspase-3. TNF-α and IL-1β were used to evaluate inflammation levels.Results: GF deteriorated inflammation damage and apoptosis injury in MCs, while quercetin could alleviate this GF-triggered cytotoxicity. GF intensified aerobic glycolysis in MCs and quercetin could inhibit this intensification in a dose-dependent manner. Quercetin prevented activities of two FeS-dependent metabolic enzymes, aconitase, and complex I, under GF injury in MCs. The mRNA expression and protein contents of HIF-1α were increased after GF exposure, and these could be alleviated by quercetin treatment. Knockdown of ISCU by siRNA and Up-regulating of miR-210 by mimic could weaken the effects of quercetin that maintained protein levels of ISCU1/2, improved cell viability, relieved inflammation injury, decreased apoptosis, and reduced aerobic glycolysis switch in MCs.Conclusion: Quercetin antagonizes GF-induced renal injury by suppressing aerobic glycolysis via HIF-1α/miR-210/ISCU/FeS pathway in MCs cell model. Our findings contribute to a new insight into understanding the mechanism of GF-induced renal injury and protective effects of quercetin.

Short-Chain Fatty Acids Ameliorate Diabetic Nephropathy via GPR43-Mediated Inhibition of Oxidative Stress and NF-κB Signaling.

Diabetic nephropathy (DN) is a chronic low-grade inflammatory disease. Oxidative stress and nuclear factor kappa B (NF-κB) signaling play an important role in the pathogenesis of DN. Short-chain fatty acids (SCFAs) produced from carbohydrate fermentation in the gastrointestinal tract exert positive regulatory effects on inflammation and kidney injuries. However, it is unclear whether SCFAs can prevent and ameliorate DN. In the present study, we evaluated the role and mechanism of the three main SCFAs (acetate, propionate, and butyrate) in high-fat diet (HFD) and streptozotocin- (STZ-) induced type2 diabetes (T2D) and DN mouse models and in high glucose-induced mouse glomerular mesangial cells (GMCs), to explore novel therapeutic strategies and molecular targets for DN. We found that exogenous SCFAs, especially butyrate, improved hyperglycemia and insulin resistance; prevented the formation of proteinuria and an increase in serum creatinine, urea nitrogen, and cystatin C; inhibited mesangial matrix accumulation and renal fibrosis; and blocked NF-κB activation in mice. SCFAs also inhibited high glucose-induced oxidative stress and NF-κB activation and enhanced the interaction between β-arrestin-2 and I-κBα in GMCs. Specifically, the beneficial effects of SCFAs were significantly facilitated by the overexpression GPR43 or imitated by a GPR43 agonist but were inhibited by siRNA-GPR43 in GMCs. These results support the conclusion that SCFAs, especially butyrate, partially improve T2D-induced kidney injury via GPR43-mediated inhibition of oxidative stress and NF-κB signaling, suggesting SCFAs may be potential therapeutic agents in the prevention and treatment of DN.

Hyperoside Alleviates High Glucose-Induced Proliferation of Mesangial Cells through the Inhibition of the ERK/CREB/miRNA-34a Signaling Pathway.

Purpose Hyperoside, a flavonoid isolated from conventional medicinal herbs, has been demonstrated to exert a significant protective effect in diabetic nephropathy. This study aimed to determine the underlying mechanisms, by which hyperoside inhibits high glucose-(HG-) induced proliferation in mouse renal mesangial cells. Methods Mouse glomerular mesangial cells line (SV40-MES13) was used to study the inhibitory effect of hyperoside on cell proliferation induced by 30 mM glucose, which was used to simulate a diabetic condition. Viable cell count was assessed using the Cell Counting Kit-8 and by the 5-ethynyl-20-deoxyuridine incorporation assay. The underlying mechanism involving miRNA-34a was further investigated by quantitative RT-PCR and transfection with miRNA-34a agomir. The phosphorylation levels of extracellular signal-regulated kinases (ERKs) and cAMP-response element-binding protein (CREB) were measured by Western blotting. The binding region and the critical binding sites of CREB in the miRNA-34a promoter were investigated by the chromatin immunoprecipitation assay and luciferase reporter assay, respectively. Results We found that hyperoside could significantly decrease HG-induced proliferation of SV40-MES13 cells in a dose-dependent manner, without causing obvious cell death. In addition, hyperoside inhibited the activation of ERK pathway and phosphorylation of its downstream transcriptional factor CREB, as well as the miRNA-34a expression. We further confirmed that CREB-mediated regulation of miRNA-34a is dependent on the direct binding to specific sites in the promoter region of miRNA-34a. Conclusion Our cumulative results suggested that hyperoside inhibits the proliferation of SV40-MES13 cells through the suppression of the ERK/CREB/miRNA-34a signaling pathway, which provides new insight to the current investigation on therapeutic strategies for diabetic nephropathy.

P0993GYMNEMA SYLVESTRE EXTRACT ATTENUATE THE PATHOLOGICAL PROGESSION OF DIABETIC NEPHROPATHY IN RATS

Abstract Background and Aims Diabetic nephropathy is not only a common and severe microvascular complication of diabetes mellitus but also the leading cause of renal failure. Aqueous extract of leaves of Gymnema sylvestre (ALGS) has been reported to cause reversible increases in intracellular calcium, hypolipidemic activity and insulin secretion in mouse and human β cells with type 2 diabetes. The present study aimed to investigate the antidiabetic and renoprotective effects of ALGS in a rat model of type 2 diabetic mellitus. Method Male Sprague-Dawley rats with type 2 diabetes induced by a high-fat diet (HFD)/streptozotocin (STZ) were treated with ALGS at dosages of 0.5% and 1% (w/w) daily for 4 weeks. At the end of the experimental period, body weight, serum glucose levels, insulin levels, and kidney function were assessed. Furthermore, antioxidant enzyme and lipid peroxide levels were determined in the kidney with histopathological examination Molecular mechanism underlying the functioning of ALGS, mouse glomerular mesangial cells (MES-13) treated with high glucose (HG, 25 mM glucose) were chosen as a model for an examination of the signal transduction pathway of ALGS. Results The results revealed that ALGS improved diabetic kidney injury by reducing blood glucose, serum creatinine, and blood urea nitrogen levels and enhanced antioxidant enzyme activities in kidney tissue. Treatment with ALGS significantly reduced the malondialdehyde and 8-hydroxy-2-deoxyguanosine levels and increased serum insulin levels; expression of renal superoxide dismutase, catalase, and glutathione peroxidase activities; and glutathione content. Histological studies have also demonstrated that ALGS treatment inhibited the dilation of Bowman's capsule, which confirmed its renoprotective action in diabetes. In addition, treatment with ALGS attenuated 25 mM HG-induced suppressed nuclear factor erythroid 2-related factor 2 and antioxidant enzyme expression in MES-13 cells. Conclusion The results of present study reveal that ALGS extract could be useful intervention in the treatment of diabetes mellitus and, through antioxidative pathways.

Novel biphenyl diester derivative AB-38b inhibits NLRP3 inflammasome through Nrf2 activation in diabetic nephropathy.

Inflammation reaction mediated by NLRP3 inflammasome and Nrf2-related oxidative stress are vital participants in the development of diabetic nephropathy (DN) and closely associated to kidney fibrosis. Nrf2, a known antioxidative transcription factor, has been reported to activate NLRP3 inflammasome through its downstream factors (HO-1, NQO1, etc.) recently. AB38b is a newly synthesized biphenyl diester derivative with a Nrf2 activation property. This research aims to evaluate the renal protective effects of AB-38b and to elucidate the anti-inflammation mechanisms involved. Type 2 diabetic mice induced by high fat diet with streptozocin (STZ) and high glucose-cultured mouse glomerular mesangial cells (GMCs) were used in current study. Results showed that administration of AB-38b improved the kidney function while attenuated renal fibrosis progression in diabetic mice together with reducing the extracellular matrix (ECM) accumulation of GMCs cultured in high glucose. Mechanistically, treatment with AB-38b significantly decreased the high level of NLRP3 inflammasome in diabetic condition by inhibiting the ROS/TXNIP/NLRP3 signaling pathway. And meanwhile, AB-38b treatment effectively improved Nrf2 signaling during diabetic condition. Furthermore, knocking down the gene expression of Nrf2 by siRNA in GMCs abolished the inhibition effect of AB-38b on NLRP3 inflammasome activation and ECM accumulation. Taken together, our data suggest that AB-38b was able to improve the renal function of diabetic mice, and the NLRP3 inflammasome inhibition effect of AB-38b was responsible for the renal protective effect. Further exploration indicate that Nrf2 plays pivotal role in AB-38b's attenuation of DN progression through inhibiting NLRP3 inflammasome activation.

A novel compound AB38b attenuates oxidative stress and ECM protein accumulation in kidneys of diabetic mice through modulation of Keap1/Nrf2 signaling.

Extracellular matrix (ECM) deposition following reactive oxygen species (ROS) overproduction has a key role in diabetic nephropathy (DN), thus, antioxidant therapy is considered as a promising strategy for treating DN. Here, we investigated the therapeutic effects of AB38b, a novel synthetic α, β-unsaturated ketone compound, on the oxidative stress (OS) and ECM accumulation in type 2 diabetes mice, and tried to clarify the mechanisms underlying the effects in high glucose (HG, 30 mm)-treated mouse glomerular mesangial cells (GMCs). Type 2 diabetes model was established in mice with high-fat diet feeding combined with streptozocin intraperitoneal administration. The diabetic mice were then treated with AB38b (10, 20, 40 mg· kg−1· d−1, ig) or a positive control drug resveratrol (40 mg· kg−1· d−1, ig) for 8 weeks. We showed that administration of AB38b or resveratrol prevented the increases in malondialdehyde level, lactate dehydrogenase release, and laminin and type IV collagen deposition in the diabetic kidney. Simultaneously, AB38b or resveratrol markedly lowered the level of Keap1, accompanied by evident activation of Nrf2 signaling in the diabetic kidney. The underlying mechanisms of antioxidant effect of AB38b were explored in HG-treated mouse GMCs. AB38b (2.5−10 μM) or resveratrol (10 μM) significantly alleviated OS and ECM accumulation in HG-treated GMCs. Furthermore, AB38b or resveratrol treatment effectively activated Nrf2 signaling by inhibiting Keap1 expression without affecting the interaction between Keap1 and Nrf2. Besides, AB38b treatment effectively suppressed the ubiquitination of Nrf2. Taken together, this study demonstrates that AB38b ameliorates experimental DN through antioxidation and modulation of Keap1/Nrf2 signaling pathway.