Passive Heymann Nephritis In Rats Research Articles

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.

POS-042 C3d-Directed Factor H Targeting Delivers Potent and Durable Complement Inhibition and Disease-Modifying Efficacy In Kidney and Skin Without Inhibiting Systemic Complement

POS-042 C3d-Directed Factor H Targeting Delivers Potent and Durable Complement Inhibition and Disease-Modifying Efficacy In Kidney and Skin Without Inhibiting Systemic Complement

Evaluation of renal injury and function biomarkers, including symmetric dimethylarginine (SDMA), in the rat passive Heymann nephritis (PHN) model

Symmetric dimethylarginine (SDMA) is a serum biomarker of excretory renal function which consistently correlates with glomerular filtration rate (GFR) across multiple species including rats, dogs, and humans. In human and veterinary clinical settings SDMA demonstrates enhanced sensitivity for detection of declining renal function as compared to other serum biomarkers, but application in preclinical study designs thus far has been limited. The purpose of this study was to determine the performance of serum SDMA in a rat passive Heyman nephritis model of glomerulopathy. In addition to SDMA other biomarkers of excretory renal function were measured including serum creatinine (sCr), blood urea nitrogen (BUN), and cystatin C along with creatinine clearance. Urinary renal biomarkers including microalbumin (μALB), clusterin (CLU), cystatin C, kidney injury marker-1 (KIM-1), neutrophil gelatinase-associated lipocalin (NGAL), and osteopontin (OPN) were also measured. PHN was induced using commercial sheep anti-Fx1A serum. Tissue, serum, and urine were collected from groups of control and anti-Fx1A-treated animals for biomarker evaluation, hematology, urinalysis, serum biochemistry, and histologic examination of kidney. Over the course of a 28-day study, concentrations of the urinary biomarkers μALB, CLU, cystatin C, NGAL, KIM-1 and the serum biomarker cystatin C increased significantly in anti-Fx1A-treated rats as compared to controls but no significant increase in serum SDMA, sCr, BUN, or creatinine clearance were noted in anti-Fx1A-treated rats. Given lack of direct GFR measurement or significant change in the renal function biomarkers sCr, BUN, and creatinine clearance, it is unclear if GFR differed significantly between control and anti-Fx1A-treated rats in this study, though urinary biomarkers and histopathologic findings supported renal injury in anti-Fx1A-treated rats over the time course investigated. This study is among the first to investigate serum SDMA in a rat model relevant to preclinical safety assessment and serves to inform future experimental designs and biomarker selection when evaluation of glomerular injury is of priority.

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.

MO042LNP023: A NOVEL ORAL COMPLEMENT ALTERNATIVE PATHWAY FACTOR B INHIBITOR FOR THE TREATMENT OF GLOMERULAR DISEASE

Abstract Background and Aims The alternative complement pathway (AP) provides the central amplification loop of complement activation and is critically involved in a number of diseases including C3 glomerulopathy (C3G), paroxysmal nocturnal haemoglobinuria (PNH) and atypical haemolytic uremic syndrome. There is also evidence for AP dysregulation in IgA and membranous nephropathies (IgAN, MN). Novartis has developed a highly selective oral low molecular weight inhibitor of complement Factor B, a key AP protease. LNP023 potently blocks AP activation in vitro and in vivo and is efficacious in passive Heymann nephritis in rats, suggesting an important role of the AP in classical-pathway mediated nephropathies. Method LNP023 was tested at increasing single or multiple ascending doses in a Phase I safety, tolerability, pharmacokinetic (PK) and pharmacodynamic study in healthy volunteers. Complement inhibition was measured by the ex vivo Wieslab assay and by quantifying fragment Bb level. Results LNP023 was well tolerated at all doses from 5 mg to 400 mg (single dose) and from 25 mg to 200 mg bid for 14 days (multiple dose). There were no deaths, SAEs or AEs leading to study drug discontinuation in LNP023-treated subjects. LNP023 dose-dependently inhibited the AP complement system. Based on the Wieslab assay, maximal inhibition of AP activity (80% or more) was observed 2 hours after a single dose of 10 mg or higher. The level of Bb decreased until 12 hours post-dose. Approximately 30% to 50% decrease in Bb, relative to baseline, was achieved for subjects receiving a single dose of LNP023 of 5mg or higher. For both assays, the duration of this inhibition was dependent upon the dose administered. Persistent inhibition was obtained in the multiple dose cohorts, the magnitude of inhibition being dose-dependent for the Wieslab assay but not for Bb. PK studies showed rapid drug absorption and no evidence of food effect. The plasma clearance was moderate and the terminal half-life was around 20h. There was an under-proportional dose-exposure relationship. Among other mechanisms, binding to highly expressed target (Factor B) is believed to affect clearance and thus exposure. Following a successful proof of concept Phase 2 study in PNH (data not shown), LNP023 is currently being investigated in three ongoing Phase 2 studies in IgAN (NCT03373461), C3G (NCT03832114) and MN (NCT04154787). The IgAN study has an adaptive seamless design; the first part has recently completed, 46 patients having received 3 months of one of three LNP023 doses or placebo. The study remains blinded; however, similar to the FIH study safety appears excellent, with only one treatment-emergent SAE (inhalation of chlorine gas) and two SAEs in the post-treatment follow-up period. No patient discontinued randomised treatment for any reason. There were no episodes of proven infection with encapsulated bacteria. The independent data safety monitoring committee have endorsed the study proceeding to the second part with the addition of a fourth LNP023 dose and recruitment is ongoing, completion being anticipated in mid-2020.

Urine exosomal ceruloplasmin: a potential early biomarker of underlying kidney disease.

Previously we found that kidney tissue and urinary exosomes from patients of diabetic kidney disease showed high levels of ceruloplasmin (CP). Because CP is an acute-phase protein of kidney origin, it could be an early marker of many other kidney diseases. To investigate this hypothesis, we first measured urine exosomal and kidney expression of CP in non-diabetic chronic kidney disease (CKD) patients (membranous nephropathy, focal segmental glomerulosclerosis, lupus nephritis and IgA nephropathy) followed by a longitudinal study in rat passive Heymann nephritis (PHN), a model of human membranous nephropathy. Urinary exosomes were isolated from urine of patients (and rats) by differential centrifugation. The exosomal extracts were used for measuring CP using ELISA. Kidney expression of CP was evaluated by immune-staining biopsy tissues. Similar techniques were applied in rat PHN model (produced by injection of anti-gp600 antiserum) to analyze urine exosomal and kidney CP. Urine exosomal CP levels were 10-20 times higher in CKD patients than in controls; consistent with this we found high immune-reactive CP localized in tubules and collecting ducts of biopsies of CKD patients. In the PHN model urinary exosomal CP level was significantly higher prior to the onset of proteinuria. Early rise of urine exosomal CP, which preceded proteinuria, correlated with high immunoreactive CP found in rat kidneys at this time. We propose that urine exosomal CP, observed to increase prior to proteinuria, makes it a potential urinary biomarker to diagnose early kidney disease.

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.

Production of heterologous IgG antibody against Heymann nephritis antigen by injections of immune complexes

Heterologous IgG antibody (ab) can be produced against Heymann nephritis (HN) antigen (ag) in rabbits by administering it in Freund's complete adjuvant. The developing abs reacted at high titre with rat kidney brush border (BB) regions of the renal proximal tubules in an indirect fluorescence ab test. A single IV injection of the heterologous ab into a susceptible strain of rat resulted in the localization of IgG ab to glomerular fixed ags, producing immune complex glomerular nephritis. The injected ab also reacted with the BB region of the renal proximal tubules. The aim of this experiment was to find out whether heterologous IgG ab against the HN ag can also be produced in recipient rabbits by injecting immune complexes (ICs) composed of a rat kidney tubular preparation [rat kidney fraction 3 (rKF3)] and donor rabbit-derived rabbit anti-rKF3 IgG ab. We found that anti-rKF3 IgG ab--against the BB region of the renal proximal tubules--could be induced in rabbits injected with ICs, and the resulting ab was able to initiate passive HN in rats. This was the first time a pathogenic IgG ab was produced against HN ag in rabbits without the use of adjuvant. Ab responses in recipient rabbits were achieved by ab information transfer. Recipient rabbits injected with the IC produced the same class of immunoglobulin with the same specificity against the target ag rKF3, as was present in the innoculum, namely rabbit anti-rKF3 IgG ab.

Role of apoptosis signal-regulating kinase 1 in complement-mediated glomerular epithelial cell injury

In the rat passive Heymann nephritis (PHN) model of membranous nephropathy, complement C5b-9 activates protein kinases in glomerular epithelial cells (GEC), and induces sublethal GEC injury and proteinuria. Complement induces production of reactive oxygen species (ROS) via the NAPDH oxidase, and stimulates phosphorylation of c-Jun N-terminal kinase (JNK) and p38 kinase in a ROS-dependent manner. In the present study, we demonstrate that apoptosis signal-regulating kinase 1 (ASK1) was activated in glomeruli of rats with PHN, and that incubation of GEC in culture with antibody and sublytic C5b-9 stimulated ASK1 activity. The latter was, in part, mediated via the NADPH oxidase and ROS. Sublytic complement induced JNK and p38 phosphorylation, which was amplified in GEC that stably overexpress ASK1, as compared with Neo (control) GEC. Complement-induced lysis was enhanced in GEC that overexpress ASK1, as compared with Neo, and was attenuated in GEC that overexpress a dominant negative ASK1 mutant. Inhibition of p38, but not JNK, attenuated complement lysis in GEC that overexpress ASK1, but not in Neo GEC. In Neo GEC, generation of ROS restricted complement-mediated GEC injury but the protective effect of ROS was lost when ASK1 was overexpressed. We propose that the level of ASK1 expression determines the functional effect of p38 activation, i.e. when ASK1 is overexpressed, p38 activation is amplified, and C5b-9 assembly leads to GEC injury via ASK1 and p38. The present study thus defines a novel role for ASK1 as a mediator of C5b-9-dependent cell injury.

Role of Rho-GTPases in complement-mediated glomerular epithelial cell injury

Visceral glomerular epithelial cells (GEC) are essential for maintenance of normal glomerular permselectivity. The actin cytoskeleton is a key determinant of GEC morphology and function. In the rat passive Heymann nephritis (PHN) model of membranous nephropathy, complement C5b-9 induces nonlytic GEC injury associated with morphological changes of GEC and proteinuria. The current study addresses the role of Rho family of small GTPases in complement-mediated GEC injury. When cultured rat GEC were stimulated with complement C5b-9 for 18 h, RhoA activity increased, whereas Rac1/Cdc42 activities decreased, compared with control cells. Similar changes in Rho-GTPase activities were observed in glomeruli from rats with PHN. The amount of active p190RhoGAP, a negative upstream regulator of RhoA, was decreased in complement-stimulated GEC, potentially contributing to increased RhoA activity. To address the functional effects of Rho-GTPases, GEC were transfected with constitutively active (CA) or dominant negative (DN) Rho-GTPase mutants. GEC transfected with CA-RhoA showed a smaller and round contour and prominent cortical F-actin. In contrast, GEC transfected with CA-Rac1 demonstrated morphological changes that resembled process formation. In addition, expression of CA-RhoA attenuated complement-mediated cytotoxicity, whereas cytotoxicity was augmented by DN-RhoA. Thus exposure of GEC to complement alters the balance of RhoA, Rac1, and Cdc42 activities. The activity of Rac1 may contribute to process formation, while activation of RhoA (e.g., in the setting of complement attack), with or without blunting of Rac1 activity, may have an opposite effect, i.e., contribute to foot process effacement. Activation of RhoA increases the resistance of GEC to complement-mediated injury.

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.

Effect of DP-1904, a thromboxane A 2 synthase inhibitor, on passive Heymann nephritis in rats

The antinephritic effect of DP-1904 [6-(1-imidazolylmethyl)-5,6,7,8-tetrahydronaphthalene-2-carboxylic acid hydrochloride], a thromboxane A 2 synthase inhibitor, was evaluated using an experimental model of membranous nephropathy, viz. accelerated passive Heymann nephritis in which the glomerular injury is mediated by immune complexes. DP-1904 markedly inhibited the development of glomerular alteration as well as the elevation of proteinuria and plasma creatinine. When the treatment was started from the 22nd day, at which time proteinuria is fully developed, DP-1904 showed beneficial effects on proteinuria and glomerular histopathological changes. DP-1904 apparently decreased the deposition of both rabbit immunoglobulin G and rat immunoglobulin G on glomerular basement membrane in nephritic rats. A single administration of DP-1904 restored the decreased renal tissue blood flow, inhibited glomerular thromboxane B 2 production and increased glomerular prostaglandin E 2 and 6-keto prostaglandin F 1α production in nephritic rats. These results suggest that DP-1904 may be an effective agent for the treatment of idiopathic membranous nephropathy and that the beneficial effect of this drug may be due to the elimination of glomerular immune deposits and to an increase in renal tissue blood flow related to amelioration of the abnormal metabolism of arachidonic acid.

Isolation of a 330-kDa glycoprotein from human kidney similar to the Heymann nephritis autoantigen (gp330).

Because of the similarities between Heymann nephritis of rat and human membranous glomerulonephritis, we searched for a protein in human kidney similar to that of the putative autoantigen of Heymann nephritis, gp330. Human gp330 was isolated by a purification scheme identical to that used for the isolation of rat gp330. The isolated human gp330 was compared with rat gp330 by peptide mapping and was found to be very similar in structure. Studies of immunologic reactivity of the rat antigen-induced Heymann nephritis nephritogenic autoantibody to human gp330 indicated that some of the rat antigen epitopes were shared with its human counterpart. The human gp330 was capable of eliciting on active Heymann nephritis response in rats when used as an immunogen. The nephritogenic autoantibody eluted from these rat kidney glomeruli showed equal binding to both rat and human antigens. Finally, a polyclonal antibody to human gp330 was able to induce passive Heymann nephritis in rats. These results demonstrate that there is a gp330 protein in human kidney that is very similar to the putative autoantigen of Heymann nephritis.

Studies on Antinephritic Effect of TJ-8014, a New Japanese Herbal Medicine (4): Effects on Accelerated Passive Heymann Nephritis in Rats

We investigated the antinephritic effect of TJ-8014, in comparison to dipyridamole, on accelerated passive Heymann nephritis in rats. TJ-8014 (4.0 g/ kg/day, p.o.) given from the heterologous phase (from the day of injection of the antiserum against Fx1A) markedly inhibited the urinary protein excretion and the elevation of plasma cholesterol levels as well as glomerular histopathological changes. When the treatment was started from the autologous phase (from the 22nd day) after proteinuria was fully developed, TJ-8014 also showed a beneficial effect. Dipyridamole (0.4 g/kg/day, p.o.) had no effect when the treatment was started either from the heterologous or autologous phase. TJ-8014 decreased glomerular rat IgG and rat C3 deposits, although it affected neither the plasma antibody titer against rabbit γ-globulin nor the plasma complement level. TJ-8014 markedly prevented the reduction of plasma and adrenal corticosterone level as well as the reduction of renal blood flow of rats with nephritis. These results suggest that TJ-8014 may be a useful drug against idiopathic membranous nephropathy and the beneficial effect of this drug may be caused by the elimination of glomerular immune deposits and C3 through the increase in renal blood flow related to the enhanced release of adrenal corticosterone.

Renal Blood Flow and Glomerular Filtration Rate during the Heterologous Phase in Passive Heymann Nephritis in Rats

When passive Heymann nephritis (PHN) is induced by infusion of antibodies (anti-Fx1A), an acute fall in renal blood flow (RBF) and glomerular filtration rate (GFR) has been reported. Activation of the complement cascade by the local antigen-antibody reaction might be involved in this reaction. We therefore studied RBF and GFR during acute infusion of anti-Fx1A and after 3 days when heterologous antibodies are no longer present in the circulation. Two groups of rats were infused with 2 mg anti-Fx1A antibodies into the left renal artery; RBF was measured by the microsphere method and GFR by 125I-Na-iothalamate clearance. In the first group, the measurements were made 40 min after the infusion, and in the second group after 3 days. A third group was studied 3 days after infusion of 1 mg anti-Fx1A. Animals infused with normal IgG were used as controls. Forty minutes after infusion of 2 mg anti-Fx1A, GFR in the left kidney was reduced from 1.16 +/- 0.07 to 0.41 +/- 0.16 ml/min/g in the controls (p less than 0.05). Three days after the infusion, GFR was 1.04 +/- 0.07, not significantly different from control. RBF was reduced to 3.97 +/- 1.11 ml/min/g after 40 min, compared to 7.53 +/- 0.73 in controls (p less than 0.05), and was normalized after 3 days. The effect of 1 and 2 mg anti-Fx1A antibodies was not significantly different after 3 days. Anti-Fx1A antibodies were detected in serum in the acute stage, but not after 3 days.(ABSTRACT TRUNCATED AT 250 WORDS)

Accelerated passive Heymann nephritis in rats as an experimental model for membranous glomerulonephritis and effects of azathioprine and prednisolone on the nephritis.

Progressive passive Heymann nephritis (group II) was induced in rats by i.v. injections of rabbit antiserum against an antigen from the brush border of the proximal tubules of rat kidney following immunization with rabbit gamma-globulin in Freund's complete adjuvant, and the process of the disease was compared with that of rats that received the antiserum alone (group I). The rats of group I showed proteinuria (30-50 mg/day) and plasma cholesterol content (80-90 mg/dl) slightly higher than the normal level from the 17th or the 22nd day after antiserum injection to the 90th day. The rats of group II revealed a heavy proteinuria (300-400 mg/day) and hypercholesterolemia (approx. 200 mg/dl) during the same period. In group II, there were the thickening of glomerular basement membrane (GBM) and spike formation. Moreover, granular deposits of rat IgG, rabbit IgG and rat C3 were observed along the GBM. These changes were weaker in group I. When given orally, daily, azathioprine (20 mg/kg) and prednisolone (1 and 3 mg/kg) showed a beneficial effect on the nephritis in group II. The group II model closely resembles human glomerulonephropathy and may be useful for studying the effect of medication on glomerulonephropathy.

Accelerated Passive Heymann Nephritis in Rats as an Experimental Model for Membranous Glomerulonephritis and Effects of Azathioprine and Prednisolone on the Nephritis

Progressive passive Heymann nephritis (group II) was induced in rats by i.v. injections of rabbit antiserum against an antigen from the brush border of the proximal tubules of rat kidney following immunization with rabbit γ-giobulin in Freund’s complete adjuvant, and the process of the disease was compared with that of rats that received the antiserum alone (group I), The rats of group I showed proteinuria (30-50 mg/day) and plasma cholesterol content (80-90 mg/dl) slightly higher than the normal level from the 17th or the 22nd day after antiserum injection to the 90th day. The rats of group II revealed a heavy proteinuria (300-400 mg/day) and hypercholesterolemia (approx. 200 mg/dl) during the same period. In group II, there were the thickening of glomerular basement membrane (GBM) and spike formation. Moreover, granular deposits of rat IgG, rabbit IgG and rat C3 were observed along the GBM. These changes were weaker in group I. When given orally, daily, azathioprine (20 mg/kg) and prednisolone (1 and 3 mg/kg) showed a beneficial effect on the nephritis in group II. The group II model closely resembles human glomerulonephropathy and may be useful for studying the effect of medication on glomerulonephropathy.

Evidence suggesting a role for hydroxyl radical in passive Heymann nephritis in rats.

We examined the effect of scavengers of reactive oxygen metabolites on proteinuria in the passive Heymann nephritis model of membranous nephropathy. Passive Heymann nephritis was induced by a single intravenous injection of anti-Fx1A IgG in a dose of 10 mg/100 g body weight. Superoxide dismutase, a scavenger of superoxide or catalase which destroys hydrogen peroxide, did not affect the proteinuria. In contrast, dimethylthiourea (DMTU, 500 mg/kg followed by 125 mg/kg ip twice a day), a scavenger of hydroxyl radical, markedly reduced the proteinuria (day 5: anti-Fx1A 53 +/- 13, n = 18; anti-Fx1A + DMTU, 21 +/- 6 mg/24 h, n = 15, P less than 0.001). Experiments with 125I-labeled anti-Fx1A antibody demonstrated that DMTU did not affect the amount of antibody deposited in the kidney. Semiquantitative estimation of IgG and complement deposition in the kidney showed no differences between the DMTU-treated and control rats. A second hydroxyl radical scavenger, sodium benzoate (150 mg/kg ip twice a day), also resulted in marked reduction in proteinuria (day 5: anti-Fx1A 56 +/- 7, n = 9; anti-Fx1A + benzoate, 14 +/- 4 mg/24 h, n = 8, P less than 0.01). Because of the participation of iron in biological systems to generate hydroxyl radical, we also examined the effect of deferoxamine (DFO, 35 mg/day), an iron chelator, on the anti-Fx1A-induced proteinuria. There was a significant reduction in proteinuria in rats treated concurrently with DFO (day 5: anti-Fx1A 67 +/- 13, n = 15; anti-Fx1A + DFO, 29 +/- 4 mg/24 h, n = 15, P less than 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)