LncRNA NNT-AS1 regulates proliferation, ECM accumulation and inflammation of human mesangial cells induced by high glucose through miR-214-5p/smad4.

BACKGROUND: LncRNA NNT-AS1 (NNT-AS1) has been extensively studied as the causative agent in propagation and progression of lung and bladder cancers, and cholangiocarcinoma. However, its significance in proliferation and inflammation of diabetic nephropathy is enigmatic. This study focuses on the molecular mechanisms followed by NNT-AS1 to establish diabetic nephropathy (DN) and its potential miRNA target. METHODS: Bioinformatics analysis to identify potential miRNA target of NNT-AS1 and smad4 transcription factor was conducted using LncBase and TargetScan, and was subsequently confirmed by luciferase reporter assay. Relative quantitative expression of NNT-AS1 in human glomerular mesangial cells (HGMCs) was detected through quantitative real-time PCR and WB analysis. Cell proliferation was detected through CCK-8 assay, whereas, ELISA was conducted to evaluate the expression of inflammatory cytokines. Following this, relative expression of miR-214-5p and smad4 were confirmed through qRT-PCR and western blot analysis. RESULTS: Results from the experiments manifested up-regulated levels of NNT-AS1 and smad4 in the blood samples of DN patients as well as in HGMCs, whereas, downregulated levels of miR-214-5p were measured in the HGMCs suggesting the negative correlation between NNT-AS1 and miR-214-5p. Potential binding sites of NNT-AS1 showed miR-214-5p as its direct target and NNT-AS1 as potential absorber for this microRNA, in turn increasing the expression of transcription factor smad4. CONCLUSION: The data suggests that NNT-AS1 can be positively used as a potential biomarker and indicator of DN and causes extracellular matrix (ECM) accumulation and inflammation of human mesangial cells.

miR-485 suppresses inflammation and proliferation of mesangial cells in an in vitro model of diabetic nephropathy by targeting NOX5.

Diabetic nephropathy (DN) is among the common complications of diabetes and is a major cause of end-stage kidney disease. Emerging data indicate that renal inflammation is involved in DN progression and aggravation. Still, the exact cellular mechanisms remain unclear. Dysregulated expression of microRNAs (miRNAs) is associated with multiple diseases, including DN. The relationship between miRNAs and inflammation in DN is also unexplored. Here, we evaluated the role of miR-485 in mediating the response of human mesangial cells (HMCs) to a high glucose (HG) concentration, and the potential underlying mechanism. We found that miR-485 expression is significantly decreased in HG-stimulated HMCs. Overexpression of miR-485 suppressed HG-induced proliferation of HMCs. Lower production of proinflammatory cytokines (i.e., TNF-α, IL-1ß, and IL-6) was observed in miR-485-overexpressing HMCs. Overexpression of miR-485 markedly suppressed the overexpression of extracellular-matrix proteins, e.g., collagen IV (Col IV) and fibronectin (FN), in HG-stimulated HMCs. Furthermore, miR-485 suppressed the expression of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 5 (NOX5), restrained the HG-induced HMC proliferation, downregulated the expression of proinflammatory cytokines, and inhibited the production of extracellular-matrix proteins in HMCs. These results provide new insights into the involvement of the miR-485-NOX5 signaling pathway in DN progression.

IgA1 isolated from Henoch-Schönlein purpura children promotes proliferation of human mesangial cells in vitro.

Previous studies show that the proliferation of human mesangial cells (HMCs) played a significant part in the pathogenesis of Henoch-Schönlein purpura nephritis (HSPN). The aim of this study was to explore the proliferation of HMCs induced by IgA1 isolated from the sera of HSP patients. HMCs were cultured in three different types of media, including IgA1 from patients with HSP (HSP IgA1 group), healthy children (healthy IgA1 group) and medium (control group). The proliferation of HMCs incubated with IgA1 was determined by cell counting kit-8 assay and bromodeoxyuridine incorporation. The expression of ERK1/2 and phosphatidylinositol 3 kinase/protein kinase B/mammalian targets of the rapamycin (PI3K/AKt/mTOR) signals and transferrin receptor (TfR/CD71) was detected with the methods of immunoblotting. The results indicated that the proliferation of HMCs significantly increased in the HSP IgA1 group compared with that in the control group or the healthy IgA1 group (P < 0.001). Moreover, we found that IgA1 isolated from HSP patients activated ERK and PI3K/AKt/mTOR signals, and markedly increased TfR/CD71 expression in HMCs. These effects induced by IgA1 isolated from patients with HSP were inhibited by human TfR polyclonal antibody (hTfR pAb) and soluble human transferrin receptor (sTfR), indicating that IgA1-induced HMC proliferation and ERK1/2 and PI3K/AKt/mTOR activation were dependent on TfR/CD71 engagement. Altogether, these data suggested that TfR/CD71 overexpression and ERK1/2 and PI3K/AKt/mTOR activation were engaged in HMC proliferation induced by IgA1 from HSP patients, which might be related to the mesangial injury of HSPN.

Norcantharidin Enhances High Concentrations of Fetal Bovine Serum-Induced Apoptosis in Human Mesangial Cells by Regulating the Mitogen-Activated Protein Kinase Signaling Pathway.

AIM: This study aimed to investigate the effect of norcantharidin (NCTD) on human mesangial cells (HMCs) apoptosis in vitro and further examine its molecular mechanism. METHODS: HMCs were divided into 5 groups: control group, 25% fetal bovine serum (FBS)-treated group, and NCTD groups (NCTD [2.5, 5 and 10 µg/mL] + 25% FBS, respectively). Cell proliferation was determined by MTT assay, while apoptosis was evaluated by Hoechest 33258 staining, the level of cytochrome c, immunohistochemistry, and apoptotic-related proteins/gene expression. RESULTS: Cell viability was inhibited in NCTD-treated HMCs in a dose-dependent manner. The number of apoptotic cells and the content of cytochrome c were significantly increased by NCTD treatment but that of mitochondrial membrane was decreased. Moreover, the expression of bcl-2 and caspase-3 was prompted by NCTD, but the expression of bax, MMP-2, and MMP-9 in 25% FBS-treated HMCs was inhibited. In addition, NCTD markedly unregulated the expression of apoptosis-related gene/protein, including p-Erk1/2, phosphorylated-Jun N-terminal kinase (JNK), p-p38, and p53. CONCLUSION: NCTD enhances 25% FBS-treated HMC apoptosis in vitro, and this effect may be attributed to the modulation of the ERK, JNK, and p38 mitogen-activated protein kinase signaling pathways.

Klotho down-regulates Egr-1 by inhibiting TGF-ß1/Smad3 signaling in high glucose treated human mesangial cells.

Diabetic kidney disease (DKD) has become the leading cause of end-stage renal disease worldwide and is associated with glomerular mesangial cell (MC) proliferation and excessive extracellular matrix (ECM) production. Klotho can attenuate renal fibrosis in part by inhibiting TGF-ß1/Smad3 signaling in DKD. Early growth response factor 1 (Egr-1) has been shown to play a key role in renal fibrosis in part by facilitating the formation of a positive feedback loop involving TGF-ß1. However, whether Klotho down-regulates Egr-1 by inhibiting TGF-ß1/Smad3 signaling in DKD is unclear. In the present study, we assessed human MCs that were incubated under high-glucose conditions to mimic diabetes. Then, we transfected the cells with Klotho plasmid or siRNA to overexpress or knock down Klotho gene and protein expression. Klotho, Egr-1, fibronectin (FN), collagen type I (Col I), Smad3 and phosphorylated Smad3 (p-Smad3) gene and protein expression levels were determined by RT-qPCR and western blotting respectively. High glucose time-dependently down-regulated Klotho mRNA and protein expression in cultured human MCs. pcDNA3.1-Klotho transfection-mediated Klotho overexpression down-regulated Egr-1, FN and Col I expression and the p-Smad3/Smad3 ratio in human MCs. Conversely, siRNA-mediated Klotho silencing up-regulated Egr-1, FN, and Col I expression and the p-Smad3/Smad3 ratio. Moreover, the effects of si-Klotho on Egr-1 expression were abolished by the TGF-ß1 inhibitor SB-431542. Klotho overexpression can prevent mesangial ECM production in high-glucose-treated human MCs, an effect that has been partially attributed to Egr-1 down-regulation facilitated by TGF-ß1/Smad3 signaling inhibition.

Toll-like receptor 3 signaling contributes to the expression of a neutrophil chemoattractant, CXCL1 in human mesangial cells.

BACKGROUND: Mesangial proinflammatory chemokine/cytokine expressions via innate immunity play a pivotal role in the pathogenesis of glomerulonephritis. CXCL1/GROα is a strong neutrophil chemoattractant cytokine and reportedly plays an important role in regional inflammatory reactions. However, detailed signaling of mesangial CXCL1 expression induced by viral or "pseudoviral" immunity remains to be determined. METHODS: We treated normal human mesangial cells (MCs) in culture with polyinosinic-polycytidylic acid (poly IC), an authentic double-stranded RNA, and analyzed the expression of CXCL1 by reverse transcription-polymerase chain reaction (RT-PCR), real-time quantitative RT-PCR and enzyme-linked immunosorbent assay. To elucidate the poly IC-induced signaling pathway for CXCL1 expression, we subjected the cells to RNA interference against Toll-like receptor (TLR) 3, retinoic acid-inducible gene-I (RIG-I), melanoma differentiation-associated gene 5 (MDA5), interferon (IFN)-ß, nuclear factor (NF)-κB p65 and IFN regulatory factor (IRF) 3. We also conducted an immunofluorescence study to examine mesangial CXCL1 expression in biopsy specimens from patients with lupus nephritis (LN) and IgA nephropathy (IgAN). RESULTS: We found that activation of TLR3 signaling could induce the expression of CXCL1 in MCs. NF-κB, IRF3 and IFN-ß, but neither RIG-I nor MDA5, were found to be involved in mesangial CXCL1 expression in this setting. Induction of CXCL1 by poly IC was inhibited by pretreatment of cells with dexamethasone. Intense glomerular CXCL1 expression was observed in biopsy specimens from patients with LN, whereas only a trace staining occurred in specimens from patients with IgAN. CONCLUSION: TLR3 signaling also contributes to the CXCL1 expression in MCs. These observations further support the implication of viral and "pseudoviral" immunity in the pathogenesis of inflammatory renal diseases, especially in LN.

Mizoribine selectively attenuates monocyte chemoattractant protein-1 production in cultured human glomerular mesangial cell: a possible benefit of its use in the treatment of lupus nephritis.

AIM: Mizoribine (MZR) is a selective inhibitor of the inosine monophosphate dehydrogenase - a key enzyme in the de novo pathway of guanine nucleotides - that was developed in Japan. Besides its immunosuppressive effects, MZR has recently been reported to suppress the progression of histologic chronicity via suppression of macrophage infiltration of the interstitium in selected patients with lupus nephritis. METHODS: We examine the direct effect of MZR in human mesangial cells on the expression of functional molecules including monocyte chemoattractants in cultured human mesangial cells (MCs) treated with polyinosinic-polycytidylic acid (poly IC), a synthetic analogue of viral dsRNA, that makes 'pseudoviral' infection, and analyzed the expression of target molecules by reverse transcriptase-polymerase chain reaction and Western blotting. Thereafter, the effect of MZR on the expressions was examined. RESULTS: Pretreatment of cells with MZR partially, but significantly, attenuates the expression of monocyte chemoattractant protein (MCP)-1 mRNA and protein, whereas the poly IC-induced expressions for the other functional molecules, such as CCL5, fractalkine and IL-8 were not influenced by MZR treatment. On the other hand, pretreatment of cells with tacrolimus did not suppress the expression of MCP-1 mRNA. CONCLUSION: Mizoribine itself selectively attenuated the expression of MCP-1 both mRNA and protein levels in MCs treated with poly IC; that is, a possible model of 'pseudoviral' infection, which may be involved in the pathogenesis of lupus nephritis.

The roles of autophagy in the treatment of diabetic nephropathy with rapamycin.

BACKGROUND: Rapamycin is known to induce autophagy, promote cell survival and inhibit the progression of diabetic nephropathy (DN). OBJECTIVES: The aim of this study was to examine the role of autophagy in the treatment of DN with rapamycin to provide the basis for the DN treatment with rapamycin. MATERIAL AND METHODS: Human mesangial cells (HMC) were cultured in a constant temperature incubator with 5% CO2, at 37°C and saturated humidity. Cells were divided into 5 groups and the 5-ethynyl-2-deoxyuridine (EdU) cell proliferation assay was used to determine cell proliferation. Flow cytometry was used to determine cell apoptosis, while GFP-RFP-LC3 showed autophagy flow. Western blot was employed to detect the expression of autophagy-related proteins LC3-II/LC3-I and P62. Enzyme-linked immunosorbent assay (ELISA) was used to determine the contents of type IV collagen fiber (Col4), hyaluronic acid (HA) and laminin (LA) in the extracellular matrix (ECM). RESULTS: Cell proliferation was the lowest in the hyperglycemic group. Additionally, the hyperglycemic group displayed the lowest number of autolysosomes compared to other groups. In contrast, the rapamycin group exhibited the highest number of autolysosomes. The LC3-II/LC3-I ratio was also the lowest in the hyperglycemic group, measuring 0.53 (0.50-0.58), while the expression level of P62 was significantly higher in that group at 0.98 (0.95-1.01) compared to other groups. Upon the introduction of rapamycin, the LC3-II/LC3-I ratio was significantly increased at 2.21 (1.95-2.21), and P62 was significantly decreased 0.38 (0.38-0.39) compared to the hyperglycemic group. Both changes were statistically significant, with p-values of 0.034 and 0.010, respectively. Enzyme-linked immunosorbent assay was employed to detect Col4, HA and LA content. The study findings demonstrated significantly higher levels of glucose in the hyperglycemic group in comparison to other groups. In contrast, the rapamycin group exhibited significantly lower levels of glucose than the hyperglycemic group, yet the difference was not statistically significant. CONCLUSIONS: Hyperglycemic can inhibit the autophagic activity of HMC, promote cell apoptosis, enhance ECM accumulation, and facilitate the DN progression. In contrast, rapamycin can elicit autophagy, decrease mesangial matrix proliferation, and therefore impede DN progression.

Albiflorin ameliorates mesangial proliferative glomerulonephritis by PI3K/AKT/NF-κB pathway.

Background: The most common type of glomerulonephritis in China is mesangial proliferative glomerulonephritis (MPGN) featured with mesangial cell overproliferation and inflammation, as well as fibrosis. Albiflorin (AF) is an effective composition extracted from Paeonia Alba Radix and has been administrated for various diseases. Nevertheless, there is no research reporting the effect of AF on MPGN.Purpose: Our work aims to probe into the role and possible mechanism of AF on MPGN.Research Design: We investigated the effects of AF on mesangial cell overproliferation, inflammation, and fibrosis in vitro and in vivo and identified the related signaling pathways.Study Sample: human mesangial cells (HMCs) and male Sprague Dawley (SD) rats.Data Analysis: SPSS 18.0 was used to analyze the data.Results: AF attenuated the proliferation and inflammation both in vitro and in vivo. In detail, AF decreased the ki67 expression in lipopolysaccharides (LPS)-treated HMCs and MPGN rats, and the mRNA expression or contents of inflammatory cytokines were reduced after AF treatment. The fibrosis of LPS-treated HMCs and MPGN rats was also reduced by AF. Moreover, AF effectively restrained 24 h urinary protein, improved kidney function, and mitigated dyslipidemia and pathological injury of MPGN rats. Additionally, we found that the protective effects of AF were accompanied by the blocking of PI3K/AKT/NF-κB pathway, and the inhibitory effects of AF on MPGN were reversed by insulin-like growth factor (IGF-1), the PI3K agonist.Conclusions: AF alleviates MPGN via restraining mesangial cell overproliferation, inflammation, and fibrosis via PI3K/AKT/NF-κB signaling.

Downregulation of PPARα mediates FABP1 expression, contributing to IgA nephropathy by stimulating ferroptosis in human mesangial cells.

Immunoglobulin A nephropathy (IgAN) is the commonest primary glomerulonephritis, and a major cause of end-stage renal disease; however, its pathogenesis requires elucidation. Here, a hub gene, FABP1, and signaling pathway, PPARα, were selected as key in IgAN pathogenesis by combined weighted gene correlation network analysis of clinical traits and identification of differentially expressed genes from three datasets. FABP1 and PPARα levels were lower in IgAN than control kidney, and linearly positively correlated with one another, while FABP1 levels were negatively correlated with urinary albumin-to-creatinine ratio, and GPX4 levels were significantly decreased in IgAN. In human mesangial cells (HMCs), PPARα and FABP1 levels were significantly decreased after Gd-IgA1 stimulation and mitochondria appeared structurally damaged, while reactive oxygen species (ROS) and malondialdehyde (MDA) were significantly increased, and glutathione and GPX4 decreased, relative to controls. GPX4 levels were decreased, and those of ACSL4 increased on siPPARα and siFABP1 siRNA treatment. In PPARα lentivirus-transfected HMCs stimulated by Gd-IgA1, ROS, MDA, and ACSL4 were decreased; glutathione and GPX4, and immunofluorescence colocalization of PPARα and GPX4, increased; and damaged mitochondria reduced. Hence, PPARα pathway downregulation can reduce FABP1 expression, affecting GPX4 and ACSL4 levels, causing HMC ferroptosis, and contributing to IgAN pathogenesis.

Mesangioproliferative Kidney Diseases and Platelet-Derived Growth Factor-Mediated AXL Phosphorylation.

RATIONALE & OBJECTIVE: Immunoglobulin A nephropathy (IgAN) is a common glomerular disease, with mesangial cell proliferation as a major feature. There is no disease-specific treatment. Platelet-derived growth factor (PDGF) contributes to the pathogenesis of IgAN. To better understand its pathogenic mechanisms, we assessed PDGF-mediated AXL phosphorylation in human mesangial cells and kidney tissue biopsy specimens. STUDY DESIGN: Immunostaining using human kidney biopsy specimens and in vitro studies using primary human mesangial cells. SETTING & PARTICIPANTS: Phosphorylation of AXL was assessed in cultured mesangial cells and 10 kidney-biopsy specimens from 5 patients with IgAN, 3 with minimal change disease, 1 with membranous nephropathy, and 1 with mesangioproliferative glomerulonephritis (GN). PREDICTOR: Glomerular staining for phospho-AXL in kidney biopsy specimens of patients with mesangioproliferative diseases. OUTCOMES: Phosphorylated AXL detected in biopsy tissues of patients with IgAN and mesangioproliferative GN and in cultured mesangial cells stimulated with PDGF. ANALYTIC APPROACH: t test, Mann-Whitney test, and analysis of variance were used to assess the significance of mesangial cell proliferative changes. RESULTS: Immunohistochemical staining revealed enhanced phosphorylation of glomerular AXL in IgAN and mesangioproliferative GN, but not in minimal change disease and membranous nephropathy. Confocal-microscopy immunofluorescence analysis indicated that mesangial cells rather than endothelial cells or podocytes expressed phospho-AXL. Kinomic profiling of primary mesangial cells treated with PDGF revealed activation of several protein-tyrosine kinases, including AXL. Immunoprecipitation experiments indicated association of AXL and PDGF receptor proteins. An AXL-specific inhibitor (bemcentinib) partially blocked PDGF-induced cellular proliferation and reduced phosphorylation of AXL and PDGF receptor and the downstream signals (AKT1 and ERK1/2). LIMITATIONS: Small number of kidney biopsy specimens to correlate the activation of AXL with disease severity. CONCLUSIONS: PDGF-mediated signaling in mesangial cells involves transactivation of AXL. Finding appropriate inhibitors to block PDGF-mediated transactivation of AXL may provide new therapeutic options for mesangioproliferative kidney diseases such as IgAN.

Apolipoprotein C3 aggravates diabetic nephropathy in type 1 diabetes by activating the renal TLR2/NF-κB pathway.

OBJECTIVE: Apolipoprotein C3 (ApoC3) is a regulator of triglyceride metabolism and inflammation, and its plasma levels are positively correlated with the progression of diabetic nephropathy (DN) in patients. However, the role and underlying mechanism of ApoC3 in DN remain unclear. METHODS: Diabetes was induced in ApoC3 transgenic (Tg) and knockout (KO) mice by injection of streptozotocin. We studied the effect of ApoC3 on type 1 DN after 4 months of diabetes. Plasma glucose and lipid levels, renal function parameters and inflammation- and fibrogenesis-related gene and protein expression levels were studied. In vitro, human mesangial cells (HMCs) were incubated with high levels of glucose or/and triglyceride-rich lipoproteins (TRLs) with a high or low ApoC3 content isolated from Tg or wild-type (WT) mice, respectively, to explore the mechanisms of ApoC3 on development of DN. RESULTS: We found that compared to WT mice, Tg mice exhibited hypertriglyceridemia (HTG), aggravated early renal function injury and inflammation, enlarged glomerular and mesangial surface areas, renal lipid deposition and elevated fibrogenesis-related gene expression levels after 4 months of diabetes. ApoC3 overexpression activated the renal Toll-like receptor 2 (TLR2) and nuclear factor-κB (NF-κB) signaling pathways and increased the renal gene and protein expression levels of the downstream inflammatory factors TNF-α, VCAM-1 and MCP-1. Unfortunately, we did not find that ApoC3 deficiency had an obvious protective effect against DN. In vitro, we found that TRLs with a high ApoC3 content increased the gene and protein expression levels of inflammation- and fibrogenesis-related factors in HMCs compared to those following administration of the same concentration of TRLs with a low ApoC3 content. These effects of ApoC3 were inhibited by blockade of TLR2 or NF-κB. CONCLUSIONS: These findings suggest that ApoC3 aggravates early-stage DN by activating the renal TLR2/NF-κB pathway which is partially independent of HTG.

Upregulation of microRNA­140­3p mediates dachshund family transcription factor 1 expression in immunoglobulin A nephropathy through cell cycle­dependent mechanisms.

Immunoglobulin A nephropathy (IgAN) is a kidney disease and one of the commonest forms of glomerulonephritis worldwide. The present study investigated the role of dachshund family transcription factor 1 (DACH1) in IgAN and identified one of its binding microRNAs (miRNAs). The expression of DACH1 in human mesangial cells (HMCs) incubated with polymeric IgA (pIgA) isolated and purified from the serum of patients with IgAN or healthy individuals was evaluated by reverse transcription­quantitative (RT­q) PCR and western blotting. Cell proliferation and cell cycle assays were performed in DACH1­overexpressing HMCs to identify the role of DACH1 in IgAN and enzyme­linked immunosorbent assay was carried out to verify the release of inflammatory factors from HMCs. The target miRNAs of DACH1 were predicted using bioinformatics software and miR­140­3p was identified as a target of DACH1 by luciferase report assay, RT­qPCR and western blotting. The results demonstrated that DACH1 was downregulated in HMCs cultured with pIgA­IgAN at both mRNA and protein levels. Overexpression of DACH1 suppressed HMC growth and inhibited inflammatory cytokine release from HMCs cultured with pIgA­IgAN. The expression of DACH1 was negatively regulated by miR­140­3p in IgAN and miR­140­3p inhibition suppressed HMC growth and inhibited inflammatory cytokine release from HMCs cultured with pIgA­IgAN. The findings of the present study demonstrated that DACH1 decreased HMC growth and the release of inflammatory cytokines from HMCs may be targeted by miR­140­3p. The results suggested that DACH1 could be associated with the progression of IgAN and provide a potential target for further studies related to the mechanism of IgAN.

Ochratoxin A induces glomerular injury through activating the ERK/NF-κB signaling pathway.

Ochratoxin A (OTA) was reported to induce proximal tubules nephrotoxicity in humans and animals. However, the toxicity of OTA on glomeruli has rarely been studied. We investigated OTA-induced glomerular injury and the underlying mechanisms. Mice were intraperitoneally treated with OTA (0, 0.5, 1.5 and 2.5 mg/kg b.w.) on alternate day for 3 weeks. OTA exposure decreased the weight gain ratio, the kidney index and increased the levels of serum creatinine and blood urea nitrogen. It induced also fragmentation and atrophy in glomeruli, and increased the expression of TNF-α, IL-6, COX-2, TGF-ß, α-SMA and vimentin in a dose-dependent manner. Human mesangial cells (HMC) were treated with OTA (0-8 µM) for 48 h. Treatment of HMC cells with OTA increased cell inhibition rate, up-regulated the expression of IL-6, TGF-ß, α-SMA and vimentin in a dose-dependent manner. Additionally, it enhanced the phosphorylation of ERK1/2 and p65, degradation of IκB-α and translocation of p65 into the nucleus. OTA-induced toxicity was attenuated by NF-κB and ERK1/2 inhibitors. In conclusion, these results suggest that OTA exposure induces glomerular injury via activation of the ERK/NF-κB signaling pathway, and provide novel insights into the research of OTA induced nephrotoxicity.

Anaphylatoxins enhance Th9 cell recruitment via the CCL20-CCR6 axis in IgA nephropathy.

BACKGROUND: CD4+ T cells are involved in the pathogenesis of immunoglobulin A nephropathy (IgAN); T helper (Th) 1, Th17 and Th22 cells promote the occurrence and amplification of inflammatory reactions, while regulatory T (Treg) cells produce the opposite effects. However, whether Th9 cells, a subset of CD4+ T cells, participate in IgAN development is still unknown. METHODS: Human peripheral blood mononuclear cells (PBMCs) were isolated from IgAN patients for Th9 cells detection by flow cytometry. Wild-type (WT) mouse was used to establish an IgAN mouse model while C3aR and C5aR inhibitor treated IgAN mouse. Kidney disease and function was assessed by histology and albumin-to-creatinine ratio. C3aR and C5aR expression was examined by immunohistochemical (IHC) assay. Th9 cell proportions in the blood of IgAN mouse was detected. C3a, C5a and interleukin (IL)-9 levels were tested by ELISA. Moreover, co-culture system between human mesangial cells (HMCs) and CD4+ T cells were constructed with or without C3a, C5a and anti-CCL20 mAb stimulation for transwell assay to examine Th9 cell chemotaxis. RESULTS: We observed the numbers of Th9 cell and the levels of IL-9 were increased in IgAN patients and IgAN mice. Furthermore, C3a and C5a level in serum and kidney, C3aR and C5aR expression was increased in IgAN mice compared to WT mice. Most interestingly, C3aR and C5aR inhibitor could reduce kidney damage, Th9 cell numbers and IL-9 levels. We also observed that C3a and C5a enhanced CCL20 production in HMCs. Notably, C3a and C5a also increased the recruitment of Th9 cells and IL-9 levels by HMCs through enhancing the CCL20-CCR6 pathway. CONCLUSIONS: Our results support that C3a and C5a increase the production of CCL20 by HMCs and consequently augment Th9 cell recruitment and IL-9 levels, resulting in IgAN exacerbation.

Rhein-8-O-ß-D-glucopyranoside inhibited high glucose-induced apoptosis of human mesangial cells by regulating the lincRNA ANRIL/let-7a/TGF-ß1/Smad signaling pathway.

Diabetic nephropathy is one of most frequent complications of diabetes, and is the major cause of end-stage disease in diabetic patients. The present study investigated the roles and mechanisms of Rhein-8-O-ß-D-glucopyranoside (Rg) protecting human mesangial cells (HMCs) from high glucose (HG)-induced apoptosis. Using a Cell Counting Kit-8 assay the proliferation of HMCs was analyzed, and flow cytometry was applied to detect apoptosis. The apoptosis-associated protein Bcl-2, caspase-3 and members of the transforming growth factor-ß1 (TGF-ß1)/Smad signaling pathway were analyzed using a western blotting assay. HG significantly induced HMC apoptosis, and Rg markedly attenuated the HG-induced apoptosis. HG decreased the Bcl-2 expression and increased the caspase-3 expression, and Rg treatment recovered the expressions of Bcl-2 and caspase-3 affected by HG. The underlying mechanisms were further analyzed, and it was demonstrated that HG significantly upregulated the long intervening non-coding RNA (lincRNA) ANRIL expression level, downregulated let-7a expression and activated the TGF-ß1/Smad signaling pathway; Rg treatment recovered the expressions of lincRNA ANRIL and let-7a, and inhibited the TGF-ß1/Smad signaling pathway in the condition of HG. In conclusion, the present results suggested that Rg attenuated HG-induced apoptosis of HMCs by regulating the lincRNA ANRIL/let-7a/TGF-ß1/Smad signaling pathway.

Long non-coding RNA MEG3 impacts diabetic nephropathy progression through sponging miR-145.

Long non-coding RNA MEG3 has been reported to implicate in the progression of several cancers. Nevertheless, few studies have focused on the role of MEG3 in the development of diabetic nephropathy. Here, we demonstrated MEG3 was differently expressed by > 4 fold and was elevated significantly using lncRNA microarray in DN patient serum. Besides, MEG3 knockdown alleviated proliferation, fibrosis and induced apoptosis of mesangial cells under high glucose condition. Furthermore, bioinformatics predictions showed that MEG3 is a direct target of miR-145. In the vivo experiment, we found MEG3 silencing decreased the laboratory indicators and fibrosis-related protein secretion in db/db mice. Altogether, our study suggests MEG3 may play as an important role in progression of diabetic nephropathy, contributing to a novel understanding of pathogenesis and underlying therapeutic strategies for diabetic nephropathy.

Corrigendum: Respiratory Syncytial Virus Exacerbates Kidney Damages in IgA Nephropathy Mice via the C5a-C5aR1 Axis Orchestrating Th17 Cell Responses.

[This corrects the article DOI: 10.3389/fcimb.2019.00151.].

Respiratory Syncytial Virus Exacerbates Kidney Damages in IgA Nephropathy Mice via the C5a-C5aR1 Axis Orchestrating Th17 Cell Responses.

Respiratory viral infections can directly lead to kidney damage such as IgA nephropathy (IgAN), partly due to mucosal immune system dysfunction. Although the activated C5a-C5aR1 axis results in increased Th1 and Th17 frequencies but reduced Treg frequencies in Respiratory syncytial virus (RSV) infection, how this axis affects Th cell disorders in RSV-induced IgAN exacerbation remains unknown. Here, we used a mouse model to dissect the activation of C5a-C5aR1 by RSV and the consequences on the regulation of Th1, Th17, and Treg immune responses in IgA nephropathy. RSV fusion protein was clearly deposited not only in the pulmonary interstitium but also in the glomerulus in RSV-IgAN mice, and RSV infection led to more severe pathological changes in the kidneys in IgAN mice. Blocking the C5a-C5aR1 axis resulted in a decrease in the albumin-to-creatinine ratio, and the attenuation of kidney damage in IgAN and RSV-IgAN mice might be partly attributed to the inhibition of Th cell and cytokine dysfunction. Th1, Th17 and Treg immune responses and their corelative cytokines were disrupted by RSV infection and rescued by C5aR1 inhibition. Moreover, we constructed a coculture system of human mesangial cells and CD4+ T cells and found that RSV infection might lead to CD4+ T cell production via human mesangial cells-enhanced CD4+ T cell proliferation, consequently increasing IL-17 levels. These pathological behaviors were augmented by C5a stimulation and decreased by C5aR1 inhibition. Thus, C5aR1 inhibition alters both kidney damage and Th1, Th17, and Treg cell dysfunction in RSV-induced IgAN exacerbation and locally regulates HMC antigen presentation function in the kidney. Taken together, our data offer profound evidence that blocking the C5a-C5aR1 axis might be a potential therapy for RSV-induced IgAN.

Rutin suppresses high glucose-induced ACTA2 and p38 protein expression in diabetic nephropathy.

The present study investigated the effect of rutin on high glucose-induced actin, α2, smooth muscle, aorta (ACTA2) and p38 protein expression in diabetic nephropathy (DN). Human mesangial cells were divided into a control group, high glucose-induced mesangial cell group, high glucose + captopril group, and high glucose + rutin group (low, middle and high doses of rutin). Cell viability, adenosine 5'-triphosphate (ATP) content, cell cycle, and ACTA2 and p38 protein expression were examined using MTT assay, ATP assay kit, flow cytometry and immunofluorescence staining in cultured human mesangial cells, respectively. Cell viability, ATP content, and ACTA2 and p38 expression increased significantly in high glucose-induced mesangial cells (P<0.05). However, at concentrations of 0.2, 0.4 and 0.8 µmol/l rutin was able to inhibit high glucose-induced human mesangial cell viability, ATP content, and ACTA2 and p38 expression and improve the cell cycle progression of mesangial cells. In conclusion, ACTA2 and p38 proteins may have important roles in DN. Rutin may inhibit the expression of ACTA2 and p38 and may be utilized in the prevention and treatment of DN.