Proliferation Of Human Mesangial Cells Research Articles

Dysregulation of miR-25-3p in Diabetic Nephropathy and Its Role in Inflammatory Response.

To investigate the expression level of miR-25-3p in patients with type 2diabetes mellitus (T2DM) and diabetic nephropathy (DN), and its effect on proliferation, apoptosis and inflammatory response of mesangial cells cultured with high glucose. Blood samples of all clinical subjects were collected for RT-qPCR analysis to detect serum miR-25-3p levels. Human mesangial cells (HMCs) cultured with high glucose were used to construct DN model in vitro. MTT assay, flow cytometry and ELISA were used to evaluate the effects of miR-25-3p on the proliferation, apoptosis, and inflammatory response of DN cell models. Serum miR-25-3p was decreased in both T2DM group and DN group, but more in DN group. Serum miR-25-3p was positively correlated with eGFR and negatively correlated with UAER. The expression of miR-25-3p was reduced in HMCs induced by high glucose. Transfection of miR-25-3p mimic could significantly up-regulate the miR-25-3p level in HMCs. Besides, high glucose culture resulted in abnormal proliferation of HMCs, reduced apoptotic cells, and increased inflammation. The addition of miR-25-3p mimic significantly inhibited cell proliferation and promoted cell apoptosis and reduced the production of inflammatory factors. The abnormal reduction of serum miR-25-3p in DN indicates that it may be a potential biomarker for clinical diagnosis of DN. In in vitro experiments, miR-25-3p was involved in the progression of DN by regulating cell proliferation, apoptosis, and inflammatory response.

Long non-coding RNA MEG3 silencing weakens high glucose-induced mesangial cell injury by decreasing LIN28B expression by sponging and sequestering miR-23c.

Diabetic nephropathy (DN) is a common kidney disease in diabetic patients. Long non-coding RNA maternally expressed gene 3 (MEG3) and microRNA (miR)-23c are reported to be implicated in DN development. Nevertheless, it is unclear that the molecular mechanism between MEG3 and miR-23c in DN remains unclear. Human mesangial cells (HMCs) were treated with high glucose (HG) to simulate the DN status in vitro. Expression of MEG3 and miR-23c was measured. Effects of MEG3 silencing on HG-stimulated HMC injury were determined. The relationship between MEG3 and miR-23c was verified by the dual-luciferase reporter and RNA immunoprecipitation assays. MEG3 was overexpressed in serums from DN patients and HG-stimulated HMCs. MEG3 knockdown weakened HG-stimulated HMC proliferation, extracellular matrix (ECM) accumulation, and inflammation. MEG3 regulated lin-28 homolog B (LIN28B) expression through adsorbing miR-23c. MiR-23c inhibitor reversed MEG3 knockdown-mediated effects on HG-stimulated HMC proliferation, ECM accumulation, and inflammation. LIN28B overexpression overturned miR-23c mimic-mediated effects on HG-stimulated HMC proliferation, ECM accumulation, and inflammation. MEG3 regulated HMC injury via regulation of the miR-23c/LIN28B axis in DN, which can help us better understand the mechanism of DN mediated by MEG3.

Overexpression of FTO inhibits excessive proliferation and promotes the apoptosis of human glomerular mesangial cells by alleviating FOXO6 m6A modification via YTHDF3-dependent mechanisms.

Background: N6-methyladenosine (m6A) is a prevalent post-transcriptional modification presented in messenger RNA (mRNA) of eukaryotic organisms. Chronic glomerulonephritis (CGN) is characterised by excessive proliferation and insufficient apoptosis of human glomerular mesangial cells (HGMCs) but its underlying pathogenesis remains undefined. Moreover, the role of m6A in CGN is poorly understood. Methods: The total level of m6A modification was detected using the m6A quantification assay (Colorimetric). Cell proliferation was assessed by EdU cell proliferation assay, and cell apoptosis was detected by flow cytometry. RNA sequencing was performed to screen the downstream target of fat mass and obesity-associated protein (FTO). MeRIP-qPCR was conducted to detect the m6A level of forkhead box o6 (FOXO6) in HGMCs. RIP assay was utilized to indicate the targeting relationship between YTH domain family 3 (YTHDF3) and FOXO6. Actinomycin D assay was used to investigate the stability of FOXO6 in HGMCs. Results: The study found that the expression of FTO was significantly reduced in lipopolysaccharide (LPS)-induced HGMCs and renal biopsy samples of patients with CGN. Moreover, FTO overexpression and knockdown could regulate the proliferation and apoptosis of HGMCs. Furthermore, RNA sequencing and cellular experiments revealed FOXO6 as a downstream target of FTO in regulating the proliferation and apoptosis of HGMCs. Mechanistically, FTO overexpression decreases the level of FOXO6 m6A modification and reduces the stability of FOXO6 mRNA in a YTHDF3-dependent manner. Additionally, the decreased expression of FOXO6 inhibits the PI3K/AKT signaling pathway, thereby inhibiting the proliferation and promoting apoptosis of HGMCs. Conclusion: This study offers insights into the mechanism through which FTO regulates the proliferation and apoptosis of HGMCs by mediating m6A modification of FOXO6 mRNA. These findings also suggest FTO as a potential diagnostic marker and therapeutic target for CGN.

RNA sequencing reveals the mechanism of FTO in inhibiting inflammation and excessive proliferation of lipopolysaccharide-induced human glomerular mesangial cells.

Chronic glomerulonephritis (CGN) is a leading cause of end-stage renal disease in China; thus, there is an urgent need for effective therapeutic targets and strategies for CGN treatment. However, studies on CGN pathogenesis are limited. In this study, we found that the fat mass and obesity-associated protein (FTO) was significantly decreased in the lipopolysaccharide (LPS)-induced human glomerular mesangial cells (HGMCs) (P < 0.01) and kidney tissues of CGN patients (P < 0.05). Moreover, double-labeling immunofluorescence and flow cytometry assays demonstrated that the overexpression of FTO could inhibit inflammation and excessive proliferation of HGMCs. Furthermore, RNA-sequencing (RNA-seq) and real-time quantitative polymerase chain reaction (RT-qPCR) analyses revealed that FTO overexpression induced differential expression of 269 genes (absolute fold change ≥ 2 and P-value < 0.05), including 143 upregulated and 126 downregulated genes. Further functional analysis of these differentially expressed genes by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses suggested that FTO possibly mediates its inhibitory function by regulating the mammalian target of rapamycin (mTOR) signaling pathway and substance metabolism. Lastly, analysis of the PPI network and further identification of the top 10 hub genes (RPS15, RPS18, RPL18A, GNB2L1, RPL19, EEF1A1, RPS25, FAU, UBA52, and RPS6) indicated that FTO mediates its function by affecting the ribosomal proteins. Therefore, in this study, we elucidated the important role of FTO in the regulation of inflammation and excessive proliferation of HGMCs, suggesting FTO administration as a suitable therapeutic intervention for CGN.

Spiroleiferthione A and Oleiferthione A: Two Unusual Isothiocyanate-Derived Thioketone Alkaloids from Moringa oleifera Lam. Seeds.

Spiroleiferthione A (1), with a 2-thiohydantoin a heterocyclic spiro skeleton, and oleiferthione A (2), an imidazole-2-thione derivative, were isolated from the aqueous extract of Moringa oleifera Lam. seeds. The unprecedented structures of 1 and 2 were elucidated by extensive spectroscopic data, X-ray diffraction, and gauge-independent atomic orbital (GIAO) NMR calculation, as well as electronic circular dichroism (ECD) calculation. The structures of 1 and 2 were determined to be (5R,7R,8S)-8-hydroxy-3-(4'-hydroxybenzyl)-7-methyl-2-thioxo-6-oxa-1, 3-diazaspiro [4.4] nonan-4-one, and 1-(4'-hydroxybenzyl)-4,5-dimethyl-1,3-dihydro-2H-imidazole-2-thione, respectively. Biosynthetic pathways for 1 and 2 have been proposed. Compounds 1 and 2 are considered to have originated from isothiocyanate and then undergone a series of oxidation and cyclization reactions to form 1 and 2. Compounds 1 and 2 demonstrated weak inhibition rates of NO production, 42.81 ± 1.56% and 33.53 ± 2.34%, respectively, at a concentration of 50 μM. Additionally, Spiroleiferthione A demonstrated moderate inhibitory activity against high glucose-induced human renal mesangial cell proliferation in a dosage-dependent manner. A wider range of biological activities, and the diabetic nephropathy protective activity of Compound 1 in vivo and its mechanism of action, need further investigation after the sufficient enrichment of Compound 1 or total synthesis.

Network Pharmacology and Experimental Validation to Explore That Celastrol Targeting PTEN is the Potential Mechanism of Tripterygium wilfordii (Lév.) Hutch Against IgA Nephropathy.

Accumulating clinical evidence showed that Tripterygium hypoglaucum (Lév.) Hutch (THH) is effective against IgA nephropathy (IgAN), but the mechanism is still unclear. This study is to evaluate the renal protective effect and molecular mechanism of THH against IgAN via network pharmacology, molecular docking strategy and experimental validation. Several databases were used for obtaining the active ingredients of THH, the corresponding targets, as well as the IgAN-related genes. The critical active ingredients, functional pathways, and potential for the combination of the hub genes and their corresponding active components were determined through bioinformatics analysis and molecular docking. The IgAN mouse model was treated with celastrol (1 mg/kg/d) for 21 days, and the aggregated IgA1-induced human mesangial cell (HMC) was treated with various concentrations of celastrol (25, 50 or 75 nM) for 48 h. The immunohistochemistry and Western blot techniques were applied to evaluate the protein expression of the predicted target. The cell counting kit 8 (CCK8) was used to detect HMC proliferation. A total of 17 active ingredients from THH were screened, covering 165 IgAN-related targets. The PPI network identified ten hub targets, including PTEN. The binding affinity between the celastrol and PTEN was the highest (-8.69 kJ/mol). The immunohistochemistry showed that celastrol promoted the expression of PTEN in the glomerulus of IgAN mice. Furthermore, the Western blot techniques showed that celastrol significantly elevated the expression of PTEN and inhibited PCNA and Cyclin D1 in vitro and in vivo. The CCK8 assay determined that celastrol decreased HMC proliferation in a concentration-dependent manner. This study suggests that activating PTEN by celastrol may play a pivotal role in THH alleviating IgAN renal injury.

Salviolone protects against high glucose-induced proliferation, oxidative stress, inflammation, and fibrosis of human renal mesangial cells by upregulating membrane metalloendopeptidase expression.

As one of complications of diabetes mellitus, diabetic nephropathy is related to renal dysfunction. Membrane metalloendopeptidase (MME) is associated with the pathogenesis of diabetic nephropathy and exerts a protective function in high glucose (HG)-treated podocytes. Salviolone, one of important bioactive components from Salvia miltiorrhiza, possesses an anti-inflammatory activity. However, the roles of salviolone in renal mesangial cell dysfunction under HG condition remain unknown. The targets of salviolone in diabetic nephropathy were predicted by bioinformatics analysis. Relative mRNA level of MME was detected by qPCR in HG-treated human renal mesangial cells (HRMCs). Cell viability was analyzed using CCK-8 assay. Cell proliferation was investigated by EdU staining. Oxidative stress was evaluated by detection of ROS generation and levels of oxidative stress-related biomarkers. The inflammatory cytokines and fibrosis-related biomarkers were examined by ELISA. Our results showed that MME expression was decreased in diabetic nephropathy and HG-treated HRMCs. Salviolone increased MME level in HG-treated HRMCs. Salviolone mitigated HG-induced HRMC proliferation by increasing MME expression. Salviolone attenuated HG-induced ROS generation, MDA level increase, and SOD activity decrease through upregulating MME expression. Moreover, salviolone suppressed HG-induced increase of levels of TNF-α, IL-1β, IL-6, fibronectin, and collagen IV through upregulating MME expression. In conclusion, salviolone attenuates proliferation, oxidative stress, inflammation, and fibrosis in HG-treated HRMCs through upregulating MME expression.

Identification and characterization of circulating immune complexes in IgA nephropathy.

The underlying pathology of immunoglobulin A (IgA) nephropathy (IgAN), the most common glomerulonephritis worldwide, is driven by the deposition of immune complexes containing galactose-deficient IgA1 [Tn(+)IgA1] in the glomerular mesangium. Here, we report that novel anti-Tn circulating immune complexes (anti-Tn CICs) contain predominantly IgM, representing large macromolecular complexes of ~1.2 megadaltons to several megadalton sizes together with Tn(+)IgA1 and some IgG. These complexes are significantly elevated in sera of patients with IgAN, which contains higher levels of complement C3, compared to healthy individuals. Anti-Tn CICs are bioactive and induce specific proliferation of human renal mesangial cells. We found that these anti-Tn CICs can be dissociated with small glycomimetic compounds, which mimic the Tn antigen of Tn(+)IgA1, releasing IgA1 from anti-Tn CICs. This glycomimetic compound can also significantly inhibit the proliferative activity of anti-Tn CICs of patients with IgAN. These findings could enhance both the diagnosis of IgAN and its treatment, as specific drug treatments are now unavailable.

1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) Induces Cell Apoptosis of Human Mesangial Cells

In China, primary glomerulonephritis is the leading cause of end-stage renal disease (ESRD), and patients who require kidney transplantation often with ESRD. Glomerulonephritis primary’s most prevalent subtype is Mesangial proliferative glomerulonephritis (MsPGN). Although the cell apoptosis of human mesangial cells (HMCs) may be harmful in MsPGN, it plays a major role in the elimination of excessive HMCs and reparative glomerular remodelling after inflammation. A powerful mitogen for growing mesangial cells is epidermal growth factor (EGF). Therefore, it is possible to induce the proliferation of HMCs by incubating with EGF in vitro. 1,25(OH)2D3(VD3) may cause cell death in HMCs via activating AKT, caspase-3, caspase-9, JNK, and ERK, as well as elevating Bax and Bad levels.

Triptolide promotes autophagy to inhibit mesangial cell proliferation in IgA nephropathy via the CARD9/p38 MAPK pathway.

BackgroundMesangial cell proliferation is the most basic pathological feature of immunoglobulin A nephropathy (IgAN); however, the specific underlying mechanism and an appropriate therapeutic strategy are yet to be unearthed. This study aimed to investigate the therapeutic effect of triptolide (TP) on IgAN and the mechanism by which TP regulates autophagy and proliferation of mesangial cells through the CARD9/p38 MAPK pathway.MethodsWe established a TP‐treated IgAN mouse model and produced IgA1‐induced human mesangial cells (HMC) and divided them into control, TP, IgAN, and IgAN+TP groups. The levels of mesangial cell proliferation (PCNA, cyclin D1, cell viability, and cell cycle) and autophagy (P62, LC3 II, and autophagy flux rate) were measured, with the autophagy inhibitor 3‐Methyladenine used to explore the relationship between autophagy and proliferation. We observed CARD9 expression in renal biopsies from patients and analyzed its clinical significance. CARD9 siRNA and overexpression plasmids were constructed to investigate the changes in mesangial cell proliferation and autophagy as well as the expression of CARD9 and p‐p38 MAPK/p38 MAPK following TP treatment.ResultsAdministering TP was safe and effectively alleviated mesangial cell proliferation in IgAN mice. Moreover, TP inhibited IgA1‐induced HMC proliferation by promoting autophagy. The high expression of CARD9 in IgAN patients was positively correlated with the severity of HMC proliferation. CARD9/p38 MAPK was involved in the regulation of HMC autophagy and proliferation, and TP promoted autophagy to inhibit HMC proliferation by downregulating the CARD9/p38 MAPK pathway in IgAN.ConclusionTP promotes autophagy to inhibit mesangial cell proliferation in IgAN via the CARD9/p38 MAPK pathway.

Stimulated phosphorylation of ERK in mouse kidney mesangial cells is dependent upon expression of Cav3.1

BackgroundT-type calcium channels (TTCC) are low voltage activated channels that are widely expressed in the heart, smooth muscle and neurons. They are known to impact on cell cycle progression in cancer and smooth muscle cells and more recently, have been implicated in rat and human mesangial cell proliferation. The aim of this study was to investigate the roles of the different isoforms of TTCC in mouse mesangial cells to establish which may be the best therapeutic target for treating mesangioproliferative kidney diseases. MethodsIn this study, we generated single and double knockout (SKO and DKO) clones of the TTCC isoforms CaV3.1 and CaV3.2 in mouse mesangial cells using CRISPR-cas9 gene editing. The downstream signals linked to this channel activity were studied by ERK1/2 phosphorylation assays in serum, PDGF and TGF-β1 stimulated cells. We also examined their proliferative responses in the presence of the TTCC inhibitors mibefradil and TH1177.ResultsWe demonstrate a complete loss of ERK1/2 phosphorylation in response to multiple stimuli (serum, PDGF, TGF-β1) in CaV3.1 SKO clone, whereas the CaV3.2 SKO clone retained these phospho-ERK1/2 responses. Stimulated cell proliferation was not profoundly impacted in either SKO clone and both clones remained sensitive to non-selective TTCC blockers, suggesting a role for more than one TTCC isoform in cell cycle progression. Deletion of both the isoforms resulted in cell death.ConclusionThis study confirms that TTCC are expressed in mouse mesangial cells and that they play a role in cell proliferation. Whereas the CaV3.1 isoform is required for stimulated phosphorylation of ERK1/2, the Ca V3.2 isoform is not. Our data also suggest that neither isoform is necessary for cell proliferation and that the anti-proliferative effects of mibefradil and TH1177 are not isoform-specific. These findings are consistent with data from in vivo rat mesangial proliferation Thy1 models and support the future use of genetic mouse models to test the therapeutic actions of TTCC inhibitors.

Circular ribonucleic acid nucleoporin 98 knockdown alleviates high glucose-induced proliferation, fibrosis, inflammation and oxidative stress in human glomerular mesangial cells by regulating the microribonucleic acid-151-3p-high mobility group AT-hook 2 axis.

Aims/IntroductionThis study aimed to investigate the role and mechanism of circular ribonucleic acid nucleoporin 98 (circNUP98) in diabetic nephropathy (DN).Materials and MethodsHuman 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.ResultsCircNUP98 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.ConclusionCircNUP98 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.

Salvianolic acid B attenuates membranous nephropathy by activating renal autophagy via microRNA-145-5p/phosphatidylinositol 3-kinase/AKT pathway

ABSTRACT The abnormal proliferation and inflammatory response of the mesangial cells play a crucial role in the progression of membranous nephropathy (MN). Herein, this study aimed to investigate the therapeutic effect of Salvianolic acid B (SalB) on MN-induced mesangial abnormalities and its underlying mechanisms. MN models were established in cationic bovine serum albumin-induced Sprague-Dawley rats and lipopolysaccharide-induced human mesangial cells (HMCs). Following SalB and microRNA-145-5p antagomir treatment, kidney function was investigated by 24-hours urine protein, serum creatinine, and blood urea nitrogen. Pathological changes of kidney were investigated by Periodic acid Schiff staining. CD68 and IgG were detected by immunofluorescence in glomerulus. Mesangial autophagosomes were observed by transmission electron microscope. MicroRNA-145-5p inhibitor, mimic, LY294002, and SalB were used to treat with HMCs. In kidney and HMCs, IL-1 , IL-2, IL-6, TNF- and microRNA-145-5p was detected by quantitative real-time PCR. Phosphatidylinositol 3-kinase (PI3K), phosphorylated AKT, AKT, beclin1, and microtubule-associated protein light chain 3 (LC3) levels were detected by Western blot. HMCs proliferation and cycle were detected by Cell Counting Kit-8 and flow cytometry. LC3 were detected by LC3-dual-fluorescent adenovirus in HMCs. Our results showed that SalB significantly ameliorated kidney function and pathological changes. Furthermore, it significantly alleviated proliferation, inflammation and activated autophagy in mesangial cells. Moreover, microRNA-145-5p antagomir accentuated MN while microRNA-145-5p inhibitor and LY294002 encouraged proliferation and inflammation through PI3K/AKT pathway in HMCs. Collectively, our study demonstrated that SalB activated renal autophagy to reduce cell proliferation and inflammation of MN, which was mediated by microRNA-145-5p to inhibit PI3K/AKT pathway, and ultimately attenuated MN.

Retracted: Resistin-Like Molecule Beta (RELM-β) Regulates Proliferation of Human Diabetic Nephropathy Mesangial Cells via Mitogen-Activated Protein Kinases (MAPK) Signaling Pathway.

This publication has been retracted by the Editor due to the identification of non-original figure images and manuscript content that raise concerns regarding the credibility and originality of the study and the manuscript. Reference: Yun-Qian Wang, Cong-Cong Fan, Bao-Ping Chen, Jun Shi. Resistin-Like Molecule Beta (RELM-ß) Regulates Proliferation of Human Diabetic Nephropathy Mesangial Cells via Mitogen-Activated Protein Kinases (MAPK) Signaling Pathway.Med Sci Monit 2017; 23:3897-3903. DOI: 10.12659/MSM.905381.

LncRNA HOTAIR facilitates high glucose-induced mesangial cell proliferation, fibrosis and oxidative stress in diabetic nephropathy via regulating miR-147a/WNT2B axis

BackgroundLong non-coding RNAs (lncRNAs) have been shown to be involved in the regulation of many disease progression. However, the role of lncRNA HOX transcript antisense RNA (HOTAIR) in diabetic nephropathy (DN) remains unclear.MethodsHigh glucose (HG)-induced human mesangial cells (HMC) was used to construct DN cell models in vitro. HMC proliferation was evaluated by CCK8 assay and EDU staining. Protein levels of proliferation markers, fibrosis markers, and wingless-type family member 2B (WNT2B) were measured using western blot analysis. HMC oxidative stress was assessed by determining the levels of oxygen species and malondialdehyde, as well as superoxide dismutase activity. Relative expression levels of lncRNA HOTAIR, microRNA (miR)-147a, and WNT2B were examined using quantitative real-time PCR. The interaction between miR-147a and lncRNA HOTAIR or WNT2B was confirmed by dual-luciferase reporter assay and RIP assay.ResultsOur data showed that lncRNA HOTAIR knockdown could inhibit the proliferation, fibrosis, and oxidative stress in HG-induced HMC. LncRNA HOTAIR could serve as a sponge of miR-147a. The inhibition effect of lncRNA HOTAIR silencing on the biological functions of HG-induced HMC could be reversed by miR-147a inhibitor. WNT2B was targeted by miR-147a, and its overexpression also overturned the suppressive effect of miR-147a on the proliferation, fibrosis, and oxidative stress of HG-induced HMC.ConclusionIn total, our research pointed out that lncRNA HOTAIR could mediate miR-147a/WNT2B axis to promote DN progression.

Trigonelline reverses high glucose-induced proliferation, fibrosis of mesangial cells via modulation of Wnt signaling pathway

BackgroundDiabetic nephropathy (DN) is the leading cause of the end-stage renal disease (ESRD). The proliferation and apoptosis of mesangial cells induced by the activated Wnt/β-catenin pathway is crucial in DN. Trigonelline (TRL) is an alkaloid that has been shown to decrease proteinuria and protect the renal function in DN. However, the effect of TRL on the Wnt/β-catenin pathway of mesangial cells is unclear.MethodsAs a cellular DN model, human mesangial cells (HMCs) were treated with high-glucose (HG). β-Catenin plasmid and control knockdown plasmids were transfected into HG-treated HMCs as β-catenin pcDNA and β-catenin siRNA groups, respectively. Cell viability was measured by MTT assay. Flow cytometry was used to detect the cell cycle. Cell apoptosis was evaluated by flow cytometry and terminal dUTP transferase nick end labeling (TUNEL) assay. mRNA expression of Wnt1, Wnt3a, Wnt4, Wnt5a, β-catenin, TCF4, Cyclin D1, and CDK4 were detected by qRT-PCR. Protein expression of Wnt4, Wnt5a, nucleus-β-catenin, TCF4, Cyclin D1, and CDK4 were detected by western blotting.ResultsTRL significantly inhibited HG-induced HMCs viability over three-time points measured (24, 48, and 72 h). In addition, TRL suppressed the levels of fibronectin (FN) and collagen IV (Col IV) in HG-stimulated HMCs. Furthermore, TRL efficiently inhibited the activation of the Wnt/β-catenin signaling pathway in HG-stimulated HMCs. Taken together, these data indicated that TRL inhibited HG-induced HMCs proliferation and ECM expression via the modulation of the Wnt signaling pathway.ConclusionsTRL reduces HG-induced cell injury by regulating the Wnt/β-catenin signaling pathway.

LncRNA ZFAS1 regulates the proliferation, oxidative stress, fibrosis, and inflammation of high glucose-induced human mesangial cells via the miR-588/ROCK1 axis

BackgroundDiabetic nephropathy (DN) is a critical and the most common microvascular complication and its pathogenesis is still faintly understood. Thus, this study was performed to examine the long non-coding RNA ZNFX1 Antisense Gene Protein 1 (lncRNA ZFAS1) biological function and mechanism of regulation in DN.MethodHuman glomerular mesangial cells (HGMC) were induced with high glucose (HG, 25 mM) to establish HG-induced cell viability, pro-inflammation observed in DN. After, target miRNA and mRNA were predicted through Lncbase and Targetscan. Subsequently, the expression of ZFAS1, miR-588, and ROCK1 in DN clinical samples and cell-model was examined through qRT-PCR and western blot analysis. We upheld the targeted interaction between miR-588 and ZFAS1 or ROCK1 through a dual-luciferase reporter assay. The proliferation of the cell was also examined through CCK-8 assay, while the level of HG-induced oxidative stress was established by measuring reactive oxygen species (ROS) level, and also the activities of antioxidant enzymes in the cell. Lastly, the level of accumulated extracellular matrix (ECM) protein-fibronectin and collagen type IV, and inflammatory cytokines produced by the cell was analyzed through western blot analysis and ELISA.ResultsZFAS1 was significantly upregulated in the DN blood samples and HG-induced HGMC. Prediction result revealed that the ZFAS1 endogenously targets the miR-588 seed sequence while miR-588 plays a role in post-transcriptional regulation of ROCK1 mRNA. Moreover, we found that miR-588 expression was significantly downregulated in DN blood samples and negatively correlates with ZFAS1 expression. Further results show that silencing ZFAS1 had a protective effect on HG-induced proliferation, oxidative stress, fibrosis, and inflammation in HGMC while miR-588 inhibition and ROCK1 overexpression reversed this effect.ConclusionsAltogether, our data suggest that ZFAS1 regulates the proliferation, oxidative stress, fibrosis, and inflammation of high glucose-induced diabetic nephropathy through the miR-588/ROCK1 axis.

Quercetin attenuates the proliferation, inflammation, and oxidative stress of high glucose-induced human mesangial cells by regulating the miR-485-5p/YAP1 pathway.

BackgroundDiabetic nephropathy (DN) is a kidney damage caused by diabetes and the main cause of end-stage renal disease. However, the current treatment of DN has many limitations. Quercetin is a bioflavonoid compound with therapeutic benefits in metabolic diseases. This study aims to determine the therapeutic potentials and underlying mechanism of quercetin on DN.MethodsWe collected blood samples from DN patients and healthy controls and treated human mesangial cells (HMCs) with high glucose (HG) to establish an in vitro model of DN. Then we assessed the expression difference of miR-485-5p as well as YAP1 in serum of DN patients and healthy controls and between HG-induced HMCs and control cells. qRT-PCR and western blot were performed to assess miR-485-5p and YAP1 expression levels; CCK-8 and ELISAs were used to examine cell proliferation, inflammation, and oxidative stress. Dual luciferase reporter assay was implemented to detect the binding of miR-485-5p and YAP1 mRNA sequence.ResultsQuercetin suppressed proliferation, inflammation, and oxidative stress of HMCs induced by HG. As for mechanism, miR-485-5p directly bound to YAP1 and inhibited YAP1 expression. The downregulation of miR-485-5p and upregulation of YAP1 were also observed in the serum of DN patients. Quercetin modulated miR-485-5p/YAP1 axis to regulate HG-induced inflammation and oxidative stress.Conclusion:Quercetin inhibits the proliferation, inflammation, and oxidative stress of HMCs induced by HG through miR-485-5p/YAP1 axis, which might provide a novel treatment strategy for DN.

Based on Network Pharmacology Tools to Investigate the Mechanism of Tripterygium wilfordii Against IgA Nephropathy.

Background: Immunoglobulin A nephropathy (IgAN) is the most common primary glomerular disease and poses a global major public health burden. The preparation of Tripterygium wilfordii Hook F (TwHF) is widely applied for treating patients with Immunoglobulin A nephropathy in China, while the molecular mechanisms remain unclear. This study aimed to verify the therapeutic mechanism of TwHF on IgAN by undertaking a holistic network pharmacology strategy in combination with in vitro and in vivo experiments.Methods: TwHF active ingredients and their targets were obtained via the Traditional Chinese Medicine Systems Pharmacology Database. The collection of IgAN-related target genes was collected from GeneCards and OMIM. TwHF-IgAN common targets were integrated and visualized by Cytoscape. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were performed to determine the predominant molecular mechanisms and pathways of TwHF on the treatment of IgAN. The protein-protein interaction network was constructed by the STRING online search tool, and hub genes were identified using R software. The expression of hub gene and related signaling were evaluated in TwHF-treated mice through immunohistochemistry and western blot and further validated in human mesangial cells (HMCs). In addition, Cell counting kit 8 (CCK8) and flow cytometry were used to detect the effects of TwHF on cell proliferation and cell cycle of mesangial cells.Results: A total of 51 active ingredients were screened from TwHF and 61 overlapping targets related to IgAN were considered potential therapeutic targets, GO functions and KEGG analyses demonstrated that these genes were primarily associated with DNA-binding transcription factor binding, lipid and atherosclerosis pathway. Genes with higher degrees including AKT1, CXCL8, MMP9, PTGS2, CASP3, JUN are hub genes of TwHF against IgAN. Verification of hub gene JUN both in vitro and in vivo showed that TwHF significantly attenuated JUN phosphorylation in the kidneys of IgAN mice and aIgA1-activated HMCs, meanwhile suppressing HMCs proliferation and arresting G1-S cell cycle progression.Conclusion: Our research strengthened the mechanisms of TwHF in treating IgAN, inhibition of JUN activation may play a pivotal role in TwHF in alleviating IgAN renal injury.

Endosulfan promotes cell proliferation and extracellular matrix accumulation through TGF-β/Smad signaling pathway in HRMCs

Endosulfan is an organochlorine pesticide, which poses a potential danger to human health and safety. It is known that dysfunction of glomerular mesangial cells causes glomerular sclerosis, associated with chronic kidney diseases. In the present study, we investigated the effects of endosulfan on cell proliferation and extracellular matrix accumulation (ECM) in human renal mesangial cells (HRMCs). Cells were treated with endosulfan, endosulfan (10 μM) plus specific inhibitor of TGF-β signaling (LY2109761) or antioxidant (NAC). The results showed that endosulfan significantly promoted cell proliferation, accompanied with the decrease of p27 mRNA expression and the increase in the mRNA expression levels of p21 and inflammatory factors IL-6/IL-8. qRT-PCR results showed that matrix metalloproteinase-2 (MMP2) and tissue metalloproteinase-3 (TIMP3) were down-regulated whereas laminin was up-regulated when exposure to endosulfan. Western blot results showed that p-Smad2/3 was up-regulated, while Smad7 was down-regulated when exposure to endosulfan, which were reversed in the presence of LY2109761. Endosulfan significantly decreased the activity of SOD and increased the MDA level and CAT activity, which were reversed in the presence of NAC. These findings suggest that endosulfan can cause excessive proliferation and massive accumulation of ECM through TGF-β/Smad signaling pathway, and also induced oxidative stress and inflammation in HRMCs.