Heymann Nephritis Research Articles

Transplantation of Cultured Mononuclear Fraction of Bone Marrow Cells in the Treatment of Heymann Nephritis in Rats.

In an experiment on Wistar rats, a Heymann's active nephritis model was reproduced. After the chronic course of the disease was confirmed, we compared the effectiveness of single systemic and local transplantation of allogeneic cultured stromal cells of the mononuclear fraction of the bone marrow. Both methods of cell therapy reduced clinical manifestations of active Heymann's nephritis: proteinuria decreased and glomerular filtration rate increased 30 days after cell administration. At the histological level, signs of the inflammatory process manifested in an increase in the number of CD68+ cells. Thus, in the case of autoimmune glomerulonephritis, two transplantation methods provide a comparable level of correction of functional disorders in Heymann's active nephritis model.

Animal models of membranous nephropathy: more choices and higher similarity.

Membranous nephropathy (MN) is an antibody-mediated autoimmune glomerular disease in which PLA2R1 is the main autoantibody. It has become the most common cause of adult nephrotic syndrome, and about one-third of patients can progress to end-stage kidney disease, but its pathogenesis is still unclear. Animal models can be used as suitable tools to study the pathogenesis and treatment of MN. The previous Heymann nephritis rat model and C-BSA animal model are widely used to study the pathogenesis of MN. However, the lack of target antigen expression in podocytes of model animals (especially rodents) restricts the application. In recent years, researchers constructed animal models of antigen-specific MN, such as THSD7A, PLA2R1, which more truly simulate the pathogenesis and pathological features of MN and provide more choices for the follow-up researchers. When selecting these MN models, we need to consider many aspects, including cost, difficulty of model preparation, labor force, and whether the final model can answer the research questions. This review is to comprehensively evaluate the mechanism, advantages and disadvantages and feasibility of existing animal models, and provide new reference for the pathogenesis and treatment of MN.

Differential Expression of Renal and Hepatic PCSK9 during Development of Hypercholesterolemia in the Passive Heymann Nephritis Rat Model of Nephrotic Syndrome

Differential Expression of Renal and Hepatic PCSK9 during Development of Hypercholesterolemia in the Passive Heymann Nephritis Rat Model of Nephrotic Syndrome

Target Protein of Passive Heymann Nephritis and Its Role in the Mechanism of Proteinuria in Membranous Nephropathy (MN)

Target Protein of Passive Heymann Nephritis and Its Role in the Mechanism of Proteinuria in Membranous Nephropathy (MN)

Expansion of T follicular helper cells is associated with disease progression in rat experimental membranous nephropathy model.

T follicular helper (Tfh) cells drive humoral immunity by facilitating B cell responses, but the functional role of Tfh cells in the pathogenesis of idiopathic membranous nephropathy (IMN) remains unclear. This study aimed to establish a rat experimental membranous nephropathy model, investigate the phenotypic characteristics of Tfh cells, and analyze a clinically significant correlation between Tfh cells. Passive Heymann nephritis (PHN) rats were induced by immunizing Sprague Dawley rats with anti-Fx1A serum. The frequency of Tfh and B cell subsets was analyzed with flow cytometry (FC). The serum concentration of interleukin-21 (IL-21), the relative mRNA expression levels of IL-21 and B cell lymphoma 6 (Bcl-6) in spleen mononuclear cells (MNCs), and the kidney infiltration of CD4+ T cells and IL-21 were assessed. The potential correlations among these measures were analyzed. In comparison with the control group, significantly increased percentages of Tfh cells, inducible T cell co-stimulator-positive (ICOS+) Tfh cells, and mRNA expression of Bcl-6 were detected in the spleen of PHN rats. Elevated IL-21 expression was detected in the serum and kidneys. Remarkably, the percentage of splenic ICOS+ Tfh cells was positively correlated with 24 h urine protein concentrations (r = 0.676, p = 0.011) in PHN rats. These data indicate that ICOS+ Tfh cells contribute to development of IMN, and they might be potential therapeutic targets for IMN.

Astragaloside IV relieves passive heymann nephritis and podocyte injury by suppressing the TRAF6/NF-κb axis

The pathogenesis of membranous nephropathy (MN) involves podocyte injury that is attributed to inflammatory responses induced by local immune deposits. Astragaloside IV (AS-IV) is known for its robust anti-inflammatory properties. Here, we investigated the effects of AS-IV on passive Heymann nephritis (PHN) rats and TNF-α-induced podocytes to determine the underlying molecular mechanisms of MN. Serum biochemical parameters, 24-h urine protein excretion and renal histopathology were evaluated in PHN and control rats. The expression of tumor necrosis factor receptor associated factor 6 (TRAF6), the phosphorylation of nuclear factor kappa B (p-NF-κB), the expression of associated proinflammatory cytokines (TNF-α, IL-6 and IL-1β) and the ubiquitination of TRAF6 were measured in PHN rats and TNF-α-induced podocytes. We detected a marked increase in mRNA expression of TNF-α, IL-6 and IL-1β and in the protein abundance of p-NF-κB and TRAF6 within the renal tissues of PHN rats and TNF-α-induced podocytes. Conversely, there was a reduction in the K48-linked ubiquitination of TRAF6. Additionally, AS-IV was effective in ameliorating serum creatinine, proteinuria, and renal histopathology in PHN rats. This effect was concomitant with the suppression of NF-κB pathway activation and decreased expression of TNF-α, IL-6, IL-1β and TRAF6. AS-IV decreased TRAF6 levels by promoting K48-linked ubiquitin conjugation to TRAF6, which triggered ubiquitin-mediated degradation. In summary, AS-IV averted renal impairment in PHN rats and TNF-α-induced podocytes, likely by modulating the inflammatory response through the TRAF6/NF-κB axis. Targeting TRAF6 holds therapeutic promise for managing MN.

Rapamycin ameliorates renal pathology in anti-Fx1A induced passive Heymann nephritis

Rapamycin ameliorates renal pathology in anti-Fx1A induced passive Heymann nephritis

Activation of the IL-6/STAT3 pathway contributes to the pathogenesis of membranous nephropathy and is a target for Mahuang Fuzi and Shenzhuo Decoction (MFSD) to repair podocyte damage

BackgroundPrimary membranous nephropathy (PMN) is an autoimmune glomerular disease. IL-6 is a potential therapeutic target for PMN. Previous clinical studies have demonstrated the effectiveness of Mahuang Fuzi and Shenzhuo Decoction (MFSD) in treating membranous nephropathy. However, the mechanism of action of MFSD remains unclear. MethodsSerum IL-6 levels were measured in patients with PMN and healthy subjects. The passive Heymann nephritis (PHN) rat model was established, and high and low doses of MFSD were used for intervention to observe the repair effect of MFSD on renal pathological changes and podocyte injury. RNA-seq was used to screen the possible targets of MFSD, and the effect of MFSD targeting IL-6/STAT3 was further verified by combining the experimental results. Finally, the efficacy of tocilizumab in PHN rats was observed. ResultsSerum IL-6 levels were significantly higher in PMN patients than in healthy subjects. These levels significantly decreased in patients in remission after MFSD treatment. MFSD treatment improved laboratory indicators in PHN rats, as well as glomerular filtration barrier damage and podocyte marker protein expression. Renal transcriptome changes showed that MFSD-targeted differential genes were enriched in JAK/STAT and cytokine-related pathways. MFSD inhibits the IL6/STAT3 pathway in podocytes. Additionally, MFSD significantly reduced serum levels of IL-6 and other cytokines in PHN rats. However, treatment of PHN with tocilizumab did not achieve the expected effect. ConclusionThe IL-6/STAT3 signaling pathway is activated in podocytes of experimental membranous nephropathy. MFSD alleviates podocyte damage by inhibiting the IL-6/STAT3 pathway.

Imbalance of Tfollicular helper cell subsets trigger the differentiation of pathogenic B cells in idiopathic membranous nephropathy.

This study aims to elucidate the role of T follicular helper (Tfh) cells and their subsets in idiopathic membranous nephropathy (IMN). The frequencies of Tfh cell subsets and B cell subsets in peripheral blood (PB) were detected in both IMN patients and healthy controls (HCs). The involvement of Tfh cells in the disease pathogenesis was examined by coculturing human Tfh cells with B cells. The dynamic changes of Tfh cells in PB or spleen were monitored in passive Heymann nephritis (PHN) rats. The frequencies of circulating Tfh (cTfh) cells, cTfh2 cells, and plasmablasts were enriched in the PB of patients with IMN. cTfh cells expressed higher ICOS, and lower BTLA than healthy counterparts. The frequency of ICOS + cTfh2 was associated with the severity of IMN, including 24h urine protein, IgG4 concentration and the IgG4: IgG ratio. Positive correlations were also observed between the frequency of cTfh2 cells with plasmablasts, serum IL-21 and IL-4 levels. Importantly, cTfh cells isolated from IMN patients were able to induce the differentiation of B cells to memory B cells (MBC) and plasmablasts, this process could be substantially attenuated by blocking the IL-21. Similar increases of ICOS + cTfh cells were also detected in spleen of PHN rats, concomitant with elevated urine protein levels. Collectively, our results demonstrate that the imbalance of cTfh cell subsets play a crucial pathogenic role in IMN by inducing the differentiation of B cells through IL-21, and cTfh2 cells might serve as useful markers to evaluate the progression of IMN.

Klotho Stabilizes the Podocyte Actin Cytoskeleton in Idiopathic Membranous Nephropathy through Regulating the TRPC6/CatL Pathway

Introduction: The aim of this study was to explore the renoprotective effects of Klotho on podocyte injury mediated by complement activation and autoantibodies in idiopathic membranous nephropathy (IMN). Methods: Rat passive Heymann nephritis (PHN) was induced as an IMN model. Urine protein levels, serum biochemistry, kidney histology, and podocyte marker levels were assessed. In vitro, sublytic podocyte injury was induced by C5b-9. The expression of Klotho, transient receptor potential channel 6 (TRPC6), and cathepsin L (CatL); its substrate synaptopodin; and the intracellular Ca2+ concentration were detected via immunofluorescence. RhoA/ROCK pathway activity was measured by an activity quantitative detection kit, and the protein expression of phosphorylated-LIMK1 (p-LIMK1) and p-cofilin in podocytes was detected via Western blotting. Klotho knockdown and overexpression were performed to evaluate its role in regulating the TRPC6/CatL pathway. Results: PHN rats exhibited proteinuria, podocyte foot process effacement, decreased Klotho and Synaptopodin levels, and increased TRPC6 and CatL expression. The RhoA/ROCK pathway was activated by the increased phosphorylation of LIMK1 and cofilin. Similar changes were observed in C5b-9-injured podocytes. Klotho knockdown exacerbated podocyte injury, while Klotho overexpression partially ameliorated podocyte injury. Conclusion: Klotho may protect against podocyte injury in IMN patients by inhibiting the TRPC6/CatL pathway. Klotho is a potential target for reducing proteinuria in IMN patients.

The fate of immune complexes in membranous nephropathy.

The most characteristic feature of membranous nephropathy (MN) is the presence of subepithelial electron dense deposits and the consequential thickening of the glomerular basement membrane. There have been great advances in the understanding of the destiny of immune complexes in MN by the benefit of experimental models represented by Heymann nephritis. Subepithelial immune complexes are formed in situ by autoantibodies targeting native autoantigens or exogenous planted antigens such as the phospholipase A2 receptor (PLA2R) and cationic BSA respectively. The nascent immune complexes would not be pathogenic until they develop into immune deposits. Podocytes are the major source of autoantigens in idiopathic membranous nephropathy. They also participate in the modulation and removal of the immune complexes to a large extent. The balance between deposition and clearance is regulated by a wide range of factors such as the composition and physicochemical properties of the immune complexes and the complement system. Complement components such as C3 and C1q have been reported to be precipitated with the deposits whereas a complement regulatory protein CR1 expressed by podocytes is involved in the phagocytosis of immune complexes by podocytes. Podocytes regulate the dynamic change of immune complexes which is disturbed in membranous nephropathy. To elucidate the precise fate of the immune complexes is essential for developing more rational and novel therapies for membranous nephropathy.

Prediction of biomarkers associated with membranous nephropathy: Bioinformatic analysis and experimental validation

Membranous nephropathy (MN), the most prevalent form of nephrotic syndrome in non-diabetic adults globally, is currently the second most prevalent and fastest-increasing primary glomerular disease in China. Numerous renal disorders are developed partly due to ferroptosis. However, its relationship to the pathogenesis of MN has rarely been investigated in previous studies; actually, ferroptosis is closely linked to the immune microenvironment and inflammatory response, which might affect the entire process of MN development. In this study, we aimed to identify ferroptosis-related genes that are potentially related to immune cell infiltration, which can further contribute to MN pathogenesis.The microarray datasets were downloaded from the Gene Expression Omnibus (GEO) database. Ferroptosis-related differentially expressed genes (FDEGs) were identified, which were further used for functional enrichment analysis. The common genes identified using the Least Absolute Shrinkage and Selection Operator (LASSO) logistic regression algorithm and the support vector machine recursive feature elimination (SVM-RFE) algorithm were used to identify the characteristic genes related to ferroptosis. The feasibility of the 7 genes as a distinguishing factor was assessed using the receiver operating characteristic (ROC) curve, with the area under the curve (AUC) score serving as the evaluation metric. Gene set enrichment analysis (GSEA) and correlation analysis of these genes were further performed. The correlation between the expression of these genes and immune cell infiltration inferred by single sample gene set enrichment analysis (ssGSEA) algorithm was explored. As a result, 7 genes, including NR1D1, YTHDC2, EGR1, ZFP36, RRM2, RELA and PDK4, which were most relevant to immune cell infiltration, were identified to be potential diagnostic genes in MN patients. Next, the signature genes were validated with other GEO datasets. In the subsequent steps, we conducted quantitative real-time fluorescence PCR (qRT-PCR) analysis and immunohistochemistry (IHC) method on the cationic bovine serum albumin (C-BSA) induced membranous nephropathy (MN) rat model and the passive Heymann nephritis (pHN) rat model to examine characteristic genes. Finally, we analysed the mRNA expression patterns of hub genes in MN patients and normal controls using the Nephroseq V5 online platform. In concise terms, our study successfully identified biomarkers specific to MN patients and delved into the potential interplay between these markers and immune cell infiltration. This knowledge bears significance for the diagnosis and prospective treatment strategies for individuals affected by MN.

Ferroptosis is involved in passive Heymann nephritis in rats

Ferroptosis is found to be involved in some experimental models of kidney diseases, but its role in membrane nephropathy (MN) is still unclear. The purpose of this study is to explore whether ferroptosis occurred in MN, and the role of ferritinophagy. In this study, passive Heymann nephritis (PHN) rats were induced by single tail vein injection of anti-Fx1A serum, and normal rats were used as control. The changes of 24 h urinary protein, serum biochemical parameters, renal pathological damage, iron content, lipid peroxidation parameters, ferroptosis markers, and ferritinophagy markers were evaluated in the two groups. Compared with the control group, PHN rats showed obvious proteinuria, hypoproteinemia, and hyperlipidemia. Besides, more severe renal pathological damage and higher Fe2+ levels were observed in PHN rats, and the levels of malondialdehyde (MDA) increased significantly, while the levels of superoxide Dismutase (SOD) and glutathione (GSH) decreased. In addition, the expression of glutathione peroxidase 4 (GPX4) in renal tissues of PHN rats decreased significantly, while the expression of transferrin receptor (TFR) and acyl-CoA synthetase long-chain family member 4 (ACSL4) increased. The expression of microtubule associated protein 1 light chain 3 (LC3) II/LC3I and nuclear receptor coactivator 4 (NCOA4) increased significantly. Therefore, our study shows that ferroptosis is involved in the pathological damage of MN, and companied by activation of ferritinophagy.

Crocin improves the renal autophagy in rat experimental membranous nephropathy via regulating the SIRT1/Nrf2/HO-1 signaling pathway

Membranous nephropathy (MN) is a glomerular disease. Crocin is isolated from saffron and gardenia. Its antioxidant, anti-inflammatory, anti-hyperlipidemic, anti-atherosclerotic, anti-tumor, free-radical scavenging and neuroprotective activities have been well established. We investigated the biological functions of crocin and its related mechanisms in MN. We established an experimental passive Heymann nephritis (PHN) rat model induced by anti-Fx1A antiserum. The rats were divided into sham, sham + crocin, PHN, PHN + crocin, and PHN + enalapril groups. Blood samples and kidneys of rats were collected for estimation of biochemical parameters in serum and oxidative stress indicators in kidney tissues. Histopathological changes of renal tissues were evaluated by hematoxylin and eosin, periodic acid-Schiff (PAS) and Masson staining. The podocyte number was estimated by immunohistochemistry staining of Wilms tumor type 1 (WT1). The deposition of rat anti-rabbit IgG antibodies, complement C3 and C5b-9 was detected by immunofluorescence staining. Western blotting was performed to measure the levels of Sirtuin 1 (Sirt1), nuclear factor erythroid 2-related factor 2 (Nrf2), heme oxygenase 1 (HO-1) and apoptosis-related proteins. The total cholesterol, triglycerides, creatinine, blood urea nitrogen, urine volume and urine albumin of PMN rats were significantly reduced by crocin. Additionally, crocin attenuated the renal histopathological changes. Moreover, the oxidative stress damage and podocyte loss and immune injury were relieved by crocin in PHN rats. Mechanistically, crocin administration activated the Sirt1/Nrf2/HO-1 pathways. The results provide a scientific basis that crocin could alleviate MN by inhibiting immune injury and podocyte damage through activating the Sirt1/Nrf2/HO-1 pathways.

Negative Modulation of B Cell Activation by Melanocortin 1 Receptor Signaling Protects against Membranous Nephropathy.

Emerging evidence suggests that melanocortin neuropeptides-specifically adrenocorticotropic hormone-offer a novel, steroidogenic-independent therapeutic modality for membranous nephropathy (MN). The molecular mechanism underlying this beneficial effect, however, remains largely elusive. To investigate whether melanocortins modulate humoral immunity, the authors induced passive Heymann nephritis, a model of human MN, in wild-type and melanocortin 1 receptor (MC1R) knockout rats and treated them with melanocortin agents. Additional rats received adoptive transfer of bone marrow-derived cells beforehand from wild-type or MC1R knockout rats. The findings indicate that MC1R signaling plays a key role in negative modulation of B-cell activation and thereby suppresses humoral immune responses in passive Heymann nephritis, and suggest that MC1R signaling might offer a novel B cell-targeted therapeutic strategy for MN. Emerging evidence suggests that the pituitary neuropeptide melanocortins-specifically, adrenocorticotropic hormone-offer a novel nonsteroidogenic therapeutic modality for membranous nephropathy (MN). However, the mechanism(s) of action remains elusive. To investigate whether melanocortins modulate humoral immunity, we induced passive Heymann nephritis (PHN), a model of MN, in wild-type (WT) and melanocortin 1 receptor (MC1R) knockout (KO) rats. We treated the animals with melanocortin agents-repository corticotropin injection, the nonsteroidogenic pan-melanocortin receptor agonist [Nle 4 , DPhe 7 ]-α-melanocyte stimulating hormone, the selective MC1R agonist MS05, vehicle gel, or phosphate-buffered saline-and evaluated kidney function, histology, and molecular changes. Additional rats received adoptive transfer of syngeneic bone marrow-derived cells beforehand from WT or MC1R KO rats. KO of MC1R worsened PHN and this was associated with increased deposition of autologous immunoglobulin G (IgG) and complement C5b-9 in glomeruli and higher circulating levels of autologous IgG-evidence of a sensitized humoral immune response. Melanocortin therapy ameliorated PHN in WT rats, coinciding with reduced glomerular deposition of autologous IgG and C5b -9. The beneficial efficacy of melanocortins was blunted in KO rats but restored by adoptive transfer of syngeneic bone marrow-derived cells derived from WT rats. Mechanistically, MC1R was expressed in B lymphocytes and was negatively associated with B cell activation. MC1R agonism triggered the expression of microphthalmia-associated transcription factor in activated B cells in a cAMP-dependent mode and also repressed the expression of interferon regulatory factor 4 (a lymphoid transcription factor essential for B-cell development and maturation), resulting in suppressed plasma cell differentiation and IgG production. MC1R signaling negatively modulates B cell activation and suppresses humoral immune responses in PHN, suggesting that MC1R signaling might offer a novel therapeutic target for MN.

Membranous nephropathy: Clearer pathology and mechanisms identify potential strategies for treatment.

Primary membranous nephropathy (PMN) is one of the common causes of adult-onset nephrotic syndrome and is characterized by autoantibodies against podocyte antigens causing in situ immune complex deposition. Much of our understanding of the disease mechanisms underpinning this kidney-limited autoimmune disease originally came from studies of Heymann nephritis, a rat model of PMN, where autoantibodies against megalin produced a similar disease phenotype though megalin is not implicated in human disease. In PMN, the major target antigen was identified to be M-type phospholipase A2 receptor 1 (PLA2R) in 2009. Further utilization of mass spectrometry on immunoprecipitated glomerular extracts and laser micro dissected glomeruli has allowed the rapid discovery of other antigens (thrombospondin type-1 domain-containing protein 7A, neural epidermal growth factor-like 1 protein, semaphorin 3B, protocadherin 7, high temperature requirement A serine peptidase 1, netrin G1) targeted by autoantibodies in PMN. Despite these major advances in our understanding of the pathophysiology of PMN, treatments remain non-specific, often ineffective, or toxic. In this review, we summarize our current understanding of the immune mechanisms driving PMN from animal models and clinical studies, and the implications on the development of future targeted therapeutic strategies.

Integrated Network Pharmacology Analysis and Experimental Validation to Investigate the Molecular Mechanism of Triptolide in the Treatment of Membranous Nephropathy.

Triptolide, a major active ingredient isolated from Tripterygium wilfordii Hook f., is effective in the treatment of membranous nephropathy (MN); however, its pharmacological mechanism of action has not yet been clarified. We applied an approach that integrated network pharmacology and experimental validation to systemically reveal the molecular mechanism of triptolide in the treatment of MN. First, potential targets of triptolide and the MN-related targets were collected from publicly available database. Then, based on a protein-protein interaction network as well as GO and KEGG pathway enrichment analyses, we constructed target-pathway networks to unravel therapeutic targets and pathways. Moreover, molecular docking was applied to validate the interactions between the triptolide and hub targets. Finally, we induced passive Heymann nephritis (PHN) rat models and validated the possible molecular mechanisms of triptolide against MN. The network pharmacology results showed that 118 intersected targets were identified for triptolide against MN, including mTOR, STAT3, CASP3, EGFR and AKT1. Based on enrichment analysis, signaling pathways such as PI3K/AKT, MAKP, Ras and Rap1 were involved in triptolide treatment of MN. Furthermore, molecular docking confirmed that triptolide could bind with high affinity to the PIK3R1, AKT1 and mTOR, respectively. Then, in vivo experiments indicated that triptolide can reduce 24 h urine protein (P < 0.01) and protect against renal damage in PHN. Serum albumin level was significantly increased and total cholesterol, triglycerides, and low-density lipoprotein levels were decreased by triptolide (P < 0.05). Compared with PHN group, triptolide treatment regulated the PI3K/AKT/mTOR pathway according to Western blot analyses. Triptolide could exert antiproteinuric and renoprotective effects in PHN. The therapeutic mechanism of triptolide may be associated with the regulation of PI3K/AKT/mTOR signaling pathway. This study demonstrates the pharmacological mechanism of triptolide in the treatment of MN and provides scientific evidence for basic and clinical research.

Studies on the metabolism and mechanism of acteoside in treating chronic glomerulonephritis

Ethnopharmacological relevanceActeoside (ACT) is the main ingredient derived from the leaves of Rehmannia glutinosa (Dihuangye). Dihuangye has the function of clearing heat, replenishing qi and activating blood, nourishing yin and tonifying kidney in traditional Chinese medicine. Recent studies have demonstrated that Dihuangye can be used to treat nephritis and ACT is a promising antinephritic agent. Aim of the studyTo clarify the metabolites of ACT in biological samples and investigate the renoprotective effect and mechanism of ACT in rats with chronic glomerulonephritis (CGN). Materials and methodsIn this study, the biotransformation of ACT in rat biological samples was clarified by quadrupole time-of-flight tandem mass spectrometry. The metabolites were validated by urine samples in nephropathy model rats. The effect of ACT and its metabolites was evaluated by glomerular podocyte injury due to high glucose. Based on an analysis of the ingredients in vivo, the potential therapeutic targets in the treatment of CGN were investigated by using network pharmacological analysis and molecular docking. Then, the renoprotective effect and mechanism of ACT were determined in rats in a passive Heymann nephritis (PHN) model. ResultsA total of 49 metabolites of ACT were detected and identified. Meanwhile, 21 metabolites were detected in nephropathy model rats. ACT was absorbed rapidly and transferred from the kidney, and the metabolites were eliminated via urine. The whole process lasted approximately 8 h. ACT had a significant protective effect on glomerular podocytes damaged by high glucose and 3,4-dihydroxyphenylacetic acid might be the main metabolite of ACT underlying its functions in vivo. The network pharmacology and molecular docking results showed 84 ACT-CGN targets, among which MAPK1, HRAS, AKT1, EGFR, and others were a highly correlated. In the PHN rat model, ACT significantly reduced the 24-h urine protein and serum creatinine concentrations, suppressed the leukocyte CD18 expression levels, decreased the serum tumor necrosis factor α (TNF-α) levels and tended to reduce serum interleukin 6 (IL-6) levels. ACT significantly reduced the platelet aggregation rate and inhibited the proliferative activity of splenic lymphocytes in response to the mitogen concanavalin A. Meanwhile, ACT inhibited transforming growth factor-β and fibronectin expression in renal tissues and dose-dependently inhibited TNF-α and IL-6 production in RAW264.7 mouse macrophages at doses ranging from 1.8 to 1330 μg/mL. ConclusionsACT had therapeutic effects on PHN rats, and its mechanism might be related to the inhibition of intercellular or intercellular-matrix adhesion, suppression of inflammatory response, regulation of immune function, improvement of tissue hemodynamics and hemorheology, and relief of fibrotic lesions.

VEGFA promotes the occurrence of PLA2R-associated idiopathic membranous nephropathy by angiogenesis via the PI3K/AKT signalling pathway

BackgroundThe M-type phospholipase A2 receptor (PLA2R)-associated idiopathic membranous nephropathy (IMN) is a common immune-related disease in adults. Vascular endothelial growth factor A (VEGFA) is the key mediator of angiogenesis, which leads to numerous kidney diseases. However, the role of VEGFA in IMN is poorly understood.MethodsIn the present study, we downloaded the microarray data GSE115857 from Gene Expression Omnibus (GEO). The differentially expressed genes (DEGs) were identified with R software. The cytoHubba plug-in were used to identify hub genes from the protein–protein interaction network. Gene set enrichment analysis (GSEA) was used to identify signalling pathway in IMN. CCK8 was performed to assess the cell viability in human vascular endothelial cells (HVECs). Then, passive Heymann nephritis (PHN) was induced in rats by a single tail vein injection of anti-Fx1A antiserum. Animals treated with VEGFA inhibitor bevacizumab (BV), with saline as a positive control. Proteinuria was evaluated by biochemical measurements. Immunohistochemistry and immunofluorescence was used to evaluate relative proteins expression. Electron microscopy was performed to observe the thickness of the glomerular basement membrane (GBM).ResultsWe revealed 3 hub genes, including one up-regulated gene VEGFA and two down-regulated genes JUN and FOS, which are closely related to the development of PLA2R-associated IMN. Pathway enrichment analysis found that the biological process induced by VEGFA is associated with PI3K/Akt signalling. GSEA showed that the signalling pathway of DEGs in GSE115857 was focused on angiogenesis, in which VEGFA acts as a core gene. We confirmed the high expression of VEGFA, PI3K, and AKT in IMN renal biopsy samples with immunohistochemistry. In HVECs, we found that BV suppresses cell viability in a time and dose dependent manner. In vivo, we found low dose of BV attenuates proteinuria via inhibiting VEGFA/PI3K/AKT signalling. Meanwhile, low dose of BV alleviates the thickening of the GBM.ConclusionVEGFA/PI3K/AKT signalling may play significant roles in the pathogenesis of IMN, which may provide new targets for the treatment of IMN.

Complement C3a and C3a Receptor Activation Mediates Podocyte Injuries in the Mechanism of Primary Membranous Nephropathy.

The complement system is highly activated in primary membranous nephropathy (MN). Identifying the complement components that damage podocytes has important therapeutic implications. This study investigated the role of C3a and the C3a receptor (C3aR) in the pathogenesis of MN. C3aR expression in kidneys and circulating levels of C3a of MN patients were examined. Human podocyte damage was assessed after exposure to MN plasma +/- C3aR blockade (SB290157, JR14a). C3aR antagonists were administered to rats with Heymann nephritis on day 0 or after proteinuria. Clinical and pathologic parameters, specific IgG and complement activation, and podocyte injuries were then assessed. In the glomeruli, C3aR staining merged well with podocin. Overexpression of C3aR correlated positively with proteinuria, serum creatinine, and no response to treatments. Human podocytes exposed to MN plasma showed increased expression of PLA2R, C3aR, and Wnt3/β-catenin, reduced expression of synaptopodin and migration function, downregulated Bcl-2, and decreased cell viability. C3aR antagonists could block these effects. In Heymann nephritis rats, C3aR blockade attenuated proteinuria, electron-dense deposition, foot process width, and glomerular basement membrane thickening in glomeruli. The increased plasma C3a levels and overexpression of C3aR were also alleviated. Specific, but not total, IgG levels decreased, with less deposition of rat IgG in glomeruli and subsequent reduction of C1q, factor B, and C5b-9. C3a anaphylatoxin is a crucial effector of complement-mediated podocyte damage in MN. The C3aR antagonist may be a potentially viable treatment for this disease.