Crystalline silica-induced pulmonary inflammation and autoimmunity in mature adult NZBW/f1 mice: age-related sensitivity and impact of omega-3 fatty acid intervention.

OBJECTIVE: Occupational exposure to respirable crystalline silica (cSiO2) has been linked to lupus development. Previous studies in young lupus-prone mice revealed that intranasal cSiO2 exposure triggered autoimmunity, preventable with docosahexaenoic acid (DHA). This study explores cSiO2 and DHA effects in mature lupus-prone adult mice, more representative of cSiO2-exposed worker age. METHODS: Female NZBWF1 mice (14-week old) were fed control (CON) or DHA-supplemented diets. After two weeks, mice were intranasally instilled saline (VEH) or 1 mg cSiO2 weekly for four weeks. Cohorts were then analyzed 1- and 5-weeks postinstillation for lung inflammation, cell counts, chemokines, histopathology, B- and T-cell infiltration, autoantibodies, and gene signatures, with results correlated to autoimmune glomerulonephritis onset. RESULTS: VEH/CON mice showed no pathology. cSiO2/CON mice displayed significant ectopic lymphoid tissue formation in lungs at 1 week, increasing by 5 weeks. cSiO2/CON lungs exhibited elevated cellularity, chemokines, CD3+ T-cells, CD45R + B-cells, IgG + plasma cells, gene expression, IgG autoantibodies, and glomerular hypertrophy. DHA supplementation mitigated all these effects. DISCUSSION: The mature adult NZBWF1 mouse used here represents a life-stage coincident with immunological tolerance breach and one that more appropriately represents the age (20-30 yr) of cSiO2-exposed workers. cSiO2-induced robust pulmonary inflammation, autoantibody responses, and glomerulonephritis in mature adult mice, surpassing effects observed previously in young adults. DHA at a human-equivalent dosage effectively countered cSiO2-induced inflammation/autoimmunity in mature mice, mirroring protective effects in young mice. CONCLUSION: These results highlight life-stage significance in this preclinical lupus model and underscore omega-3 fatty acids' therapeutic potential against toxicant-triggered autoimmune responses.

Rab4A-directed endosome traffic shapes pro-inflammatory mitochondrial metabolism in T cells via mitophagy, CD98 expression, and kynurenine-sensitive mTOR activation.

Activation of the mechanistic target of rapamycin (mTOR) is a key metabolic checkpoint of pro-inflammatory T-cell development that contributes to the pathogenesis of autoimmune diseases, such as systemic lupus erythematosus (SLE), however, the underlying mechanisms remain poorly understood. Here, we identify a functional role for Rab4A-directed endosome traffic in CD98 receptor recycling, mTOR activation, and accumulation of mitochondria that connect metabolic pathways with immune cell lineage development and lupus pathogenesis. Based on integrated analyses of gene expression, receptor traffic, and stable isotope tracing of metabolic pathways, constitutively active Rab4AQ72L exerts cell type-specific control over metabolic networks, dominantly impacting CD98-dependent kynurenine production, mTOR activation, mitochondrial electron transport and flux through the tricarboxylic acid cycle and thus expands CD4+ and CD3+CD4-CD8- double-negative T cells over CD8+ T cells, enhancing B cell activation, plasma cell development, antinuclear and antiphospholipid autoantibody production, and glomerulonephritis in lupus-prone mice. Rab4A deletion in T cells and pharmacological mTOR blockade restrain CD98 expression, mitochondrial metabolism and lineage skewing and attenuate glomerulonephritis. This study identifies Rab4A-directed endosome traffic as a multilevel regulator of T cell lineage specification during lupus pathogenesis.

Application of nanotechnology in the treatment of glomerulonephritis: current status and future perspectives.

Glomerulonephritis (GN) is the most common cause of end-stage renal failure worldwide; in most cases, it cannot be cured and can only delay the progression of the disease. At present, the main treatment methods include symptomatic therapy, immunosuppressive therapy, and renal replacement therapy. However, effective treatment of GN is hindered by issues such as steroid resistance, serious side effects, low bioavailability, and lack of precise targeting. With the widespread application of nanoparticles in medical treatment, novel methods have emerged for the treatment of kidney diseases. Targeted transportation of drugs, nucleic acids, and other substances to kidney tissues and even kidney cells through nanodrug delivery systems can reduce the systemic effects and adverse reactions of drugs and improve treatment effectiveness. The high specificity of nanoparticles enables them to bind to ion channels and block or enhance channel gating, thus improving inflammation. This review briefly introduces the characteristics of GN, describes the treatment status of GN, systematically summarizes the research achievements of nanoparticles in the treatment of primary GN, diabetic nephropathy and lupus nephritis, analyzes recent therapeutic developments, and outlines promising research directions, such as gas signaling molecule nanodrug delivery systems and ultrasmall nanoparticles. The current application of nanoparticles in GN is summarized to provide a reference for better treatment of GN in the future.

The therapeutic effect of Shenhua tablet against mesangial cell proliferation and renal inflammation in mesangial proliferative glomerulonephritis.

Shenhua tablet (SH), a formulation of traditional Chinese medicine, exerts renoprotective effect on chronic kidney diseases, and it has been found to restrain inflammation, but the mechanism is still unclear. Here, we explored the potential renoprotection of SH in mesangial proliferative glomerulonephritis (MsPGN) rat model induced by anti-Thy1 antibody. Administration of SH reduced urinary albumin/creatinine ratio (UACR) and significantly attenuated mesangial cell proliferation and renal inflammation. Notably, SH protected rats against renal inflammation, which was associated with decreasing macrophage infiltration and promoting macrophage anti-inflammatory activity. Network analysis combined with arrays identified the Janus kinase signal transducer and activator of transcription (JAK-STAT) signaling pathway as the main pathways of SH could target inflammation. Furthermore, it was confirmed that mesangial cell proliferation, which response to inflammation, were alleviated by ASS1 expression enhanced after SH administration both in vivo and in vitro. Collectively, SH has the beneficial on relieving the progression of MsPGN to alleviate inflammation and mesangial proliferation by inhibiting STAT3 phosphorylation and maintains the expression level of ASS1, might be an effective strategy for treating MsPGN.

Effects of electroacupuncture at KI3 and ST36 on the hypothalamic paraventricular nucleus in a rat model of chronic glomerulonephritis.

OBJECTIVE: The hypothalamic paraventricular nucleus (PVN) acts as a critical integrating center of endocrine/autonomic responses and regulates visceral functional activities. However, its involvement in electroacupuncture (EA) treatment of chronic glomerulonephritis (CGN) remains unclear. METHODS: Over four experiments, we randomized 111 rats into: control, untreated model (CGN) or EA-treated model (CGN + EA) groups, a model group receiving EA after PVN damage (CGN + EA + Lesion) or untreated model groups injected with adeno-associated viral vectors encoding human M4 muscarinic receptor (CGN + hM4D) or enhanced green fluorescent protein (CGN + EGFP). CGN was modeled by intraperitoneal injection of bovine serum albumin for 2 weeks. Rats in the CGN + EA and CGN + EA + Lesion groups received EA at bilateral ST36 and KI3 for 14 days. Urine/serum samples were collected to evaluate inflammatory factors and changes in renal function. RESULTS: EA inhibited the release of interleukin (IL)-6, tumor necrosis factor (TNF)-α and IL-1ß, and decreased urine protein (PRO), creatinine (Cre) and blood urea nitrogen (BUN) levels. PVN damage influenced the effect of EA on the levels of these parameters. EA appeared to inhibit the firing frequency and spectral energy of PVN neurons. In the viral vector experiment, levels of PRO, Cre, IL-6, IL-1ß and TNF-α in the CGN group were increased in CGN versus control groups (p < 0.0001), decreased in CGN + hM4D versus CGN groups (p < 0.05) and did not differ between CGN + EGFP and control groups (p > 0.05). CONCLUSION: Our findings indicate that EA at ST36 and KI3 improves CGN in this rat model by weakening the activity of PVN neurons, alleviating impairment of renal function impairment and restricting the release of inflammatory factors.

Intrarenal renin-angiotensin system activation and macrophage infiltrations in pediatric chronic glomerulonephritis.

BACKGROUND: The current study tested the hypothesis that urinary angiotensinogen (UAGT) and urinary monocyte chemoattractant protein-1 (UMCP-1) levels provide a specific index of intrarenal renin-angiotensin system (RAS) status and the degree of infiltration of macrophages associated with RAS blockade and immunosuppressant treatment in pediatric patients with chronic glomerulonephritis. METHODS: We measured baseline UAGT and UMCP-1 levels to examine the correlation between glomerular injury in 48 pediatric chronic glomerulonephritis patients before treatment. Furthermore, we performed immunohistochemical analysis of angiotensinogen (AGT) and CD68 in 27 pediatric chronic glomerulonephritis patients treated with RAS blockades and immunosuppressants for 2 years. Finally, we examined the effects of angiotensin II (Ang II) on monocyte chemoattractant protein-1 (MCP-1) expression in cultured human mesangial cells (MCs). RESULTS: Baseline UAGT and UMCP-1 levels positively correlated with urinary protein levels, scores for mesangial hypercellularity, rate of crescentic formation, and expression levels of AGT and CD68 in renal tissues (p < 0.05). UAGT and UMCP-1 levels were significantly decreased after RAS blockade and immunosuppressant treatment (p < 0.01), which was accompanied by AGT and CD68 (p < 0.01), as well as the magnitude of glomerular injury. Cultured human MCs showed increased MCP-1 messenger ribonucleic acid and protein levels after Ang II treatment (p < 0.01). CONCLUSIONS: The data indicates that UAGT and UMCP-1 are useful biomarkers of the degree of glomerular injury during RAS blockade and immunosuppressant treatment in pediatric patients with chronic glomerulonephritis.

Down-regulation of human long non-coding RNA LINC01187 is associated with nephropathies.

Chronic kidney diseases affect a substantial percentage of the adult population worldwide. This observation emphasizes the need for novel insights into the molecular mechanisms that control the onset and progression of renal diseases. Recent advances in genomics have uncovered a previously unanticipated link between the non-coding genome and human kidney diseases. Here we screened and analysed long non-coding RNAs (lncRNAs) previously identified in mouse kidneys by genome-wide transcriptomic analysis, for conservation in humans and differential expression in renal tissue from healthy and diseased individuals. Our data suggest that LINC01187 is strongly down-regulated in human kidney tissues of patients with diabetic nephropathy and rapidly progressive glomerulonephritis, as well as in murine models of kidney diseases, including unilateral ureteral obstruction, nephrotoxic serum-induced glomerulonephritis and ischemia/reperfusion. Interestingly, LINC01187 overexpression in human kidney cells in vitro inhibits cell death indicating an anti-apoptotic function. Collectively, these data suggest a negative association of LINC01187 expression with renal diseases implying a potential protective role.

CD4+ T cells produce GM-CSF and drive immune-mediated glomerular disease by licensing monocyte-derived cells to produce MMP12.

GM-CSF in glomerulonephritisDespite glomerulonephritis being an immune-mediated disease, the contributions of individual immune cell types are not clear. To address this gap in knowledge, Paust et al. characterized pathological immune cells in samples from patients with glomerulonephritis and in samples from mice with the disease. The authors found that CD4+ T cells producing granulocyte-macrophage colony-stimulating factor (GM-CSF) licensed monocytes to promote disease by producing matrix metalloproteinase 12 and disrupting the glomerular basement membrane. Targeting GM-CSF to inhibit this axis reduced disease severity in mice, implicating this cytokine as a potential therapeutic target for patients with glomerulonephritis. -CM.

[Effective substances and mechanism of Yishen Guluo Mixture in treatment of chronic glomerulonephritis based on metabolomics and serum pharmacochemistry].

This study aimed to investigate the effective substances and mechanism of Yishen Guluo Mixture in the treatment of chronic glomerulonephritis(CGN) based on metabolomics and serum pharmacochemistry. The rat model of CGN was induced by cationic bovine serum albumin(C-BSA). After intragastric administration of Yishen Guluo Mixture, the biochemical indexes related to renal function(24-hour urinary protein, serum urea nitrogen, and creatinine) were determined, and the efficacy evaluations such as histopathological observation were carried out. The serum biomarkers of Yishen Guluo Mixture in the treatment of CGN were screened out by ultra-performance liquid chromatography-quadrupole time-of-flight/mass spectrometry(UPLC-Q-TOF-MS) combined with multivariate statistical analysis, and the metabolic pathways were analyzed. According to the mass spectrum ion fragment information and metabolic pathway, the components absorbed into the blood(prototypes and metabolites) from Yishen Guluo Mixture were identified and analyzed by using PeakView 1.2 and MetabolitePilot 2.0.4. By integrating metabolomics and serum pharmacochemistry data, a mathematical model of correlation analysis between serum biomarkers and components absorbed into blood was constructed to screen out the potential effective substances of Yishen Guluo Mixture in the treatment of CGN. Yishen Guluo mixture significantly decreased the levels of 24-hour urinary protein, serum urea nitrogen, and creatinine in rats with CGN, and improved the pathological damage of the kidney tissue. Twenty serum biomarkers of Yishen Guluo Mixture in the treatment of CGN, such as arachidonic acid and lysophosphatidylcholine, were screened out, involving arachidonic acid metabolism, glycerol phosphatide metabolism, and other pathways. Based on the serum pharmacochemistry, 8 prototype components and 20 metabolites in the serum-containing Yishen Guluo Mixture were identified. According to the metabolomics and correlation analysis of serum pharmacochemistry, 12 compounds such as genistein absorbed into the blood from Yishen Guluo Mixture were selected as the potential effective substances for the treatment of CGN. Based on metabolomics and serum pharmacochemistry, the effective substances and mechanism of Yishen Guluo Mixture in the treatment of CGN are analyzed and explained in this study, which provides a new idea for the development of innovative traditional Chinese medicine for the treatment of CGN.

[Hepatitis B virus X protein promotes podocyte pyroptosis in hepatitis B virus-associated glomerulonephritis by down-regulating microRNA -223 targeting NLRP3 inflammasome].

Objective: To investigate the potential function and related mechanism of microRNA-223 (miRNA-223) in the podocyte pyroptosis of hepatitis B virus (HBV)-associated glomerulonephritis induced by HBV X protein (HBx). Methods: HBx-overexpressing lentivirus was transfected into human renal podocytes to mimic the pathogenesis of HBV-GN. Real-time fluorescence quantitative PCR and Western blotting experiments were used to detect the mRNA and protein expression of pyroptosis-related proteins [nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3), apoptosis-associated speck-like protein containing a CARD (ASC) and caspase-1], and inflammatory factors (interleukin-1ß and interleukin-18), respectively.TUNEL staining and flow cytometry were used to detect the number of pyroptosis cells. Immunofluorescence staining was used to detect the expression of podocytes biomarkers desmin and nephrin; Hoechst 33342 staining was used to observe the morphological and quantitative changes of podocyte nuclei. Enzyme-linked immunosorbent assay was used to measure caspase-1 activity. The dual luciferase reporter gene assay was used to verify the downstream target of miRNA-223. Podocytes were divided into the following nine groups: control group (no special treatment), empty plasmid group (transfected with empty plasmid), HBx overexpression group (transfected with HBx overexpression lentivirus), HBx overexpression+miRNA-223 mimic group (transfected with HBx overexpression lentivirus and miRNA-223 mimic), HBx overexpression+miRNA-223 inhibitor group (transfected with HBx overexpression lentivirus and miRNA-223 inhibitor), HBx overexpression+miRNA-223 mimic+NLRP3 group (transfected with HBx overexpression lentivirus, miRNA-223 mimic and NLRP3 overexpression plasmid), HBx overexpression+miRNA-223 mimic+ NLRP3 siRNA group (transfected with HBx overexpression lentivirus, miRNA-223 mimic and NLRP3 siRNA), HBx overexpression+miRNA-223 inhibitor+NLRP3 group (transfected with HBx overexpression lentivirus, miRNA-223 inhibitor and NLRP3 overexpression plasmid), HBx overexpression+miRNA-223 inhibitor+NLRP3 siRNA group (transfected with HBx overexpression lentivirus, miRNA-223 inhibitor and NLRP3 siRNA). Results: miRNA-223 was down-regulated in HBx overexpression group compared with the control group (P < 0.05). TUNEL and immunofluorescence staining showed that NLRP3 knockdown attenuated podocyte injury and pyroptosis induced by HBx overexpression (P < 0.05). Dual luciferase reporter gene assay demonstrated that NLRP3 was one of the downstream targets of miRNA-223. Rescue experiments revealed that NLRP3 overexpression weakened the protective effect of miRNA-223 in podocyte injury (P < 0.05). The addition of miRNA-223 mimic and NLRP3 siRNA decreased the expression of NLRP3 inflammasome and cytokines, and reduced the number of pyroptosis cells induced by HBx overexpression (all P < 0.05); The addition of miRNA-223 inhibitor and NLRP3 overexpression plasmid significantly increased the expression of NLRP3 inflammasome and cytokines, caspase-1 activity, and the number of pyroptosis cells (all P < 0.05). Conclusion: HBx may promote podocyte pyroptosis of HBV-GN via downregulating miRNA-223 targeting NLRP3 inflammasome, suggesting that miRNA-223 is expected to be a potential target for the treatment of HBV-GN.

Engineered Bone Marrow Stem Cell-Sheets Alleviate Renal Damage in a Rat Chronic Glomerulonephritis Model.

Although mesenchymal stem cell (MSC)-based regenerative therapy is being developed for the treatment of kidney diseases, cell delivery and engraftment still need to be improved. Cell sheet technology has been developed as a new cell delivery method, to recover cells as a sheet form retaining intrinsic cell adhesion proteins, which promotes its transplantation efficiency to the target tissue. We thus hypothesized that MSC sheets would therapeutically reduce kidney disease with high transplantation efficiency. When the chronic glomerulonephritis was induced by two injections of the anti-Thy 1.1 antibody (OX-7) in rats, the therapeutic efficacy of rat bone marrow stem cell (rBMSC) sheet transplantation was evaluated. The rBMSC-sheets were prepared using the temperature-responsive cell-culture surfaces and transplanted as patches onto the surface of two kidneys of each rat at 24 h after the first injection of OX-7. At 4 weeks, retention of the transplanted MSC-sheets was confirmed, and the animals with MSC-sheets showed significant reductions in proteinuria, glomerular staining for extracellular matrix protein, and renal production of TGFß1, PAI-1, collagen I, and fibronectin. The treatment also ameliorated podocyte and renal tubular injury, as evidenced by a reversal in the reductions of WT-1, podocin, and nephrin and by renal overexpression of KIM-1 and NGAL. Furthermore, the treatment enhanced gene expression of regenerative factors, and IL-10, Bcl-2, and HO-1 mRNA levels, but reduced TSP-1 levels, NF-kB, and NAPDH oxidase production in the kidney. These results strongly support our hypothesis that MSC-sheets facilitated MSC transplantation and function, and effectively retarded progressive renal fibrosis via paracrine actions on anti-cellular inflammation, oxidative stress, and apoptosis and promoted regeneration.

Toll-like Receptor 9 Induced Dendritic Cell Activation Promotes Anti-Myeloperoxidase Autoimmunity and Glomerulonephritis.

ANCA-associated vasculitis (AAV) is intricately linked with infections. Toll-like receptors (TLR) provide a potential link between infection and anti-myeloperoxidase (MPO) autoimmunity. TLR9 ligation has been shown to promote anti-MPO autoimmunity and glomerular vasculitis in murine MPO-AAV. This study investigates dendritic cell TLR9 ligation in murine experimental anti-MPO glomerulonephritis. We analyzed autoimmune responses to MPO following transfer of TLR9 stimulated, MPO pulsed dendritic cells and kidney injury following a sub-nephritogenic dose of sheep anti-mouse glomerular basement membrane globulin. TLR9 ligation enhanced dendritic cell activation upregulating CD40 and CD80 expression, promoting systemic anti-MPO autoimmunity and T cell recall responses and exacerbating kidney injury. CD40 upregulation by TLR9 was critical for the induction of nephritogenic autoimmunity. The presence of DEC205, which transports the TLR9 ligand to TLR9 located in the endosome, also promoted kidney injury. This confirms TLR9 mediated dendritic cell activation as a mechanism of anti-MPO autoimmunity in AAV and further defines the link between infection and the generation of MPO specific autoimmune inflammation.

Generation of Conditional KO Mice of CCN2 and Its Function in the Kidney.

CCN2 has been shown to be closely involved in the progression of renal fibrosis, indicating the potential of CCN2 inhibition as a therapeutic target. Although the examination of the renal disease phenotypes of adult CCN2 knockout mice has yielded valuable scientific insights, perinatal death has limited studies of CCN2 in vivo. Conditional knockout technology has become widely used to delete genes in the target cell populations or time points using cell-specific Cre recombinase-expressing mice. Therefore, several lines of CCN2-floxed mice have been developed to assess the functional role of CCN2 in adult mice.CCN2 levels are elevated in renal fibrosis and proliferative glomerulonephritis, making them suitable disease models for assessing the effects of CCN2 deletion on the kidney. Renal fibrosis is characterized by glomerulosclerosis and tubulointerstitial fibrosis and transforming growth factor-ß. CCN2 is increased in fibrosis and modulates a number of downstream signaling pathways involved in the fibrogenic properties of TGF-ß. Unilateral ureteral obstruction is one of the most widely used models of renal tubulointerstitial fibrosis. In addition, anti-glomerular basement membrane antibody glomerulonephritis has become the most widely used model for evaluating the effect of increased renal CCN2 expression. Herein, we describe the construction of CCN2-floxed mice and inducible systemic CCN2 conditional knockout mice and methods for the operation of unilateral ureteral obstruction and the induction of anti-glomerular basement membrane antibody glomerulonephritis.

Hepatitis B virus X protein promotes podocyte pyroptosis in hepatitis B virus-associated glomerulonephritis by down-regulating microRNA -223 targeting NLRP3 inflammasome / 中华肝脏病杂志

Objective: To investigate the potential function and related mechanism of microRNA-223 (miRNA-223) in the podocyte pyroptosis of hepatitis B virus (HBV)-associated glomerulonephritis induced by HBV X protein (HBx). Methods: HBx-overexpressing lentivirus was transfected into human renal podocytes to mimic the pathogenesis of HBV-GN. Real-time fluorescence quantitative PCR and Western blotting experiments were used to detect the mRNA and protein expression of pyroptosis-related proteins [nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3), apoptosis-associated speck-like protein containing a CARD (ASC) and caspase-1], and inflammatory factors (interleukin-1β and interleukin-18), respectively.TUNEL staining and flow cytometry were used to detect the number of pyroptosis cells. Immunofluorescence staining was used to detect the expression of podocytes biomarkers desmin and nephrin; Hoechst 33342 staining was used to observe the morphological and quantitative changes of podocyte nuclei. Enzyme-linked immunosorbent assay was used to measure caspase-1 activity. The dual luciferase reporter gene assay was used to verify the downstream target of miRNA-223. Podocytes were divided into the following nine groups: control group (no special treatment), empty plasmid group (transfected with empty plasmid), HBx overexpression group (transfected with HBx overexpression lentivirus), HBx overexpression+miRNA-223 mimic group (transfected with HBx overexpression lentivirus and miRNA-223 mimic), HBx overexpression+miRNA-223 inhibitor group (transfected with HBx overexpression lentivirus and miRNA-223 inhibitor), HBx overexpression+miRNA-223 mimic+NLRP3 group (transfected with HBx overexpression lentivirus, miRNA-223 mimic and NLRP3 overexpression plasmid), HBx overexpression+miRNA-223 mimic+ NLRP3 siRNA group (transfected with HBx overexpression lentivirus, miRNA-223 mimic and NLRP3 siRNA), HBx overexpression+miRNA-223 inhibitor+NLRP3 group (transfected with HBx overexpression lentivirus, miRNA-223 inhibitor and NLRP3 overexpression plasmid), HBx overexpression+miRNA-223 inhibitor+NLRP3 siRNA group (transfected with HBx overexpression lentivirus, miRNA-223 inhibitor and NLRP3 siRNA). Results: miRNA-223 was down-regulated in HBx overexpression group compared with the control group (P < 0.05). TUNEL and immunofluorescence staining showed that NLRP3 knockdown attenuated podocyte injury and pyroptosis induced by HBx overexpression (P < 0.05). Dual luciferase reporter gene assay demonstrated that NLRP3 was one of the downstream targets of miRNA-223. Rescue experiments revealed that NLRP3 overexpression weakened the protective effect of miRNA-223 in podocyte injury (P < 0.05). The addition of miRNA-223 mimic and NLRP3 siRNA decreased the expression of NLRP3 inflammasome and cytokines, and reduced the number of pyroptosis cells induced by HBx overexpression (all P < 0.05); The addition of miRNA-223 inhibitor and NLRP3 overexpression plasmid significantly increased the expression of NLRP3 inflammasome and cytokines, caspase-1 activity, and the number of pyroptosis cells (all P < 0.05). Conclusion: HBx may promote podocyte pyroptosis of HBV-GN via downregulating miRNA-223 targeting NLRP3 inflammasome, suggesting that miRNA-223 is expected to be a potential target for the treatment of HBV-GN.

Effective substances and mechanism of Yishen Guluo Mixture in treatment of chronic glomerulonephritis based on metabolomics and serum pharmacochemistry / 中国中药杂志

This study aimed to investigate the effective substances and mechanism of Yishen Guluo Mixture in the treatment of chronic glomerulonephritis(CGN) based on metabolomics and serum pharmacochemistry. The rat model of CGN was induced by cationic bovine serum albumin(C-BSA). After intragastric administration of Yishen Guluo Mixture, the biochemical indexes related to renal function(24-hour urinary protein, serum urea nitrogen, and creatinine) were determined, and the efficacy evaluations such as histopathological observation were carried out. The serum biomarkers of Yishen Guluo Mixture in the treatment of CGN were screened out by ultra-performance liquid chromatography-quadrupole time-of-flight/mass spectrometry(UPLC-Q-TOF-MS) combined with multivariate statistical analysis, and the metabolic pathways were analyzed. According to the mass spectrum ion fragment information and metabolic pathway, the components absorbed into the blood(prototypes and metabolites) from Yishen Guluo Mixture were identified and analyzed by using PeakView 1.2 and MetabolitePilot 2.0.4. By integrating metabolomics and serum pharmacochemistry data, a mathematical model of correlation analysis between serum biomarkers and components absorbed into blood was constructed to screen out the potential effective substances of Yishen Guluo Mixture in the treatment of CGN. Yishen Guluo mixture significantly decreased the levels of 24-hour urinary protein, serum urea nitrogen, and creatinine in rats with CGN, and improved the pathological damage of the kidney tissue. Twenty serum biomarkers of Yishen Guluo Mixture in the treatment of CGN, such as arachidonic acid and lysophosphatidylcholine, were screened out, involving arachidonic acid metabolism, glycerol phosphatide metabolism, and other pathways. Based on the serum pharmacochemistry, 8 prototype components and 20 metabolites in the serum-containing Yishen Guluo Mixture were identified. According to the metabolomics and correlation analysis of serum pharmacochemistry, 12 compounds such as genistein absorbed into the blood from Yishen Guluo Mixture were selected as the potential effective substances for the treatment of CGN. Based on metabolomics and serum pharmacochemistry, the effective substances and mechanism of Yishen Guluo Mixture in the treatment of CGN are analyzed and explained in this study, which provides a new idea for the development of innovative traditional Chinese medicine for the treatment of CGN.

Axl regulated survival/proliferation network and its therapeutic intervention in mouse models of glomerulonephritis.

BACKGROUND: Lupus nephritis (LN) is the most common and serious complication of systemic lupus erythematosus (SLE). LN pathogenesis is not fully understood. Axl receptor tyrosine kinase is upregulated and contributes to the pathogenic progress in LN. We have reported that Axl disruption attenuates nephritis development in mice. METHODS: In this study, we analyzed the gene expression profiles with RNA-seq using renal cortical samples from nephritic mice. Axl-KO mice were bred onto a B6.lpr spontaneous lupus background, and renal disease development was followed and compared to the Axl-sufficient B6.lpr mice. Finally, anti-glomerular basement membrane (anti-GBM) Ab-induced nephritic mice were treated with Axl small molecule inhibitor, R428, at different stages of nephritis development. Blood urine nitrogen levels and renal pathologies were evaluated. RESULTS: Transcriptome analysis revealed that renal Axl activation contributed to cell proliferation, survival, and motility through regulation of the Akt, c-Jun, and actin pathways. Spontaneous lupus-prone B6.lpr mice with Axl deficiency showed significantly reduced kidney damages and decreased T cell infiltration compared to the renal damage and T cell infiltration in Axl-sufficient B6.lpr mice. The improved kidney function was independent of autoAb production. Moreover, R428 significantly reduced anti-GBM glomerulonephritis at different stages of GN development compared to the untreated nephritic control mice. R428 administration reduced inflammatory cytokine (IL-6) production, T cell infiltration, and nephritis disease activity. CONCLUSIONS: Results from this study emphasize the important role of Axl signaling in LN and highlight Axl as an attractive target in LN.

Ntrk1 promotes mesangial cell proliferation and inflammation in rat glomerulonephritis model by activating the STAT3 and p38/ERK MAPK signaling pathways.

BACKGROUND: Mesangial proliferative glomerulonephritis (MsPGN) accounts for a main cause of chronic kidney disease (CKD), chronic renal failure and uremia. This paper aimed to examine the effect of Ntrk1 on MsPGN development, so as to identify a novel therapeutic target for MsPGN. METHODS: The MsPGN rat model was constructed by single injection of Thy1.1 monoclonal antibody via the tail vein. Additionally, the Ntrk1 knockdown rat model was established by injection of Ntrk1-RNAi lentivirus via the tail vein. Periodic acid-schiff staining and immunohistochemistry (IHC) were performed on kidney tissues. Moreover, the rat urinary protein was detected. Mesangial cells were transfected and treated with p38 inhibitor (SB202190) and ERK inhibitor (PD98059). Meanwhile, the viability and proliferation of mesangial cells were analyzed by cell counting kit-8 (CCK-8) and 5-Ethynyl-2'-deoxyuridine assays. Gene expression was detected by quantitative real-time polymerase chain reaction (qRT-PCR) and Western-blot (WB) assays. RESULTS: The proliferation of mesangial cells was enhanced in glomerulus and Ki67 expression was up-regulated in renal tubule of MsPGN rats. The urine protein level increased in MsPGN rats. Pro-inflammatory factors and Ntrk1 expression were up-regulated in glomerulus of MsPGN rats. Ntrk1 up-regulation promoted the viability, proliferation, expression of pro-inflammatory factors and activation of the STAT3, p38 and ERK signaling pathways in mesangial cells. Ntrk1 knockdown reduced mesangial cell proliferation, urine protein, pro-inflammatory factors, activation of STAT3, p38 and ERK signaling pathways in glomerulus, and decreased Ki67 expression in renal tubule of MsPGN rats. Treatment with SB202190 and PD98059 reversed the effect of Ntrk1 on promoting the viability, proliferation and inflammatory response of mesangial cells. CONCLUSION: Ntrk1 promoted mesangial cell proliferation and inflammation in MsPGN rats by activating the STAT3 and p38/ERK MAPK signaling pathways.

Treating crescentic glomerulonephritis by targeting macrophages.

Macrophage accumulation in the kidney is associated with the progression of crescentic glomerulonephritis (GN) and is mostly derived from circulating monocytes. FROUNT, a C-C motif chemokine receptor 2 (CCR2)-interacted protein, which is strongly expressed in monocytes/macrophages, enhances macrophage infiltration through CCR2-mediated chemotaxis. In this issue of the journal, Toda et al. reported that disulfiram, an inhibitor of FROUNT, attenuates GN by inhibition of the FROUNT-CCR2 interaction and macrophage migration and activation, suggesting a potential therapeutic role for crescentic GN.

CD206+CD68+ mono-macrophages and serum soluble CD206 level are increased in antineutrophil cytoplasmic antibodies associated glomerulonephritis.

BACKGROUND: Antineutrophil Cytoplasmic Antibodies (ANCA) associated glomerulonephritis (AGN) is a group of autoimmune diseases and mono-macrophages are involved in its glomerular injuries. In this study, we aim to investigate the role of CD206+ mono-macrophages in AGN. METHODS: 27 AGN patients (14 active AGN, 13 remissive AGN) together with healthy controls (n = 9), disease controls (n = 6) and kidney function adjusted controls (n = 9) from Department of Nephrology, Ruijin hospital were recruited. Flow cytometry was used to study proportion of CD206+ cells in peripheral blood. Immunohistochemistry for CD206 staining was performed and CD206 expression was scored in different kidney regions. Serum soluble CD206 (sCD206) was measured by enzyme-linked immunosorbent assay (ELISA). We also generated murine myeloperoxidase (MPO) (muMPO) ANCA by immunizing Mpo-/- mice. Mouse bone marrow-derived macrophages (BMDMs) from wild C57BL/6 mice and peripheral blood mononuclear cell (PBMC) derived macrophages from healthy donors were treated with MPO ANCA with or without its inhibitor AZD5904 to investigate the effects of MPO-ANCA on CD206 expression. RESULTS: The proportion of peripheral CD206+CD68+ cells in active AGN patients were significantly higher than that in remissive patients (p < 0.001), healthy controls (p < 0.001) and kidney function adjusted controls (p < 0.001). Serum sCD206 level in active AGN patients was higher than that in healthy controls (p < 0.05) and remissive patients (p < 0.01). Immunohistochemistry showed CD206 was highly expressed in different kidney regions including fibrinoid necrosis or crescent formation, glomeruli, periglomerular and tubulointerstitial compartment in active AGN patients in comparison with disease controls. Further studies showed MPO ANCA could induce CD206 expression in BMDMs and PBMC derived macrophages and such effects could be reversed by its inhibitor AZD5904. CONCLUSION: ANCA could induce CD206 expression on mono-macrophages and CD206+ mono-macrophages are activated in AGN. CD206 might be involved in the pathogenesis of AAV and may be a potential target for the disease.