ABSTRACT
Objectives: Astaxanthin (ATX) is a strong antioxidant drug. This study aimed to investigate the effects of ATX on podocytes in diabetic nephropathy and the underlying renal protective mechanism of ATX, which leads to pathological crosstalk with mesangial cells.Methods: In this study, diabetic rats treated with ATX exhibited reduced 24-h urinary protein excretion and decreased blood glucose and lipid levels compared to vehicle-treated rats. Glomerular mesangial matrix expansion and renal tubular epithelial cell injury were also attenuated in ATX-treated diabetic rats compared to control rats.Results: ATX treatment markedly reduced the α-SMA and collagen IV levels in the kidneys of diabetic rats. Additionally, ATX downregulated autophagy levels. In vitro, compared with normal glucose, high glucose inhibited LC3-II expression and increased p62 expression, whereas ATX treatment reversed these changes. ATX treatment also inhibited α-SMA and collagen IV expression in cultured podocytes. Secreted factors (vascular endothelial growth factor B and transforming growth factor-ß) generated by high glucose-induced podocytes downregulated autophagy in human mesangial cells (HMCs); however, this downregulation was upregulated when podocytes were treated with ATX.Conclusions: The current study revealed that ATX attenuates diabetes-induced kidney injury likely through the upregulation of autophagic activity in podocytes and its antifibrotic effects. Crosstalk between podocytes and HMCs can cause renal injury in diabetes, but ATX treatment reversed this phenomenon.
Subject(s)
Autophagy , Diabetes Mellitus, Experimental , Diabetic Nephropathies , Mesangial Cells , Podocytes , Up-Regulation , Xanthophylls , Podocytes/drug effects , Podocytes/metabolism , Podocytes/pathology , Autophagy/drug effects , Diabetic Nephropathies/metabolism , Diabetic Nephropathies/drug therapy , Diabetic Nephropathies/pathology , Animals , Mesangial Cells/drug effects , Mesangial Cells/metabolism , Mesangial Cells/pathology , Xanthophylls/pharmacology , Xanthophylls/therapeutic use , Rats , Diabetes Mellitus, Experimental/complications , Diabetes Mellitus, Experimental/metabolism , Diabetes Mellitus, Experimental/drug therapy , Male , Humans , Up-Regulation/drug effects , Rats, Sprague-Dawley , Actins/metabolism , Collagen Type IV/metabolism , Cells, Cultured , Antioxidants/pharmacologyABSTRACT
ETHNOPHARMACOLOGICAL RELEVANCE: Toona sinensis (A. Juss.) Roem. Is a deciduous woody plant native to Eastern and Southeastern Asia. Different parts of this plant have a long history of being applied as traditional medicines to treat various diseases. The fruits have been used for antidiabetic, antidiabetic nephropathy (anti-DN), antioxidant, anti-inflammatory, and other activities. AIM OF THE STUDY: The purpose of this study was to investigate the effects of EtOAc (PEAE) and n-BuOH extracts (PNBE) from T. sinensis pericarps (TSP) on kidney injury in high-fat and high-glucose diet (HFD)/streptozotocin (STZ)-induced DN mice by network pharmacology and pharmacological investigations, as well as to further discover active compounds that could ameliorate oxidative stress and inflammation, thereby delaying DN progression by regulating the Nrf2/NF-κB pathway in high glucose (HG)-induced glomerular mesangial cells (GMCs). MATERIALS AND METHODS: The targets of TSP 1-16 with DN were analyzed by network pharmacology. HFD/STZ-induced DN mouse models were established to evaluate the effects of PEAE and PNBE. Six groups were divided into normal, model, PEAE100, PEAE400, PNBE100, and PNBE400 groups. Fasting blood glucose (FBG) levels, organ indices, plasma MDA, SOD, TNF-α, and IL-6 levels, as well as renal tissue Nrf2, HO-1, NF-κB, TNF-α, and TGF-ß1 levels were determined, along with hematoxylin-eosin (H&E) and immunohistochemical (IHC) analysis of kidney sections. Furthermore, GMC activity screening combined with molecular docking was utilized to discover active compounds targeting HO-1, TNF-α, and IL-6. Moreover, western blotting assays were performed to validate the mechanism of Nrf2 and NF-κB in HG-induced GMCs. RESULTS: Network pharmacology predicted that the main targets of PEAE and PNBE in the treatment of DN include IL-6, INS, TNF, ALB, GAPDH, IL-1ß, TP53, EGFR, and CASP3. Additionally, major pathways include AGE-RAGE and IL-17. In vivo experiments, treatment with PEAE and PNBE effectively reduced FBG levels and organ indices, while plasma MDA, SOD, TNF-α, and IL-6 levels, renal tissue Nrf2, HO-1, NF-κB, TNF-α, and TGF-ß1 levels, and renal function were significantly improved. PEAE and PNBE significantly improved glomerular and tubule injury, and inhibited the development of DN by regulating the levels of oxidative stress and inflammation-related factors. In vitro experiments, compound 11 strongly activated HO-1 and inhibited TNF-α and IL-6. The molecular docking results revealed that compound 11 exhibited a high binding affinity towards the targets HO-1, TNF-α, and IL-6 (<-6 kcal/mol). Western blotting results showed compound 11 effectively regulated Nrf2 and NF-κB p65 protein levels, and significantly improved oxidative stress damage and inflammatory responses in HG-induced GMCs. CONCLUSION: PEAE, PNBE, and their compounds, especially compound 11, may have the potential to prevent and treat DN, and are promising natural nephroprotective agents.
Subject(s)
Diabetic Nephropathies , NF-E2-Related Factor 2 , Network Pharmacology , Plant Extracts , Animals , Diabetic Nephropathies/drug therapy , Diabetic Nephropathies/metabolism , Diabetic Nephropathies/pathology , Male , Mice , Plant Extracts/pharmacology , Plant Extracts/chemistry , NF-E2-Related Factor 2/metabolism , Mice, Inbred C57BL , Diabetes Mellitus, Experimental/drug therapy , Meliaceae/chemistry , Oxidative Stress/drug effects , Mesangial Cells/drug effects , Mesangial Cells/metabolism , NF-kappa B/metabolism , Hypoglycemic Agents/pharmacology , Hypoglycemic Agents/isolation & purification , Fruit/chemistry , Diet, High-Fat , Anti-Inflammatory Agents/pharmacology , Anti-Inflammatory Agents/isolation & purification , Streptozocin , Antioxidants/pharmacology , Antioxidants/isolation & purificationABSTRACT
Dihydromyricetin (DHM) is a flavonoid from vine tea with broad pharmacological benefits, which improve inflammation by blocking the NF-κB pathway. A growing body of research indicates that chronic kidney inflammation is vital to the pathogenesis of diabetic renal fibrosis. Sphingosine kinase-1 (SphK1) is a key regulator of diabetic renal inflammation, which triggers the NF-κB pathway. Hence, we evaluated whether DHM regulates diabetic renal inflammatory fibrosis by acting on SphK1. Here, we demonstrated that DHM effectively suppressed the synthesis of fibrotic and inflammatory adhesion factors like ICAM-1, and VCAM-1 in streptozotocin-treated high-fat diet-induced diabetic mice and HG-induced glomerular mesangial cells (GMCs). Moreover, DHM significantly suppressed NF-κB pathway activation and reduced SphK1 activity and protein expression under diabetic conditions. Mechanistically, the results of molecular docking, molecular dynamics simulation, and cellular thermal shift assay revealed that DHM stably bound to the binding pocket of SphK1, thereby reducing sphingosine-1-phosphate content and SphK1 enzymatic activity, which ultimately inhibited NF-κB DNA binding, transcriptional activity, and nuclear translocation. In conclusion, our data suggested that DHM inhibited SphK1 phosphorylation to prevent NF-κB activation thus ameliorating diabetic renal fibrosis. This supported the clinical use and further drug development of DHM as a potential candidate for treating diabetic renal fibrosis.
Subject(s)
Diabetes Mellitus, Experimental , Diabetic Nephropathies , Fibrosis , Flavonols , NF-kappa B , Phosphotransferases (Alcohol Group Acceptor) , Signal Transduction , Animals , Flavonols/pharmacology , Flavonols/therapeutic use , NF-kappa B/metabolism , Phosphotransferases (Alcohol Group Acceptor)/metabolism , Phosphotransferases (Alcohol Group Acceptor)/antagonists & inhibitors , Mice , Male , Diabetic Nephropathies/drug therapy , Diabetic Nephropathies/pathology , Diabetic Nephropathies/metabolism , Diabetes Mellitus, Experimental/drug therapy , Diabetes Mellitus, Experimental/complications , Diabetes Mellitus, Experimental/metabolism , Diabetes Mellitus, Experimental/pathology , Signal Transduction/drug effects , Mice, Inbred C57BL , Mesangial Cells/drug effects , Mesangial Cells/metabolism , Mesangial Cells/pathology , Molecular Docking Simulation , Intercellular Adhesion Molecule-1/metabolism , Phosphorylation/drug effects , Kidney/drug effects , Kidney/pathology , Kidney/metabolismABSTRACT
Diabetic nephropathy (DN) is a complication of diabetes and is mainly characterized by renal fibrosis, which could be attributed to chronic kidney inflammation. Stimulator of interferon genes (STING), a linker between immunity and metabolism, could ameliorate various metabolic and inflammatory diseases. However, the regulatory role of STING in DN remains largely unexplored. In this study, knockdown of STING decreased extracellular matrix (ECM), pro-inflammatory, and fibrotic factors in high glucose (HG)-induced glomerular mesangial cells (GMCs), whereas overexpression of STING triggered the inflammatory fibrosis process, suggesting that STING was a potential target for DN. Polydatin (PD) is a glucoside of resveratrol and has been reported to ameliorate DN by inhibiting inflammatory responses. Nevertheless, whether PD improved DN via STING remains unclear. Here, transcriptomic profiling implied that the STING/NF-κB pathway might be an important target for PD. We further found that PD decreased the protein expression of STING, and subsequently suppressed the activation of downstream targets including TBK1 phosphorylation and NF-κB nuclear translocation, and eventually inhibited the production of ECM, pro-inflammatory and fibrotic factors in HG-induced GMCs. Notably, results of molecular docking, molecular dynamic simulations, surface plasmon resonance, cellular thermal shift assay and Co-immunoprecipitation assay indicated that PD directly bound to STING and restored the declined proteasome-mediated degradation of STING induced by HG. In diabetic mice, PD also inhibited the STING pathway and improved the pathological changes of renal inflammatory fibrosis. Our study elucidated the regulatory role of STING in DN, and the novel mechanism of PD treating DN via inhibiting STING expression.
Subject(s)
Diabetes Mellitus, Experimental , Diabetic Nephropathies , Fibrosis , Glucosides , Membrane Proteins , Mice, Inbred C57BL , Stilbenes , Glucosides/pharmacology , Glucosides/therapeutic use , Animals , Diabetic Nephropathies/metabolism , Diabetic Nephropathies/drug therapy , Diabetic Nephropathies/pathology , Mice , Membrane Proteins/metabolism , Membrane Proteins/genetics , Fibrosis/drug therapy , Male , Stilbenes/pharmacology , Stilbenes/therapeutic use , Diabetes Mellitus, Experimental/drug therapy , Diabetes Mellitus, Experimental/metabolism , Signal Transduction/drug effects , Mesangial Cells/drug effects , Mesangial Cells/metabolism , Mesangial Cells/pathology , HumansABSTRACT
BACKGROUND: Ginsenoside Rg3 is an active saponin isolated from ginseng, which can reduce renal inflammation. However, the role and mechanism of Rg3 in diabetic kidney disease (DKD) are far from being studied. METHODS: The effects of Rg3 and miR-216a-5p on the proliferation, apoptosis, and MAPK pathway in high glucose (HG)-induced SV40 MES 13 were monitored by CCK-8, TUNEL staining, and western blot. RESULTS: Rg3 treatment could accelerate proliferation and suppress apoptosis in HG-induced SV40 MES. Moreover, miR-216a-5p inhibition also could alleviate renal injury, prevent apoptosis, and activate the MAPK pathway in kidney tissues of diabetic model mice. CONCLUSION: Rg3 could attenuate DKD progression by downregulating miR-216a-5p, suggesting Rg3 and miR-216a-5p might be the potential drug and molecular targets for DKD therapy.
Subject(s)
Apoptosis , Cell Proliferation , Diabetes Mellitus, Experimental , Diabetic Nephropathies , Ginsenosides , MAP Kinase Signaling System , Mesangial Cells , MicroRNAs , Ginsenosides/pharmacology , MicroRNAs/metabolism , MicroRNAs/genetics , Animals , Diabetic Nephropathies/metabolism , Diabetic Nephropathies/genetics , Diabetic Nephropathies/pathology , Diabetic Nephropathies/drug therapy , Apoptosis/drug effects , Cell Proliferation/drug effects , Mice , Mesangial Cells/drug effects , Mesangial Cells/metabolism , MAP Kinase Signaling System/drug effects , Diabetes Mellitus, Experimental/metabolism , Male , Cell LineABSTRACT
Lupus nephritis (LN) occurs in 50% of cases of systemic lupus erythematosus (SLE) and is one of the most serious complications that can occur during lupus progression. Mesangial cells (MCs) are intrinsic cells in the kidney that can regulate capillary blood flow, phagocytose apoptotic cells, and secrete vasoactive substances and growth factors. Previous studies have shown that various types of inflammatory cells can activate MCs for hyperproliferation, leading to disruption of the filtration barrier and impairment of renal function in LN. Here, we characterized the heterogeneity of kidney cells of LN mice by single-nucleus RNA sequencing (snRNA-seq) and revealed the interaction between macrophages and MCs through the CXC motif chemokine ligand 12 (CXCL12)/dipeptidyl peptidase 4 (DPP4) axis. In culture, macrophages modulated the proliferation and migration of MCs through this ligand-receptor interaction. In LN mice, treatment with linagliptin, a DPP4 inhibitor, effectively inhibited MC proliferation and reduced urinary protein levels. Together, our findings indicated that targeting the CXCL12/DPP4 axis with linagliptin treatment may serve as a novel strategy for the treatment of LN via the CXCL12/DPP4 axis.
Subject(s)
Cell Proliferation , Chemokine CXCL12 , Dipeptidyl Peptidase 4 , Lupus Nephritis , Macrophages , Mesangial Cells , Lupus Nephritis/pathology , Lupus Nephritis/metabolism , Animals , Dipeptidyl Peptidase 4/metabolism , Chemokine CXCL12/metabolism , Mesangial Cells/metabolism , Mesangial Cells/pathology , Mesangial Cells/drug effects , Mice , Macrophages/metabolism , Cell Proliferation/drug effects , Humans , Female , Cell Movement/drug effects , Cell Communication/drug effects , Linagliptin/pharmacology , Signal Transduction , Dipeptidyl-Peptidase IV Inhibitors/pharmacology , Mice, Inbred C57BLABSTRACT
Di-d-psicose anhydride (DPA), derived from functional rare saccharide as d-psicose, is investigated for its strong chelating ability. Methylglyoxal (MGO), an important precursor of advanced glycation end-products (AGEs), promotes obesity, and causes complications such as diabetic nephropathy. On mesangial cells, DPA can substantially reduce the negative effects of MGO. DPA effectively trapping MGO in mesangial cells. The bonding properties of the DPA-MGO adduct were discussed by mass spectrometry and nuclear magnetic resonance (NMR). The NMR spectra of the DPA-MGO adduct provide evidence for chelation bonding. The inhibition of AGE formation and the mass spectrometry results of the DPA-MGO adduct indicate that DPA can scavenge MGO at a molar ratio of 1:1. DPA suppressed 330 % of the up-regulated receptor for an AGEs protein expression to a normal level and restored the suppressed glyoxalase 1 level to 86 % of the normal group. This research provides important evidence and theoretical basis for the development of AGE inhibitors derived from rare saccharide.
Subject(s)
Diabetic Nephropathies , Glycation End Products, Advanced , Pyruvaldehyde , Pyruvaldehyde/chemistry , Diabetic Nephropathies/drug therapy , Diabetic Nephropathies/metabolism , Diabetic Nephropathies/prevention & control , Glycation End Products, Advanced/metabolism , Glycation End Products, Advanced/antagonists & inhibitors , Mesangial Cells/drug effects , Mesangial Cells/metabolism , Lactoylglutathione Lyase/antagonists & inhibitors , Lactoylglutathione Lyase/metabolism , Humans , Receptor for Advanced Glycation End Products/metabolism , Receptor for Advanced Glycation End Products/antagonists & inhibitors , Anhydrides/chemistry , Chelating Agents/chemistry , Chelating Agents/pharmacologyABSTRACT
BACKGROUND: Systemic 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. PURPOSE: This aim of this study was to investigate the impact of UA on LN and its underlying mechanism. METHODS: MRL/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. RESULTS: Our 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-1ß, 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. CONCLUSION: UA 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.
Subject(s)
Inflammasomes , Lupus Nephritis , Mice, Inbred MRL lpr , NLR Family, Pyrin Domain-Containing 3 Protein , Triterpenes , Ursolic Acid , Animals , Triterpenes/pharmacology , Lupus Nephritis/drug therapy , NLR Family, Pyrin Domain-Containing 3 Protein/metabolism , Mice , Inflammasomes/metabolism , Inflammasomes/drug effects , Disease Models, Animal , Female , Mesangial Cells/drug effects , Mesangial Cells/metabolism , Anti-Inflammatory Agents/pharmacology , Sumoylation/drug effectsABSTRACT
BACKGROUND: Phillyrin, the major lignin compound of Forsythia suspense (Thunb.) Vahl, has been shown the effects of anti-inflammatory and antioxidant. Our study was aimed to explore the protective effect of phillyrin on glomerular mesangial cells (HBZY-1) and the potential mechanism. METHODS: Cell viability, cytokine production, levels of reactive oxygen radicals (ROS), glutathione (GSH), malondialdehyde (MDA), and superoxide dismutase (SOD), as well as autophagy and apoptosis levels were determined to verify the mechanism of phillyrin on HBZY-1 cells. RESULTS: Our result indicated that phillyrin significantly inhibited HG-induced HBZY-1 proliferation by inhibiting Bcl-2 expression and upregulating Bad, cleaved caspase-3, and -9 expression. Also, phillyrin suppressed HG-induced mesangial extracellular matrix accumulation by inhibiting the expression of fibronectin and transforming growth factor-ß1. Further, phillyrin inhibited oxidative stress and inflammation by decreasing ROS, MDA, TNF-α, IL-1ß, and IL-6 contents and increasing SOD and GSH expression. Phillyrin also promoted autophagy by increasing LC3-II/LC3-I ratio and down-regulating p62 expression. Furthermore, WB assay showed that phillyrin inhibited oxidative stress caused by HG via activating Nrf2 signaling pathway, while attenuated proliferation and inflammation in HBZY-1 cells through inactivating PI3K/Akt/mTOR and NF-κB pathways. CONCLUSION: All results showed that phillyrin might be a promising therapeutic agent for the treatment of DN.
Subject(s)
Autophagy , Glucose , Inflammation , Oxidative Stress , Phosphatidylinositol 3-Kinases , Proto-Oncogene Proteins c-akt , Signal Transduction , Oxidative Stress/drug effects , Proto-Oncogene Proteins c-akt/metabolism , Signal Transduction/drug effects , Glucose/metabolism , Phosphatidylinositol 3-Kinases/metabolism , Inflammation/drug therapy , Inflammation/metabolism , Cell Line , Autophagy/drug effects , Mesangial Cells/drug effects , Mesangial Cells/metabolism , Anti-Inflammatory Agents/pharmacology , Antioxidants/pharmacology , Apoptosis/drug effects , Cell Survival/drug effects , Glucosides/pharmacology , Reactive Oxygen Species/metabolism , Humans , Cytokines/metabolism , Cell Proliferation/drug effects , NF-E2-Related Factor 2/metabolismABSTRACT
BACKGROUND: Mesangial cell fibrosis, a typical symptom of diabetic nephropathy (DN), is a major contributor to glomerulosclerosis. We previously reported that the pharmacological blockade of lysophosphatidic acid (LPA) signaling improves DN. Although LPA signaling is implicated in diabetic renal fibrosis, the underlying molecular mechanisms remain unclear. Here, the role of carbohydrate-responsive element-binding protein (ChREBP) in LPA-induced renal fibrosis and the underlying mechanisms were investigated. METHODS: Eight-week-old wild-type and db/db mice were intraperitoneally injected with the vehicle or an LPAR1/3 antagonist, ki16425 (10 mg/kg), for 8 weeks on a daily basis, following which the mice were sacrificed and renal protein expression was analyzed. SV40 MES13 cells were treated with LPA in the presence or absence of ki16425, and the expression of ChREBP and fibrotic factors, including fibronectin, TGF-ß, and IL-1ß, was examined. The role of ChREBP in the LPA-induced fibrotic response was investigated by ChREBP overexpression or knockdown. The involvement of Smad ubiquitination regulatory factor-2 (Smurf2), an E3 ligase, in LPA-induced expression of ChREBP and fibrotic factors was investigated by Smurf2 overexpression or knockdown. To identify signaling molecules regulating Smurf2 expression by LPA, pharmacological inhibitors such as A6370 (Akt1/2 kinase inhibitor) and Ly 294002 (PI3K inhibitor) were used. RESULTS: The renal expression of ChREBP increased in diabetic db/db mice, and was reduced following treatment with the ki16425. Treatment with LPA induced the expression of ChREBP and fibrotic factors, including fibronectin, TGF-ß, and IL-1ß, in SV40 MES13 cells, which were positively correlated. The LPA-induced expression of fibrotic factors increased or decreased following ChREBP overexpression and knockdown, respectively. The production of reactive oxygen species (ROS) mediated the LPA-induced expression of ChREBP and fibrotic factors, and LPA decreased Smurf2 expression via Traf4-mediated ubiquitination. The LPA-induced expression of ubiquitinated-ChREBP increased or decreased following Smurf2 overexpression and knockdown, respectively. Additionally, Smurf2 knockdown significantly increased the expression of ChREBP and fibrotic factors. The pharmacological inhibition of Akt signaling suppressed the LPA-induced alterations in the expression of ChREBP and Smurf2. CONCLUSION: Collectively, the results demonstrated that the ROS/Akt-dependent downregulation of Smurf2 and the subsequent increase in ChREBP expression might be one of the mechanisms by which LPA induces mesangial cell fibrosis in DN.
Subject(s)
Basic Helix-Loop-Helix Leucine Zipper Transcription Factors , Diabetic Nephropathies , Lysophospholipids , Mesangial Cells , Proto-Oncogene Proteins c-akt , Reactive Oxygen Species , Ubiquitin-Protein Ligases , Animals , Basic Helix-Loop-Helix Leucine Zipper Transcription Factors/metabolism , Diabetic Nephropathies/metabolism , Diabetic Nephropathies/pathology , Down-Regulation , Female , Fibronectins/metabolism , Fibrosis , Male , Mesangial Cells/drug effects , Mesangial Cells/metabolism , Mesangial Cells/pathology , Mice , Phosphatidylinositol 3-Kinases/metabolism , Proto-Oncogene Proteins c-akt/genetics , Proto-Oncogene Proteins c-akt/metabolism , Reactive Oxygen Species/metabolism , TNF Receptor-Associated Factor 4/metabolism , Transforming Growth Factor beta/metabolism , Ubiquitin-Protein Ligases/genetics , Ubiquitin-Protein Ligases/metabolism , UbiquitinationABSTRACT
AIMS/INTRODUCTION: This study aimed to investigate the role and mechanism of circular ribonucleic acid nucleoporin 98 (circNUP98) in diabetic nephropathy (DN). MATERIALS AND METHODS: Human glomerular mesangial cells (HMCs) were stimulated with high glucose (HG) to imitate the growth environment of cells under the DN condition. Levels of genes and proteins were tested by quantitative reverse transcription polymerase chain reaction and western blot. Cell proliferation, apoptosis and inflammatory response were analyzed by using cell counting kit-8, flow cytometry and enzyme-linked immunosorbent assay analysis, respectively. Oxidative stress and fibrosis were evaluated by detecting the activity of reactive oxygen species, malondialdehyde, superoxide dismutase, fibronectin and collagen IV. The binding interaction between microribonucleic acid (miR)-151-3p and high mobility group AT-hook 2 (HMGA2) or circNUP98 was confirmed using dual-luciferase reporter, pull-down and ribonucleic acid immunoprecipitation assays. Exosomes were isolated by ultracentrifugation, and qualified by transmission electron microscopy, nanoparticle tracking analysis and western blot. RESULTS: CircNUP98 expression was higher in the serum of DN patients and HG-stimulated HMCs. Functionally, circNUP98 knockdown alleviated HG-induced proliferation, fibrosis, inflammatory response and oxidative stress in HMCs. Mechanistically, circNUP98 directly sponged miR-151-3p, which targeted HMGA2. Rescue experiments showed that miR-151-3p reversed the inhibitory effects of circNUP98 knockdown on HG-induced HMC dysfunction. Furthermore, miR-151-3p re-expression also led to an inhibition of the aforementioned biological behaviors, which was attenuated by HMGA2 upregulation. Besides that, CircNUP98 was found to be packaged into exosomes of DN, and exosomal circNUP98 possessed diagnostic value for DN patients. CONCLUSION: CircNUP98 knockdown alleviates HG-induced proliferation, fibrosis inflammation and oxidative stress in HMCs by regulating the miR-151-3p-HMGA2 axis, which might provide a potential approach for DN therapeutics.
Subject(s)
Diabetic Nephropathies , HMGA2 Protein , Mesangial Cells , MicroRNAs , Oxidative Stress , RNA, Circular , Cell Proliferation , Diabetic Nephropathies/metabolism , Fibrosis , Glucose/pharmacology , HMGA2 Protein/genetics , HMGA2 Protein/metabolism , Humans , Inflammation/metabolism , Mesangial Cells/cytology , Mesangial Cells/drug effects , MicroRNAs/genetics , MicroRNAs/metabolism , Nuclear Pore Complex Proteins/metabolism , RNA, Circular/geneticsABSTRACT
BACKGROUND: Diabetic nephropathy (DN) is one of the main causes of end-stage renal disease with scantly effective treatment. Numerous evidences indicated that macrophages play an important role in the occurrence and pathogenesis of DN by secreting inflammatory cytokines. Mincle is mainly expressed in macrophages and promotes kidney inflammation and damage of acute kidney injury. However, the role of Mincle in DN is unclear. In this study, we aim to investigate the effect of Mincle-related macrophage inflammation on DN, and whether it can be identified as the therapeutic target for Astragalus mongholicus Bunge and Panax notoginseng Formula (A&P), a widely used Chinese herbal decoction for DN treatment. METHODS: In vivo experiments high-fat and high-sugar diet and streptozotocin was used to establish a diabetic nephropathy model, while in vitro experiments inflammation model was induced by high-glucose in mouse Bone Marrow-Derived Macrophages (BMDM) cells and mouse mesangial (MES) cells. Kidney pathological staining is used to detect kidney tissue damage and inflammation, Western blotting, Real-time PCR and ELISA are performed to detect Mincle signaling pathway related proteins and inflammatory cytokines. RESULTS: Mincle was mainly expressed in infiltrated macrophage of DN kidney, and was significant decreased after A&P administration. The in vitro experiments also proved that A&P effectively down-regulated the expression of Mincle in macrophage stimulated by high glucose. Meanwhile, the data demonstrated that A&P can reduce the activation of NFκB, and the expression and secretion of inflammatory cytokines in DN kidney or BMDM cells. Notably, we set up a co-culture system to conform that BMDM cells can aggravate the inflammatory response of mesangial (MES) cells under high glucose stimulation. Furthermore, we found that the anti-injury role of A&P in MES cells was dependent on inhibition of the Mincle in macrophage. CONCLUSION: In summary, our study found that A&P is effective in reducing renal pathological damage and improving renal function and inflammation in diabetic nephropathy by a mechanism mainly related to the inhibition of the Mincle/Card9/NFκB signaling pathway.
Subject(s)
Astragalus propinquus , Diabetic Nephropathies/drug therapy , Drugs, Chinese Herbal/pharmacology , Lectins, C-Type/metabolism , Macrophages/drug effects , Membrane Proteins/metabolism , Mesangial Cells/drug effects , Panax notoginseng , Animals , CARD Signaling Adaptor Proteins/metabolism , Kidney/drug effects , Male , Mice , Mice, Inbred C57BL , Models, Animal , NF-kappa B/metabolism , Signal Transduction/drug effectsABSTRACT
Diabetic nephropathy (DN) is one of the severe microvascular complications of diabetes mellitus. Oxidative stress resulting from aberrant metabolism of glucose mediates renal inflammation and fibrosis in the progression of DN. Nuclear factor erythroid 2-related factor 2 (Nrf2) is a transcription factor regulating the expression of antioxidant enzymes. Activating Nrf2 will give a promising therapy for DN. To discover novel Nrf2 activators, we have investigated caffeoylisocitric acid using mesangial cells under high glucose. The results showed at 10 µM, caffeoylisocitric acid significantly inhibited the self-limited proliferation of mesangial cells induced by high glucose. Further assessments have disclosed caffeoylisocitric acid mitigated oxidative stress, inflammation and accumulation of extracellular matrix resulting from high glucose via inactivating MAPK signalling. Meanwhile activation of Nrf2 was observed and involved in these effects through the interaction between Keap1 and caffeoylisocitric acid to disrupt Keap1-Nrf2 complex. Therefore, caffeoylisocitric acid is a promising Nrf2 activator targeting DN.
Subject(s)
Caffeic Acids/pharmacology , Diabetic Nephropathies/drug therapy , Drug Discovery , Glucose/antagonists & inhibitors , Hypoglycemic Agents/pharmacology , Kelch-Like ECH-Associated Protein 1/metabolism , Mesangial Cells/drug effects , NF-E2-Related Factor 2/metabolism , Caffeic Acids/chemistry , Cells, Cultured , Diabetic Nephropathies/metabolism , Diabetic Nephropathies/pathology , Dose-Response Relationship, Drug , Extracellular Matrix/drug effects , Glucose/metabolism , Humans , Hypoglycemic Agents/chemistry , Mesangial Cells/metabolism , Molecular Structure , Oxidative Stress/drug effects , Structure-Activity RelationshipABSTRACT
Glomerular mesangial cell (MC) proliferation and extracellular matrix deposition are the main pathological changes in diabetic nephropathy. Hydrogen sulfide (H2S) inhibits the proliferation of MCs. Dopamine 1 receptors (DR1) are expressed in MCs and serve important physiological roles. However, it is unclear whether DR1 activation inhibits MC proliferation by increasing endogenous H2S. The present study found that the production of H2S and the expression of DR1 and cystathionineγlyase (CSE) were decreased in the renal tissues of diabetic mice and high glucose (HG)induced MCs. SKF38393 (a DR1 agonist) increased the production of H2S and the expression of DR1 and CSE and NaHS (an exogenous H2S donor) only increased H2S production and CSE expression but not DR1 expression. HG increased the thickness of the glomerular basement membrane, cell viability and proliferation, the expression of cyclin D1, PCNA, collagen 1 and αsmooth muscle actin and the activity of phosphorylated ERK1/2 and decreased the expression of P21 and MMP9. SKF38393 and NaHS reversed the effects of HG. PPG (a CSE inhibitor) abolished the beneficial effects of SKF38393. The beneficial effects of SKF38393 were similar to those of PD98059 (an ERK1/2 inhibitor). Taken together, the findings suggested that the DR1CSE/H2S pathway activation attenuated diabetic MC proliferation and extracellular matrix deposition by downregulating the ERK1/2 signaling pathway.
Subject(s)
Cystathionine gamma-Lyase/metabolism , Diabetes Mellitus, Experimental/pathology , Hydrogen Sulfide/metabolism , Kidney/pathology , Receptors, Dopamine D1/metabolism , 2,3,4,5-Tetrahydro-7,8-dihydroxy-1-phenyl-1H-3-benzazepine/pharmacology , Animals , Cell Line , Cell Proliferation , Collagen/metabolism , Diabetes Mellitus, Experimental/metabolism , Diabetes Mellitus, Type 1/metabolism , Diabetes Mellitus, Type 1/pathology , Female , Fibrosis , Glucose/pharmacology , Kidney/metabolism , MAP Kinase Signaling System/physiology , Male , Mesangial Cells/drug effects , Mesangial Cells/pathology , Mice, Inbred C57BL , Receptors, Dopamine D1/agonistsABSTRACT
Emodin has been shown to exert a renoprotective effect in diabetic nephropathy (DN). In this paper, we investigated whether circular RNAs (circRNAs) might be involved in the renoprotective mechanism of emodin in DN. The levels of malondialdehyde (MDA), reactive oxygen species (ROS), superoxide dismutase (SOD), interleukin-1ß (IL-1ß), IL-6 and tumor necrosis factor-α (TNF-α) were measured using the corresponding assay kits. The expression levels of circ_0000064, microRNA (miR)-30c-5p, large multifunctional protease 7 (Lmp7), fibronectin (FN), and collagen type I (Col.1) were gauged by quantitative real-time polymerase chain reaction (qRT-PCR) or western blot. Subcellular localization assay was used to assess the cellular localization of circ_0000064. Targeted relationships among circ_0000064, miR-30c-5p and Lmp7 were confirmed by dual-luciferase reporter, RNA pull-down and RNA immunoprecipitation (RIP) assays. Our data showed the alleviative effect of emodin on HG-induced oxidative stress, inflammation and extracellular matrix (ECM) accumulation in SV-MES13 cells. Circ_0000064 was an importantly downstream effector of emodin function in HG-induced SV40-MES13 cells. Moreover, circ_0000064 directly targeted miR-30c-5p, and circ_0000064 modulated Lmp7 expression through miR-30c-5p. Circ_0000064 silencing alleviated HG-induced cell oxidative stress, inflammation and ECM accumulation via up-regulating miR-30c-5p. The enforced expression of miR-30c-5p attenuated HG-induced oxidative stress, inflammation and ECM accumulation in SV40-MES13 cells by targeting Lmp7. Our findings identified that emodin alleviated HG-induced oxidative stress, inflammation and ECM accumulation in SV40-MES13 cells at least partially by the regulation of the circ_0000064/miR-30c-5p/Lmp7 axis.
Subject(s)
Diabetic Nephropathies , Emodin , MicroRNAs , Proteasome Endopeptidase Complex , RNA, Circular , Cell Line , Diabetic Nephropathies/drug therapy , Diabetic Nephropathies/genetics , Emodin/pharmacology , Extracellular Matrix/genetics , Glucose/adverse effects , Humans , Inflammation/drug therapy , Inflammation/genetics , Mesangial Cells/drug effects , MicroRNAs/metabolism , Oxidative Stress/genetics , Proteasome Endopeptidase Complex/genetics , RNA, Circular/geneticsABSTRACT
Diabetic nephropathy (DN) is one of the major microvascular complications of diabetes. Nuclear factor erythroid 2-related factor 2 (Nrf2) is a crucial cellular defense factor to cope with oxidative stress. Silent information regulator T1 (Sirt1) is a deacetylase with antioxidative stress activity. Fucoxanthin is a marine-derived carotenoid. This study was conducted to investigate whether fucoxanthin could alleviate oxidative stress by activating Sirt1/Nrf2 signaling to alleviate DN. In streptozotocin-induced diabetic rats, fucoxanthin treatment effectively improved renal function, alleviated glomerulosclerosis. Fucoxanthin reversed the decreased protein levels of Sirt1 and Nrf2 in the kidney of diabetic rats and glomerular mesangial cells cultured in high glucose. Conversely, EX527, a Sirt1 inhibitor, counteracted the effect of fucoxanthin on the expression of Nrf2. Furthermore, in vivo and vitro results showed that fucoxanthin treatment reversed the low expression and activity of superoxide dismutase and heme oxygenase 1, depending on Sirt1 activation. Our results suggest that fucoxanthin improves diabetic kidney function and renal fibrosis by activating Sirt1/Nrf2 signaling to reduce oxidative stress.
Subject(s)
Antioxidants/pharmacology , Diabetes Mellitus, Experimental/complications , Diabetic Nephropathies/drug therapy , Mesangial Cells/pathology , Xanthophylls/pharmacology , Animals , Antioxidants/therapeutic use , Carbazoles/pharmacology , Cells, Cultured , Diabetes Mellitus, Experimental/chemically induced , Diabetes Mellitus, Experimental/drug therapy , Diabetic Nephropathies/etiology , Diabetic Nephropathies/pathology , Fibrosis , Heme Oxygenase (Decyclizing)/metabolism , Humans , Male , Mesangial Cells/drug effects , NF-E2-Related Factor 2/metabolism , Oxidative Stress/drug effects , Primary Cell Culture , Rats , Rats, Sprague-Dawley , Sirtuin 1/antagonists & inhibitors , Sirtuin 1/metabolism , Streptozocin/administration & dosage , Streptozocin/toxicity , Xanthophylls/therapeutic useABSTRACT
OBJECTIVES: Most patients with systemic lupus erythematosus (SLE) develop lupus nephritis (LN) with severe kidney manifestations. Renal fibrosis can be primarily attributed to overproliferation of mesangial cells (MCs), which are subject to drug treatment. Nevertheless, the detailed mechanisms remain elusive. We sought to identify the effect of cyclophosphamide (CTX), a drug commonly used for LN treatment, on MC proliferation and explore its underlying mechanisms. Material/Methods. Cell proliferation and fibrosis in mouse kidney tissues were determined by histopathology staining techniques. Flow cytometry was used for cell cycle analysis. Cell cycle regulators were examined in vitro following treatment of immortalized human MCs with platelet-derived growth factor subunit B (PDGF-B). Quantitative real-time PCR and western blot analyses were used to measure the mRNA and protein levels of candidate cell cycle regulators, respectively. RESULTS: CTX inhibited cell overproliferation induced by platelet-derived growth factor subunit B in vitro and in vivo. CTX (40 mg/l) was sufficient to induce G0/G1 phase cell cycle arrest. CTX treatment downregulated many critical cell cycle regulators including cyclins and cyclin-dependent kinases but upregulated cyclin-dependent kinase inhibitors. Additionally, CTX-treated samples showed significantly reduced fibrosis, as indicated by lower expression of interleukin-1ß and α-smooth muscle actin. CONCLUSION: CTX inhibits proliferation of MCs by modulating cell cycle regulator and therefore arresting them at G1 phase. CTX treatment significantly alleviates the severity of renal fibrosis. These findings provide novel insights into the mechanisms by which CTX affects LN.
Subject(s)
Cell Cycle Checkpoints , Cyclophosphamide/pharmacology , Fibrosis/drug therapy , Immunosuppressive Agents/pharmacology , Lupus Nephritis/complications , Mesangial Cells/drug effects , Animals , Apoptosis , Cell Proliferation , Cells, Cultured , Female , Fibrosis/etiology , Fibrosis/pathology , Mesangial Cells/metabolism , Mesangial Cells/pathology , Mice , Mice, Inbred C57BLABSTRACT
In a previous study, we demonstrated that melatonin prevents kidney damage in a salt-induced hypertension model by decreasing oxidative stress. We hypothesized that this effect involves melatonin's immunomodulatory properties. In vivo Study-Dahl salt-sensitive (DSS) rats were fed normal chow, a high-salt diet (HSD), or a HSD and melatonin (30 mg/kg/day) in their water for eight weeks. Kidneys were harvested for immediate lymphocyte isolation and characterization by Flow cytometry (CD3+CD4+ and CD3+CD8+) and for lymphocyte chemoattractant (mainly CXCL chemokines) gene expression studies. In vitro study-rat mesangial cells (RMC) were cultured in a high-salt medium without and with melatonin. A HSD was associated with significant renal infiltration of CD4+ and CD8+ T lymphocytes compared to control. Melatonin significantly reduced renal lymphocyte infiltration. A HSD significantly increased mRNA expression of CXCL chemokines. Adding melatonin to the HSD abolished this effect. Treating RMC cells with salt increased the expression of CXCL10 and CXCL11 but not CXCL9. Adding melatonin to the culture media prevented this increase. Treating HSD-fed rats with melatonin decreased renal lymphocyte chemoattractant mRNA expression and is associated with significantly reducing renal T lymphocyte infiltration. Salt may have a direct effect on chemokine-producing renal cells, which is blunted by melatonin treatment.
Subject(s)
Chemokines/metabolism , Hypertension/immunology , Kidney/immunology , Melatonin/pharmacology , Receptors, CXCR3/metabolism , T-Lymphocytes/immunology , Animals , Cell Line , Hypertension/prevention & control , Kidney/drug effects , Ligands , Male , Mesangial Cells/drug effects , Mesangial Cells/metabolism , Rats , Rats, Inbred Dahl , Sodium Chloride, Dietary , T-Lymphocytes/drug effects , Up-Regulation/drug effectsABSTRACT
Progressive diabetic nephropathy (DN) in diabetes leads to major morbidity and mortality. The major pathological alterations of DN include mesangial expansion, extracellular matrix alterations, tubulointerstitial fibrosis, and glomerular sclerosis. Polygoni avicularis is widely used in traditional oriental medicine and has long been used as a diuretic, astringent, insecticide and antihypertensive. However, to the best of the authors' knowledge, the effects of the ethanolic extract from rhizome of Polygoni avicularis (ER-PA) on DN have not yet been assessed. The present study aimed to identify the effect of ER-PA on renal dysfunction, which has been implicated in DN in human renal mesangial cells and db/db mice and investigate its mechanism of action. The in vivo experiment was performed using Polygoni avicularis-ethanol soluble fraction (ER-PA) and was administrated to db/db mice at 10 and 50 mg/kg dose. For the in vitro experiments, the human renal mesangial cells were induced by high glucose (HG, 25 mM). The ER-PA group showed significant amelioration in oral glucose tolerance, and insulin resistance index. ER-PA significantly improved the albumin excretion and markedly reduced plasma creatinine, kidney injury molecule-1 and C-reactive protein. In addition, ER-PA significantly suppressed inflammatory cytokines. Histopathologically, ER-PA attenuated glomerular expansion and tubular fibrosis in db/db mice. Furthermore, ER-PA suppressed the expression of renal fibrosis biomarkers (TGF and Collagen IV). ER-PA also reduced the NLR family pyrin domain containing 3 inflammatory factor level. These results suggest that ER-PA has a protective effect against renal dysfunction through improved insulin resistance as well as the inhibition of nephritis and fibrosis in DN.
Subject(s)
Diabetic Nephropathies/drug therapy , Phytotherapy , Polygonum/chemistry , Animals , Cells, Cultured , Diabetic Nephropathies/metabolism , Diabetic Nephropathies/pathology , Drugs, Chinese Herbal/chemistry , Drugs, Chinese Herbal/pharmacology , Fibrosis , Glucose/metabolism , Humans , Inflammation/drug therapy , Inflammation/metabolism , Inflammation/pathology , Insulin Resistance , Male , Membrane Proteins/metabolism , Mesangial Cells/drug effects , Mesangial Cells/metabolism , Mesangial Cells/pathology , Mice , Mice, Inbred C57BL , Plant Extracts/chemistry , Plant Extracts/pharmacology , Rhizome/chemistryABSTRACT
Doxorubicin (DOX), a category D pregnancy drug, is a chemotherapeutic agent that has been shown in animal studies to induce fetal toxicity, including renal abnormalities. Upregulation of the transient receptor potential cation (TRPC) 6 channel is involved in DOX-induced podocyte apoptosis. We have previously reported that TRPC6-mediated Ca2+ signaling promotes neonatal glomerular mesangial cell (GMC) death. However, it is unknown whether DOX alters mesangial TRPC expression or viability in the fetus. In this study, cell growth was tracked in control and DOX-treated primary GMCs derived from fetal pigs. Live-cell imaging demonstrated that exposure to DOX inhibited the proliferation of fetal pig GMCs and induced cell death. DOX did not alter the TRPC3 expression levels. By contrast, TRPC6 protein expression in the cells was markedly reduced by DOX. DOX treatment also attenuated the TRPC6-mediated intracellular Ca2+ elevation. DOX stimulated mitochondrial reactive oxygen species (mtROS) generation and mitophagy by the GMCs. The DOX-induced mtROS generation and apoptosis were reversed by the mitochondria-targeted antioxidant mitoquinone. These data suggest that DOX-induced fetal pig GMC apoptosis is independent of TRPC6 channel upregulation but requires mtROS production. The mtROS-dependent GMC death may contribute to DOX-induced fetal nephrotoxicity when administered prenatally.