Heymann Nephritis In Rats Research Articles

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.

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.

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.

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.

Optimizing the translational value of animal models of glomerulonephritis: insights from recent murine prototypes.

Animal models are indispensable for the study of glomerulonephritis, a group of diseases that destroy kidneys but for which specific therapies do not yet exist. Novel interventions are urgently needed, but their rational design requires suitable in vivo platforms to identify and test new candidates. Animal models can recreate the complex immunologic microenvironments that foster human autoimmunity and nephritis and provide access to tissue compartments not readily examined in patients. Study of rat Heymann nephritis identified fundamental disease mechanisms that ultimately revolutionized our understanding of human membranous nephropathy. Significant species differences in expression of a major target antigen, however, and lack of spontaneous autoimmunity in animals remain roadblocks to full exploitation of preclinical models in this disease. For several glomerulonephritides, humanized models have been developed to circumvent cross-species barriers and to study the effects of human genetic risk variants. Herein we review humanized mouse prototypes that provide fresh insight into mediators of IgA nephropathy and origins of antiglomerular basement membrane nephritis and Goodpasture's disease, as well as a means to test novel therapies for ANCA vasculitis. Additional and refined model systems are needed to mirror the full spectrum of human disease in a genetically diverse population, to facilitate development of patient-specific interventions, to determine the origin of nephritogenic autoimmunity, and to define the role of environmental exposures in disease initiation and relapse.

DNA vaccine encoding CD40 targeted to dendritic cells in situ prevents the development of Heymann nephritis in rats

The CD40-CD154 costimulatory pathway has been shown to be critical for both T- and B-cell activation in autoimmune disease. Here, we assessed the effects of blocking this pathway using CD40 DNA vaccine enhanced by dendritic cell targeting on the development of active Heymann nephritis, a rat model of human membranous nephropathy. DNA vaccination delivers plasmid DNA encoding the target antigen, either alone or in combination with enhancing elements, to induce both humoral and cellular immune responses. To determine whether CD40 DNA vaccine targeting the encoded CD40 directly to dendritic cells would improve the efficacy of the vaccination against self-protein CD40, we utilized a plasmid encoding a single-chain Fv antibody specific for the dendritic cell-restricted antigen-uptake receptor DEC205 (scDEC), the target gene CD40, and the adjuvant tetanus sequence p30. This vaccine plasmid was compared to a control plasmid without scDEC. Rats vaccinated with scDEC-CD40 had significantly less proteinuria and renal injury than did rats receiving scControl-CD40 and were protected from developing Heymann nephritis. Thus, CD40 DNA vaccination targeted to dendritic cells limits the development of Heymann nephritis.

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.

ヒト培養糸球体上皮細胞におけるmembrane attack complex inhibitory factor発現の意義

Membrane attack complex (MAC) inhibitory factor (MACIF) is a 20-kD membrane protein that inhibits MAC formation on homologous cells. Until recently, a functional role of MACIF had been demonstrated in erythrocytes. Therefore, we have focused on the fact that glomerular epithelial cells (GECs) are the primary target of MAC-mediated damage in rat Heymann's nephritis model of human membranous nephropathy. Using immunocytochemistry and western blotting we have shown that MACIF is expressed in cultured human GECs. Phosphatidyl-inositol (PI)-phospholipase C (PLC) reduced MACIF expression in these cells, suggesting that MACIF is a PI-linked membrane protein in GECs. In addition, we elucidated that MACIF protects GECs against complement-mediated lysis. These findings suggest that MACIF expressed on GECs plays an important role in the protection of GECs against complement mediated-cellular damage in vivo.

The effects of receptor associated protein C-terminal on nephrin in Heymann nephritis

Objective To explore the role of C-terminal antigenic determinant of Heymann nephritis receptor associated protein(RAP)in Heymann nephritis pathogenesis.Methods The full-length RAP and C-terminal polypeptide karyogamy expression vector Was constructed by PCR and molecular cloning to express fusion protein.Antiserum to the full-length RAP and C-terminal fusion protein Was prepared and orientated in kidney tissue by immunofluorescence.Two kinds of Heymann nephritis animal models were made with these antisera.We measured the 24 h urine protein quantitation,the expression and distribution of nephrin in kidney tissue and compared the data between the two groups.Results Recombinant protokaryon expression vector pGEX-dT-1-RAP1083/324 was successfully constructed and expressed the full-length RAP(70×103)and C-terminal fusion protein(40×103).We found that two kinds of prepared relevant antisera strongly expressod in the renal tubular epithelial cell by immunofluorescence.Heymann nephritis animal models were also successfully made.The 24 h urine protein quantitation in two model rats groups were (21.31±4.15)mg and(19.05±3.72)mg respectively.The 24 h urine protein quantitation in model groups were higher than that in the normal group(P<0.01).The expression of nephrin in RAP1083 induced model rats' glomeruhs Was lower than the RAP324 induced group.Conclusion Pathological antigenic determinant exists in the Heymann nephritis RAP C-terminal and Call induce rat Heymann nephritis models.Nephrin expression reduced in two kinds of Heymann nephritis.It indicated that the mechanism of RAP might be related to the low expression of nephrin in glomemhs. Key words: Heymann nephritis; Receptor associated protein; C-terminal; Podocyte; Nephrin

Glomerular T cells in Heymann nephritis.

Active Heymann nephritis (HN) is a rat model of human membranous nephropathy. The appearance of T cells within the glomeruli of HN rats suggests a role for these cells in the pathogenesis of the disease. The aims of this study were to investigate T cells infiltrating the glomerulus in HN in Lewis rats by polymerase chain reaction (PCR) of their Vbeta chains, CDR3 spectratyping and sequencing. HN was induced in Lewis rats by immunization with renal tubular antigen (Fx1A) in CFA. Kidneys were collected between 4 and 10 weeks. The glomeruli were separated, homogenized and RNA extracted. RT-PCR, CDR3 spectratyping and sequencing were used to further characterize the infiltrating T cells. Multiple Vbeta families showed restriction of their CDR3 spectratypes in each animal. Several TCR Vbeta families had identical-sized restricted spectratypes across several different animals. Four Vbeta families were sequenced. In three of those four families, the dominant clones showed identical sized CDR3 regions and a striking over-expression of Jbeta2.6. Further analysis of the CDR3 regions of the Jbeta2.6 clones showed a significant restriction of the amino acids at four of the six CDR3 positions. Glomerular T cells bearing similar CDR3 sequences, using Jbeta2.6 and expressing at least two, and possibly more, Vbeta genes are involved in the pathogenesis of HN.

A major pathogenic antigen of Heymann nephritis is present exclusively in the renal proximal tubule brush border--studies with a monoclonal antibody against pronase-digested tubular antigen.

We have isolated a nephritogenic 120-kD antigen from rat renal tubule brush border that induces rat Heymann nephritis. A MoAb that recognized this antigen reacted exclusively with the brush border on indirect immunofluorescence and immunoelectron microscopy. Rabbit antiserum against this antigen also reacted exclusively with the brush border. With the injection of this antiserum, rabbit IgG became detectable along the glomerular basement membrane (GBM) after 3 days. Our 120-kD antigen was shown to have a close relationship with gp330 based on the following: (i) this antigen can induce active Heymann nephritis as gp330; (ii) our MoAb reacted with the immune deposits of nephritic kidneys induced not only by the 120-kD antigen but also by gp330, and conversely, rabbit antiserum against gp330 reacted with those induced by the 120-kD antigen as well as gp330; and (iii) by immunoblotting, polyclonal antibodies against the 120-kD antigen reacted with gp330 and polyclonal antibodies against gp330 reacted with the 120-kD antigen. These observations indicate that antigen present exclusively in the brush border can induce active Heymann nephritis, and the common antigenic determinants shared by brush border and the coated pits of glomerular epithelium may not be a prerequisite to induce nephritis. A more precise relationship between the 120-kD antigen and reported C14 fusion protein or 40-kD alpha 2MRAP remains to be established.

Reduced Incidence of Slowly Progressive Heymann Nephritis in Rats Immunized With a Modified Vaccination Technique

A slowly progressive Heymann nephritis (SPHN) was induced in three groups of rats by weekly injections of a chemically modified renal tubular antigen in an aqueous medium. A control group of rats received the chemically unmodified version of the antigen in an aqueous solution. One group of SPHN rats were pre- and post-treated with weekly injections of IC made up of rKF3 and rarKF3 IgM antibody at antigen excess (MIC) (immune complexes [ICs] containing sonicated ultracentrifuged [u/c] rat kidney fraction 3 [rKF3] antigen and IgM antibodies specific against the antigen, at slight antigen excess). One group of SPHN rats were post-treated with MIC 3 weeks after the induction of the disease and one group of SPHN animals received no treatment. The control group of rats received pre- and post-treatment with sonicated u/c rKF3. The incidence of immune-complex glomerulonephritis (ICGN) in the untreated SPHN rats was 87%, in the pre- and post-treated animals 13%, and in the post-treated-only rats 20%. Rats receiving sonicated ultracentrifuged rKF3 antigen did not develop ICGN. The present experiment demonstrates that the development of SPHN can be not only prevented but also effectively terminated by our newly developed modified vaccination technique.

Graded Effects of Proteinuria on HDL Structure in Nephrotic Rats

Nephrotic syndrome is characterized by increased triglycerides resulting from decreased clearance of VLDL and chylomicrons. These triglyceride-rich lipoproteins are structurally altered by interaction with HDL derived from animals with proteinuria and not as a consequence of hypoalbuminemia. HDL isolated from rats with massive proteinuria is depleted in apolipoprotein E (apoE). It is unknown at what threshold of urinary albumin loss HDL structure is altered, and it is unknown what effects proteinuria has on apolipoproteins other than apoE. Two models of albuminuria were used in Sprague-Dawley rats: Adriamycin and passive Heymann nephritis (HN). The adriamycin group was divided into minimal albumin excretion (MAE) and intermediate albumin excretion (MAE, 1 to 40; intermediate albumin excretion, 60 to 210 mg/d per 100 g body wt). Urinary albumin excretion exceeded 300 mg/d per 100 g body wt in the HN rats. HDL apolipoprotein composition was analyzed with SDS-PAGE densitometry and liquid chromatography-time of flight mass spectrometer mass spectrometry. HDL apoA-IV content relative to apoA-I was reduced at all levels of albuminuria (P < 0.0001). ApoE was not reduced in MAE but was significantly reduced in IAE (72%; P < 0.001). By contrast, apoA-II and apoC-III were each significantly increased with increasing UAE. ApoA-IV and apoE were decreased to approximately 10% of control in HDL isolated from rats with HN, whereas apoA-II, apoC-II, and apoC-III were each significantly increased relative to apoA-I. HDL is structurally altered by levels of albuminuria that are insufficient to change serum albumin levels and is progressively altered as albuminuria increases.

Fucoidan inhibits the development of proteinuria in active Heymann nephritis.

Fucoidan, the sulphated polysaccharide extracted from brown seaweed, has various biological activities. The effect of fucoidan on the formation of proteinuria and renal functions in active Heymann nephritis was investigated in this study. Active Heymann nephritis was induced by administering brush border protein of rat proximal uriniferous tubules (FX1A). Fucoidan was administered by oral intubation to Heymann nephritis rats at three doses (50, 100 and 200 mg/kg) once daily for 4 weeks. The elevated urinary protein excretion and plasma creatinine due to the induction of Heymann nephritis were significantly reduced by fucoidan at doses of 100 and 200 mg/kg. The results indicated that fucoidan has a renoprotective effect on active Heymann nephritis and is a promising therapeutic agent for nephritis.

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.

Differential Expression of D-Type Cyclins in Podocytes in Vitro and in Vivo

The proliferative response of podocytes to injury determines the histological phenotype. Moreover, an apparent lack of podocyte proliferation may underlie the development of glomerulosclerosis. Podocyte proliferation is closely linked with its state of differentiation. However, the mechanisms regulating these processes are not fully elucidated. Because D-type cyclins have been shown to be important in the regulation of proliferation and differentiation, we examined their expression in podocytes in vitro and in vivo. The glomerular expression of cyclins D1 and D3 was examined in vitro in cultured immortalized podocytes by immunostaining and Western blot analysis, and in embryonic mice and rats, the passive Heymann nephritis model of experimental membranous nephropathy in rats, and human immunodeficiency virus (HIV)-transgenic mice. Kidneys from cyclin D1 knockout mice were also examined. Cyclin D1 was abundant in cultured proliferating podocytes, but not in quiescent differentiated podocytes. In contrast, cyclin D3 was abundant in differentiated, but not proliferating podocytes. Cyclin D1 was expressed in embryonic mouse and rat glomeruli during the S- and comma-shaped stages, and was absent in podocytes at the capillary loop stage and in mature rodent glomeruli. Cyclin D1 protein increased after injury in passive Heymann nephritis rats and in HIV-transgenic mice. Cyclin D3 was constitutively and specifically expressed in podocytes in normal rodent glomeruli, and decreases during dedifferentiation and proliferation in HIV-transgenic mice. Kidneys from cyclin D1-/- mice were normal with the podocytes expressing specific differentiation markers. Cyclin D1 is not necessary for the terminal differentiation of podocytes, and expression coincides with cell-cycle entry. In contrast, cyclin D3 expression coincides with podocyte differentiation and quiescence.

Endothelial chylomicron binding is altered by interaction with high-density lipoprotein in Heymann's nephritis

Very-low-density lipoprotein (VLDL) catabolism is impaired in the nephrotic syndrome, partly as a result of structural changes that impair endothelial binding in the presence of lipoprotein lipase. Previous results suggested that postsynthetic modification of VLDL by high-density lipoprotein (HDL) in nephrotic syndrome rats causes their failure to bind endothelia normally. It is unknown (1) whether the structure of secreted lipoproteins is normal before exposure to nephrotic syndrome serum and (2) whether the same structural or functional defects are imparted to chylomicrons (CMs) through their interaction with HDL from nephrotic syndrome rats. CMs were isolated from thoracic duct lymph from rats with passive Heymann's nephritis (HN) and normal controls. CMs from control rats were incubated with HDL from either HN or control rats and reisolated, and apolipoprotein E (apo E) content and endothelial binding were determined. We found that CMs secreted by HN and control rats had similar apo E/B-48 ratios. HDL from HN rats had significantly lower apo E/A-I ratios than controls. Incubation of nascent control CMs with control HDL resulted in a 4-fold increase in CM apo E content, but binding was unaffected. Incubation with HDL from HN resulted in only a 50% increase in CM apo E content but reduced binding of these treated CMs by 50% compared either with nascent control CMs or with CMs incubated with control HDL. HDL from rats with HN alters CM binding to lipoprotein lipase by a mechanism that does not involve reducing the content of apo E already present on CMs at the time of secretion. © 2001 by the National Kidney Foundation, Inc.

Ultrastructure of Glomerular Basement Membrane in Active Heymann Nephritis Rats Revealed by Tissue-Negative Staining Method

Recently, we have developed a tissue-negative staining method, and successfully visualized fine meshwork structure of the glomerular basement membrane (GBM). To clarify the mechanism of proteinuria in active Heymann nephritis, we performed tissue-negative staining and investigated the ultrastructural alterations of the GBM. Active Heymann nephritis, the animal model of human membranous nephropathy, was induced in Lewis rats by the injection of proximal tubular brush border antigen, i.e. Fx1A. Urinary protein excretion was measured and histological studies were performed over 15 weeks following the Fx1A injection. Proteinuria developed at 10 weeks after injection (38.2 ± 7.4 mg/day) and progressively increased (160.2 ± 20.6 mg/day at 15 weeks). Capillary fine deposits of IgG and C3 were seen by immunofluorescence, and subepithelial electron dense deposits (EDD) by transmission electron microscopy (TEM). Using the tissue-negative staining method, regular meshwork structure consisted of fine fibrils and pores (2.5 ± 0.7 nm in short dimension) was observed in the GBM of control rats. At 10 and 15 weeks after injection, the GBM, directly facing the endothelial side of EDD, contained enlarged pores and nephrotic tunnels. Mean values of the short dimension of enlarged pores were 2.9 ± 0.5 nm at 10 weeks and 3.1 ± 0.4 nm at 15 weeks, which were significantly larger than that of control rats (p < 0.01). The rest area of the GBM, including newly produced GBM covering the epithelial side of EDD, had no significant difference in size of the pores from control GBM and no tunnels. Although there was no significant difference in the size of enlarged pores between 10 and 15 weeks, the percentage area of GBM with impaired size barrier increased at 15 weeks (51.4 ± 8.1%) compared with 10 weeks (24.0 ± 8.3%) and related to severity of proteinuria. The density of the tunnels also increased at 15 weeks. In conclusion, immune deposits may affect the GBM biosynthesis and induce the defect of size barrier of the GBM, which is responsible for proteinuria in active Heymann nephritis.

Anti-CD69 autoantibodies cross-react with low density lipoprotein receptor-related protein 2 in systemic autoimmune diseases.

We investigated whether autoantibodies to CD69, one of the earliest markers of lymphocyte activation, exist in the sera of patients with systemic autoimmune disease. Serum samples were obtained from patients with rheumatoid arthritis (RA), systemic lupus erythematosus, and Behcet's disease, and were tested for the presence of anti-CD69 autoantibodies by ELISA and Western blotting using rCD69 fusion proteins. IgG-type autoantibodies to CD69 were detected in the sera of 38.3% of the RA patients, 14.5% of the systemic lupus erythematosus patients, and 4.0% of the patients with Behcet's disease. Among those with RA, the anti-CD69 autoantibody-positive patients had a higher serum level of rheumatoid factors and a more accelerated erythrocyte sedimentation rate than the anti-CD69 autoantibody-negative patients. Further, the predominant epitope on the CD69 molecule to which most of the anti-CD69 autoantibody-positive serum samples exclusively reacted, was mapped at the C terminus of CD69. Of interest, this epitope is homologous to a stretch of amino acids in the protein sequence of low-density lipoprotein receptor-related protein 2 (LRP2), which is a receptor for multiple ligands including beta-very low density lipoprotein and is also an autoantigen responsible for Heymann nephritis in rats. The anti-CD69 autoantibody cross-reacted to LRP2 through the homologous amino acid sequence. To our knowledge, this is the first evidence of the existence of anti-CD69 autoantibodies. This autoantibody may modulate the function of CD69- and LRP2-expressing cells.

Structural, biochemical and signaling properties of the low-density lipoprotein receptor gene family.

The low-density lipoprotein (LDL) receptor (LDL-R) family members (LDL-R, LRP, megalin, VLDL-R, apoER2) bind several extra-cellular structurally dissimilar ligands and internalize them for degradation by lysosomes by a process called receptor-mediated endocytosis. The receptor-mediated endocytosis involves immobilization of circulating ligands onto the cell-surface followed by their internalization and degradation. All the receptors can perform both of these functions. However, in the majority of the cases, other proteins immobilize ligands on to the cell-surface and subsequent internalization is mediated by these receptors. The LDL-R and LRP play important roles in plasma cholesterol homeostasis and fetal development. Megalin is an antigenic determinant for Heymann nephritis in rats and may be important for re-absorption of various molecules by the kidney. VLDL-R homologue in chicken is essential for female fertility. This receptor and apoER2 are critical for the proper development of the brain in mice. The members of the LDL-R gene family contain several complement-type and EGF precursor-like repeats, and single transmembrane and cytoplasmic domain. Cysteine-rich complement-type repeats containing DxSDE sequences at the C-termini constitute ligand-binding domains. In contrast to the ligand binding domains, receptor-binding domains in different ligands do not share sequence homology. It has been proposed that positive electrostatic surface potentials, not the primary sequences, in different ligands constitute receptor-binding domains. The EGF precursor homology repeats in receptors are important for the dissociation of ligands from receptors in endocytic vesicles. The transmembrane domain is necessary for anchoring to membranes and the cytoplasmic domain is required for their targeting to coated pits and subsequent internalization. The receptor-mediated endocytosis involves recognition of the NPXY motif by clathrin. Recently, this motif has also been implicated in signaling pathways that are crucial in brain development. The signaling process involves the recognition of the NPXY motif by Disabled-1 protein and possibly other proteins involved in intracellular signaling cascade. The LDL-R gene family has provided important insights into the mechanisms of ligand catabolism and may serve as new targets for the treatment of different cardiovascular and neuronal disorders. In the future, their role in signaling may provide novel insights into brain development and neuronal layering.