Passive Heymann Nephritis Rats Research Articles

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

<b><i>Introduction:</i></b> 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). <b><i>Methods:</i></b> 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 Ca<sup>2+</sup> 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. <b><i>Results:</i></b> 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. <b><i>Conclusion:</i></b> 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.

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.

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.

The Ameliorative Effect of Mahuang Fuzi and Shenzhuo Decoction on Membranous Nephropathy of Rodent Model is Associated With Autophagy and Wnt/β-Catenin Pathway.

The increased incidence of membranous nephropathy (MN) has made it the most common pathological type of primary nephrotic syndrome in adults in China. According to the theory of Traditional Chinese Medicine (TCM), Mahuang Fuzi (Chinese ephedra and Radix Aconiti Lateralis Preparata) and Shenzhuo Decoction (MFSD) could be used to treat such diseases. We treated patients of MN with MFSD, and observed comparable efficacy to glucocorticoid and/or immunosuppressants. In this study, we observed the therapeutic effect of MFSD on the rat model of passive Heymann nephritis (PHN), a classical MN model. Our results showed that MFSD treatment significantly reduced urinary protein level and podocyte injury in PHN rats, and correspondingly improved renal pathology, with the improvement effect on MN comparable to that of Cyclosporine A (CsA) alone. To explore the potential therapeutical mechanism of MFSD, the main chemical components of MFSD were determined by High-performance liquid chromatography-mass spectrometry (HPLC-MS). There were about 30 active components of MFSD. Next, based on network pharmacology methods, we screened related targets of MSFD on MN, which provided a preliminary understanding of the MFSD bioactive compounds. The clustering analysis showed that its active site might be in the autophagy-related protein and Wnt/β-catenin pathway, which was related to podocyte injury. Finally, we observed an improvement in renal autophagy and a down-regulation of the Wnt/β-catenin pathway after MSFD treatment in a PHN rat model. According to this study, autophagy and Wnt/β-catenin pathway may be potential targets for MFSD in the treatment of MN.

Treatment of Membranous Nephropathy by Disulfiram through Inhibition of Podocyte Pyroptosis

Introduction: Membranous nephropathy (MN) is a common chronic kidney disease in adults and a major challenge of clinical practice for its treatment. Despite major advances, since the discovery of the phospholipase A2 receptor as the major autoantigen of podocytes in MN, the mechanisms leading to glomerular damage remain elusive. Pyroptosis, a newly discovered type of programed necrotic cell death mainly mediated by gasdermin, was found to be responsible for podocyte injury in MN in our recent work. Objectives: The aim of this study was to explore the therapeutic effect of an FDA-approved drug, disulfiram (DSF), in the treatment of MN by inhibiting pyroptosis. Methods and Results: DSF significantly alleviated C3a/C5a-induced podocyte injury in vitro and renal lesions in passive Heymann nephritis (PHN) rats, as reflected by the decreased percentage of propidium iodide staining podocytes, decreased lactate dehydrogenase release from cultured podocytes and improvement in 24-h urine protein, serum albumin, serum creatinine, abnormal alterations of podocyte injury markers Desmin and WT-1 and podocyte foot process fusion in PHN rats. The protective effect of DSF on podocyte injury in vitro and in vivo can be ascribed to its inhibition of the activation and membrane translocation of the pyroptosis executor gasdermin D (GSDMD) in podocytes. DSF also inhibited the increase and activation of the pyroptosis signaling pathway NLRP3-ASC-Caspase-1/IL-18/GSDMD in C3a/C5a-treated podocytes and renal tissue of PHN rats. Conclusion: DSF is a potential drug for MN treatment, and its clinical application needs to be further investigated.

Structural modulation of gut microbiota during alleviation of experimental passive Heymann nephritis in rats by a traditional Chinese herbal formula.

Jianpi-Qushi-Heluo formula (JQHF) has been used to treat idiopathic membranous nephropathy (IMN) in hospitals for many years. Elucidating the protective effect and exploring the potential mechanism of JQHF against IMN. Passive Heymann nephritis (PHN) was induced in rats by a single tail vein injection of anti-Fx1A antiserum. Then, the animals were treated with JQHF at 16.2g/kg or 32.4g/kg, with benzepril (10mg/kg) as a positive control. Renal function was evaluated by biochemical measurements and pathological testing. Fecal samples were collected before and after treatment to analyze the gut microbiota composition by shotgun whole metagenome sequencing. JQHF exhibited potent efficacy in ameliorating PHN at both doses, as revealed by decreasing the deposition of IgG and C5b-9, relieving podocyte injury, and reducing glomerular and tubular cell apoptosis. The lower dose was corresponding to the clinical dosage and showed better therapeutic effects than the higher dose. Metagenomic analysis showed that gavage with 16.2g/kg of JQHF shifted the structure of the gut microbiota in PHN rats and significantly increased the relative abundances of Prevotella copri, Lactobacillus vaginalis and Subdoligranulum variabile. Particularly, S. variabile was strongly negatively correlated with serum levels of TC and TG, the deposition of IgG and C5b-9, and apoptosis of glomerular cells. The JQHF is an effective agent for the treatment of experimental PHN. The PHN-allevating effect of JQHF is associated with specific alternation of gut microbiota.

Sanqi Oral Solution Mitigates Proteinuria in Rat Passive Heymann Nephritis and Blocks Podocyte Apoptosis via Nrf2/HO-1 Pathway.

Idiopathic membranous nephropathy (IMN) is the most common pathological type in adult nephrotic syndrome where podocyte apoptosis was found to mediate the development of proteinuria. Sanqi oral solution (SQ), an effective Chinese herbal preparation clinically used in treatment of IMN for decades, plays an important role in reducing proteinuria, but the underlying mechanisms have not been fully elucidated yet. The current study tested the hypothesis that SQ directly lessens proteinuria in IMN by reducing podocyte apoptosis. To investigate the effects of SQ, we established the experimental passive Heymann nephritis (PHN) rat model induced by anti-Fx1A antiserum in vivo and doxorubicin hydrochloride (ADR)-injured apoptotic podocyte model in vitro. SQ intervention dramatically reduced the level of proteinuria, together with the rat anti-rabbit IgG antibodies, complement C3, and C5b-9 deposition in glomerulus of PHN rats, accompanied by an elevation of serum albumin. Protein expression of synaptopodin, marker of podocyte injury, restored after SQ administration, whereas the electron microscopic analysis indicated that fusion of foot processes, and the pachynsis of glomerular basement membrane was markedly diminished. Further studies showed that SQ treatment could significantly inhibit podocyte apoptosis in PHN rats and ADR-injured podocytes, and protein levels of Cleaved Caspase-3 or the ratio of Bax/Bcl-2 were significantly decreased with SQ treatment in vivo or in vitro. Moreover, we found that the nuclear factor erythroid 2–related factor-2/heme oxygenase 1 (Nrf2/HO-1) pathway mediated the anti-apoptosis effective of SQ in podocyte. Thus, SQ mitigates podocyte apoptosis and proteinuria in PHN rats via the Nrf2/HO-1 pathway.

Curcumin Improves the Renal Autophagy in Rat Experimental Membranous Nephropathy via Regulating the PI3K/AKT/mTOR and Nrf2/HO-1 Signaling Pathways.

Membranous nephropathy (MN, also known as membranous glomerulopathy) is one of the many glomerular diseases causing nephrotic syndrome. The literature indicates that autophagy is associated with the homeostasis of podocytes in glomeruli. Curcumin, the main active component in turmeric, has drawn attention for its effective bioactivities against chronic kidney disease. The current study was aimed at assessing the effects of curcumin and exploring the underlying mechanism that mediates autophagy in an animal model of passive Heymann nephritis (PHN) in rats. Passive Heymann nephritis (PHN) was induced in male SD rats by intraperitoneal injection of anti-Fx1A serum. The rats were divided into 3 groups: control (n = 10, normal diet), model group (n = 10, 0.5% sodium carboxymethylcellulose), and curcumin (n = 10, 300 mg/kg/d). The kidney function and oxidative stress indicators were measured using commercial diagnostic kits, and the histomorphology of renal tissues was observed. The number of podocytes was measured by immunohistochemistry. Meanwhile, the autophagosomes in podocyte were analyzed by transmission electron microscopy and the immunofluorescence assay pointing to p62, an autophagic marker. Western blot analyzed the levels of apoptosis, autophagy, PI3K/AKT/mTOR, and Nrf2/HO-1 pathway-associated proteins. The total cholesterol (TC), triglycerides (TG), creatinine (Scr), blood urea nitrogen (BUN), urine volume, and urine albumin of PHN rats were significantly reduced by the administration of curcumin and attenuated renal histomorphological changes in model rats. Meanwhile, curcumin improved the oxidative stress response by decreasing MDA and increasing SOD, GSH, and CAT levels in the kidney of PHN rats. Furthermore, curcumin significantly ameliorated the podocyte loss, along with the fusion, and increased the autophagic vacuoles compared to the PHN control rats. In addition, curcumin downregulated the expression of Bax, Caspase-3, p62, PI3K, p-AKT, and p-mTOR proteins and upregulated the Bcl-2, beclin1, LC3, Nrf2, and HO-1 levels in this animal model. The results provide a scientific basis that curcumin could significantly alleviate the development of MN by inducing autophagy and alleviating renal oxidative stress through the PI3K/AKT/mTOR and Nrf2/HO-1 pathways.

Urinary ERdj3 and mesencephalic astrocyte-derived neutrophic factor identify endoplasmic reticulum stress in glomerular disease

Podocyte injury and endoplasmic reticulum (ER) stress have been implicated in the pathogenesis of various glomerular diseases. ERdj3 (DNAJB11) and mesencephalic astrocyte-derived neurotrophic factor (MANF) are ER chaperones lacking the KDEL motif, and may be secreted extracellularly. Since podocytes reside in the urinary space, we examined if podocyte injury is associated with secretion of KDEL-free ER chaperones from these cells into the urine, and if chaperones in the urine reflect ER stress in glomerulonephritis. In cultured podocytes, ER stress increased ERdj3 and MANF intracellularly and in culture medium, whereas GRP94 (KDEL chaperone) increased only intracellularly. ERdj3 and MANF secretion was blocked by the secretory trafficking inhibitor, brefeldin A. Urinary ERdj3 and MANF increased in rats injected with tunicamycin (in the absence of proteinuria). After induction of passive Heymann nephritis (PHN) and puromycin aminonucleoside nephrosis (PAN), there was an increase in glomerular ER stress, and appearance of ERdj3 and MANF in the urine, coinciding with the onset of proteinuria. Rats with PHN were treated with the chemical chaperone, 4-phenyl butyrate (PBA), starting at the time of disease induction, or after disease was established. In both protocols, 4-PBA reduced proteinuria and urinary ER chaperone secretion, compared with PHN rats treated with saline (control). In conclusion, urinary ERdj3 and MANF reflect glomerular ER stress. 4-PBA protected against complement-mediated podocyte injury and the therapeutic response could be monitored by urinary ERdj3 and MANF.

Betulinic acid, isolated from the leaves of Syzygium cumini (L.) Skeels, ameliorates the proteinuria in experimental membranous nephropathy through regulating Nrf2/NF-κB pathways

Membranous nephropathy (MN) is associated with increased oxidative stress and inflammatory markers in the kidney. Betulinic acid (BA) is a potent antioxidant and anti-inflammatory compound isolated from the leaves of Syzygium cumini (L.) Skeels. In the present study, we investigated the effects of BA on experimental MN in rats and explored the mechanisms by which it enhances antioxidant activities and resolves inflammatory condition in experimental MN. Passive Heymann nephritis (PHN) was induced in Sprague-Dawley rats by a single tail vein injection of anti- Fx1A antiserum. The rats were orally administered BA (25 and 50 mg kg −1 d −1) or dexamethasone (DEX; 0.07 mg kg-1, reference compound) for 4 weeks after the induction of PHN. Blood, urine, and kidney tissue were collected for analysis at the end of the study. Treatment of PHN rats with BA or DEX significantly attenuated renal dysfunction, histopathological alterations and reduced immune complex deposition in the kidneys. Furthermore, BA ameliorated mRNA and protein expression of NF-κB, iNOS, TNF-α, Nrf2, HO-1 and NQO1 in the kidney. BA also restored malondialdehyde level and antioxidant enzyme activities in the kidney. In a nutshell, the protective effect of BA can be explained by its anti-inflammatory and anti-oxidant activities, which in turn is due to downregulation of NF-κB pathway and activation of Nrf2. The results indicated that BA can effectively suppress experimental PHN in rats by regulating Nrf2/NF-κB pathways.

Artemisinin analogue SM934 ameliorates the proteinuria and renal fibrosis in rat experimental membranous nephropathy.

SM934 is a novel water-soluble artemisinin derivative with immunoregulatory activities that has been used to treat murine lupus nephritis. In the current study, we investigated the effects of SM934 on rat experimental membranous nephropathy. Passive Heymann nephritis (PHN) was induced in SD rats by intraperitoneal injection of anti-Fx1A serum. The rats were orally administered SM934 (12.5 and 25 mg·kg(-1)·d(-1)) or prednisolone (5 mg·kg(-1)·d(-1)) for 28 d. Blood and urine sample, and kidney tissue were collected for analyses. Human complement C3a-induced injury of HK-2 cells was used for in vitro experiments. Treatment of PHN rats with SM934 or prednisolone attenuated the progression of glomerulonephritis and renal fibrosis, as evidenced by the reduced level of proteinuria and circulating antibodies, as well as by the reduced immune complex deposition, reversed podocyte injuries, and attenuated tubulointerstitial fibrosis in the kidneys. Furthermore, the two drugs suppressed TGF-β1 expression and Smad2/3 phosphorylation, and increased Smad7 expression in the kidneys. The two doses of SM934 produced almost identical therapeutic effects on PHN rats. Pretreatment with SM934 or a C3a receptor antagonist blocked the C3a-induced epithelial-mesenchymal transition in HK-2 cells in vitro. SM934 ameliorates kidney injury and attenuates the tubulointerstitial fibrosis in PHN rats by down-regulation of the TGF-β1/Smad signaling pathway.

The calcineurin inhibitor tacrolimus reduces proteinuria in membranous nephropathy accompanied by a decrease in angiopoietin-like-4.

Tacrolimus is an anticalcineurinic agent with potent immunosuppressive activity that has recently been shown to have the added benefit of reducing proteinuria in membranous nephropathy (MN) patients. However, its potential mechanisms remain unknown. To reveal the mechanism, rat cohorts were administered tacrolimus or vehicle from days 7 to 28 after the induction of passive Heymann nephritis (PHN). PHN induction resulted in heavy proteinuria and increased expression of desmin, a marker of injured podocytes. We also showed that the glomerular expression of angiopoietin-like-4 (Angptl4) was markedly upregulated in PHN rats and human MN followed by an increase in urine Angptl4 excretion. In addition, increased Angptl4 expression may be related to podocyte injury and proteinuria. Furthermore, upregulated Angptl4 expression primarily colocalized with podocytes rather than endothelial or mesangial cells, indicating that podocytes may be the source of Angptl4, which then gradually migrated to the glomerular basement membrane over time. However, tacrolimus treatment markedly reduced glomerular and urinary Angptl4, accompanied by a reduction in the established proteinuria and the promotion of podocyte repair. Additionally, glomerular immune deposits and circulating IgG levels induced by PHN clearly decreased following tacrolimus treatment. In conclusion, this is the first demonstration that the calcineurin inhibitor tacrolimus can reduce Angptl4 in podocytes accompanied by a decrease in established proteinuria and promotion of podocyte repair in MN.

Cytochrome P450 2B1 Mediates Complement-dependent Sublytic Injury in a Model of Membranous Nephropathy

Membranous nephropathy is a disease that affects the filtering units of the kidney, the glomeruli, and results in proteinuria accompanied by loss of kidney function. Passive Heymann nephritis is an experimental model that mimics membranous nephropathy in humans, wherein the glomerular epithelial cell (GEC) injury induced by complement C5b-9 leads to proteinuria. We examined the role of cytochrome P450 2B1 (CYP2B1) in this complement-mediated sublytic injury. Overexpression of CYP2B1 in GECs significantly increased the formation of reactive oxygen species, cytotoxicity, and collapse of the actin cytoskeleton following treatment with anti-tubular brush-border antiserum (anti-Fx1A). In contrast, silencing of CYP2B1 markedly attenuated anti-Fx1A-induced reactive oxygen species generation and cytotoxicity with preservation of the actin cytoskeleton. Gelsolin, which maintains an organized actin cytoskeleton, was significantly decreased by complement C5b-9-mediated injury but was preserved in CYP2B1-silenced cells. In rats injected with anti-Fx1A, the cytochrome P450 inhibitor cimetidine blocked an increase in catalytic iron and ROS generation, reduced the formation of malondialdehyde adducts, maintained a normal distribution of nephrin in the glomeruli, and provided significant protection at the onset of proteinuria. Thus, GEC CYP2B1 contributes to complement C5b-9-mediated injury and plays an important role in the pathogenesis of passive Heymann nephritis.

Melanocortin 1 Receptor Agonists Reduce Proteinuria

Membranous nephropathy is one of the most common causes of nephrotic syndrome in adults. Recent reports suggest that treatment with adrenocorticotropic hormone (ACTH) reduces proteinuria, but the mechanism of action is unknown. Here, we identified gene expression of the melanocortin receptor MC1R in podocytes, glomerular endothelial cells, mesangial cells, and tubular epithelial cells. Podocytes expressed most MC1R protein, which colocalized with synaptopodin but not with an endothelial-specific lectin. We treated rats with passive Heymann nephritis (PHN) with MS05, a specific MC1R agonist, which significantly reduced proteinuria compared with untreated PHN rats (P < 0.01). Furthermore, treatment with MC1R agonists improved podocyte morphology and reduced oxidative stress. In summary, podocytes express MC1R, and MC1R agonism reduces proteinuria, improves glomerular morphology, and reduces oxidative stress in nephrotic rats with PHN. These data may explain the proteinuria-reducing effects of ACTH observed in patients with membranous nephropathy, and MC1R agonists may provide a new therapeutic option for these patients.

Transcriptional Regulation of Nephrin Gene by Peroxisome Proliferator–Activated Receptor-γ Agonist

The renoprotective potential of the peroxisome proliferator-activated receptor-gamma (PPAR-gamma) agonist pioglitazone was explored in an immune model of progressive nephropathy, passive Heymann nephritis (PHN), compared with that of an angiotensin II receptor antagonist, taken as standard therapy for renoprotection. PHN rats received orally vehicle, pioglitazone (10 mg/kg twice daily), or candesartan (1 mg/kg twice daily) from months 2 to 8. Pioglitazone reduced proteinuria as effectively as candesartan and limited renal functional and structural changes. Kidneys from untreated PHN rats showed lower nephrin mRNA and protein than controls, both restored by pioglitazone. The effect was seen both early and late during the course of the disease. Whether the antiproteinuric effect of pioglitazone could be due to its effect on nephrin gene transcription also was investigated. HK-2 cells were transfected with plasmids that harbor the luciferase gene under portions (2-kb or 325-bp) of human nephrin gene promoter that contain putative peroxisome proliferator-responsive elements (PPRE) and incubated with pioglitazone (10 muM). Transcriptional activity of luciferase gene was highly increased by pioglitazone, with the strongest expression achieved with the 325-bp fragment. Increase in luciferase activity was prevented by bisphenol A diglycidyl ether, a PPAR-gamma synthetic antagonist. Electrophoretic mobility shift assay experiments showed a direct interaction of PPAR/retinoid X receptor heterodimers to PPRE present in the enhancer region of the nephrin promoter. In conclusion, pioglitazone exerts an antiproteinuric effect in immune-mediated glomerulonephritis as angiotensin II receptor antagonist does. Enhancement of nephrin gene transcription through specific PPRE in its promoter discloses a novel mechanism of renoprotection for PPAR-gamma agonists.

Inhibitory Effects of Ligustrazine, a Modulator of Thromboxane-Prostacycline-Nitric Oxide Balance, on Renal Injury in Rats with Passive Heyman Nephritis

Aims: To explore the effects of ligustrazine on proteinuria, urinary TxB₂ (metabolism of thromboxane A₂, TxA₂) and 6-keto-PGF_1α(metabolism of prostacyclines I₂, PG I₂), glomerular inducible nitric oxide(NO) synthase (iNOS) mRNA, urinary NO^–₃/NO^–₂ (decomposing products of NO) and pathological changes in rats with passive Heymann nephritis (PHN). Methods: A rat PHN model was induced by intravenous injection of rabbit anti-rat renal tubular antigen (Tub-Ag) antiserum, and ligustrazine was given intraperitoneally into PHN rats every 2 days for 1–5 weeks. Then, proteinuria, urinary TxB₂ and 6-keto-PGF_1α, glomerular iNOS mRNA, and urinary NO^–₃/NO^–₂ were measured by sulfosalicylic acid, radioimmunoassay (RIA), Northern blot and nitric acid reductase methods, respectively. Moreover, the damage to the renal tissue of the rats was observed under light and electron microscopy and immunofluorescence (IF). Results: The urinary TxB₂ in PHN rats was significantly higher than that in control rats, but the PHN rats treated with ligustrazine had significantly less proteinuria, urinary TxB₂ and tissue lesions, and more urinary 6-keto-PGF_1α, glomerular iNOS mRNA and urinary NO^–₂/NO^–₃ than the PHN rats without the administration of ligustrazine. Conclusion: These data indicate that ligustrazine has inhibitory roles on the glomerular injury of PHN rats, which may associate with modulating the balance of TxA₂-PGI₂ and elevating synthesis of NO to a certain extent.

Presence of immunoglobulin M antibodies around the glomerular capillaries and in the mesangium of normal and passive Heymann nephritis rats.

Summary Diffuse distribution of small, faintly staining, beaded deposits of rat immunoglobulin M (IgM) around the glomerular capillary blood vessels, and a more intensely staining larger deposition in the mesangium, were observed on the kidney sections of normal rats. As glomerular-fixed nephritogenic antigens are known to be present on the epithelial aspect of the glomerular basement membrane (GBM), especially at the soles of foot processes and at the slit pores, it was assumed that the IgM antibodies were directed against these antigens. Investigation by immunofluorescent antibody double-staining techniques of rat kidney sections obtained from normal and rabbit anti-FX1A-injected rats stained for the nephritogenic antigen showed that a number of antigenic sites in the glomeruli and in the mesangium shared antibody hits by heterologous rabbit IgG and autologous rat IgM antibodies. Most sites in the glomeruli stained specifically for rat IgM or rabbit IgG, but preferentially for the latter. The intensely fluorescent mesangial deposits stained mainly for rat IgM, indicating that at these sites the antigenic material was virtually saturated, while areas at the entry to the mesangial space also stained for rabbit IgG, indicating that at these locations free nephritogenic epitopes were still available for reaction with the anti-FX1A antibody. Western blot analysis have shown that the rabbit anti-rat FX1A IgG and the rat anti-rat KF3 IgM antibodies are directed against the same renal tubular-derived antigen with a molecular weight of 70,000. These experimental findings collectively demonstrate that the heterologous IgG and autologous IgM antibodies are directed against the same nephritogenic antigen, which is found in the glomeruli, the mesangium and the proximal convoluted tubules. Thus, the IgM autoantibody has a possible physiological role but, in addition, there is evidence of active immunophagocytic events, manifested in a rapid and continuous entrapment and expulsion of macromolecules after their processing by the mesangial cells of normal and passive Heymann nephritis rats.