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Several novel antigens have recently been characterized in membranous nephropathy (MN), but those involved in the rare cases of MN associated with inflammatory neuropathies (IN) remain elusive. Although several antibody specificities were identified in the serum, there is no evidence so far for their deposition in glomeruli. We report the case of a 73 y/o woman who was referred because of subacute onset of proximal asymmetric lower limb weakness together with ataxic gait. She was diagnosed with IN. Blood tests showed an eGFR of 73 ml/min/1.73m2, hypoalbuminemia (2.89 g/dl) and proteinuria (3.6 g/24h). Autoantibodies (ANA, ENA, DNA, LAC, anti-cardiolipin, ANCA) were undetectable. Serum Ig and complement levels were normal. A kidney biopsy with electron microscopy examination showed a classical picture of MN. Anti-PLA2R antibody was negative in the serum and PLA2R and THSD7A antigens were not detected in kidney tissue. Anti-contactin1 (CNTN1) antibody was found by ELISA at 1:100 serum dilution and shown to be mostly of IgG4 subclass by Western blot. CNTN1 antigen was co-localized with IgG4 within immune deposits by confocal microscopy. This observation suggests a pathophysiological link between IN and MN. CNTN1 should be considered as a potential candidate antigen involved in MN and tested in PLA2R negative forms associated with IN.

The discovery of phospholipase A2 receptor (PLA2R) antibody by Beck and associates ([1][1]) has been transformative for the diagnosis of membranous nephropathy and for the monitoring of patients affected with the disease. Less than 2 years after this discovery, serologic tests were developed: first

Membranous nephropathy can be associated with various etiologies and antigens. In this issue of Kidney International, Le Quintrec et al. described contactin-1 cell adhesion molecule as a novel target antigen shared by the peripheral nerve and podocyte in patients with neurological disease and membranous nephropathy.

Membranous nephropathy results from subepithelial antigen-antibody complex deposition along the glomerular basement membrane. Although PLA2R, THSD7A, and NELL-1 account for a majority (about 80%) of the target antigens, the target antigen in the remaining cases is not known. Using laser microdissection of PLA2R-negative glomeruli of patients with membranous nephropathy followed by mass spectrometry we identified a unique protein, Semaphorin 3B, in three cases. Mass spectrometry failed to detect Semaphorin-3B in 23 PLA2R-associated cases of membranous nephropathy and 88 controls. Semaphorin 3B in all three cases was localized to granular deposits along the glomerular basement membrane by immunohistochemistry. Next, an additional eight cases of Semaphorin 3B-associated membranous nephropathy were identified in three validation cohorts by immunofluorescence microscopy. In four of 11 cases, kidney biopsy also showed tubular basement membrane deposits of IgG on frozen sections. Confocal microscopy showed that both IgG and Semaphorin 3B co-localized to the glomerular basement membrane. Western blot analysis of five available sera showed reactivity to reduced Semaphorin 3B in four of four patients with active disease and no reactivity in one patient in clinical remission; there was also no reactivity in control sera. Eight of the 11 cases of Semaphorin 3B-associated membranous nephropathy were pediatric cases. Furthermore, in five cases, the disease started at or below the age of two. Thus, Semaphorin 3B-associated membranous nephropathy appears to be a distinct type of disease; more likely to be present in pediatric patients.

Introduction: Membranous nephropathy (MN) is the most common cause of nephrotic syndrome in Caucasian adults. Phospholipase A2 receptor (PLA2R)– and exostosin 1 (EXT1)/exostosin 2 (EXT2)–associated MN represent the most common primary and secondary forms of MN. The complement profile using a proteomics approach has not been studied in these 2 common forms of MN. Methods: We used laser microdissection and mass spectrometry (MS/MS) to dissect glomeruli and identify glomerular complement proteins in PLA2R-associated (n = 7), EXT1/EXT2-associated MN (n = 21), and 11 control cases (time 0 transplant biopsies). Results: MS/MS identified high total spectral counts for PLA2R and EXT1/EXT2 in corresponding cases of PLA2R- and EXT1/EXT2-positive MN. Both PLA2R- and EXT1/EXT2-associated MN had high spectral counts of complement proteins C3, C4, C5, C6, C7, C8, and C9. Complement protein C1 was present in low spectral counts in EXT1/EXT2-associated MN. Regulators of complement activation that were detected in MN included higher spectral counts of FH, FHR-1, FHR-5, clusterin, vitronectin and lower spectral counts of FHR-3, FHR-4, and CD59. Low spectral counts of FB and properdin, key components of the alternative pathway, also were detected. IgG4 and IgG1 were the most abundant IgG subclasses in PLA2R- and EXT1/EXT2-associated MN. Lower spectral counts for C3, C4, and C5 were detected in control cases when compared with MN. Conclusion: Significant complement activation is present in MN as evidenced by large spectral counts of complement proteins from C3- and C4-based pathways, including regulatory proteins of complement pathways. These data suggest that anticomplement drugs may be effective in treatment for MN. Keywords: complement, laser microdissection, mass spectrometry, membranous nephropathy

Background: Serum and tissue-based tests using phospholipase A2 receptor 1 (PLA2R) and thrombospondin type-1 domain containing 7A (THSD7A) are established immune biomarkers for the diagnosis of primary membranous nephropathy (PMN). This study assessed the diagnostic performance of these biomarkers in the diagnosis of PMN in South Africans. Methods This was a cross-sectional analysis from a single centre in Cape Town, South Africa. Relevant biodata was collected from all patients. Histology, including slides for PLA2R and THSD7A were processed and assessed by typical microscopic and immunohistochemical features. Biopsy tissues of patients with membranous lupus nephritis (LN-V) and diabetic nephropathy (DN) were used as controls. The diagnostic accuracy for diagnosis of PMN using positive PLA2R and THSD7A were evaluated. Results Of the 88 patients included, 41 had PMN with a mean age of 44.5 ± 17.5 years and 61.0% were female. Histologically, PLA2R and THSD7A were only positive in the PMN group (51.2% and 4.9%, respectively) but negative in both control groups. The sensitivity of PLA2R and THSD7A for identifying PMN was 51.2% and 4.9%, respectively. The sensitivity of both tests together was 53.7% while the specificity and positive predictive values (PPV) for any of the tests (alone or in combination) was 100%. There was no difference in the sensitivity and specificity when using PLA2R alone compared to combining the two tests (p=0.32). Conclusion Glomerular staining of PLA2R and THSD7A could have potential diagnostic values in South Africans. This has implications on how immunotherapies can be initiated and used in these settings.

Transcription factor 21 (TCF21) is one of the essential transcription factors in kidney development. To elucidate its influence on glomerular disease, we have investigated TCF21 expression in human and rat kidney tissue, and its urinary concentration. Immunohistological analysis suggested the highest TCF21 expression in nephrotic syndrome along with the urinary protein level. Urinary TCF21 concentration in human showed a positive correlation with its podocyte expression level. In nephrotic rat models, TCF21 expression in podocytes increased along with the severity of nephrotic syndrome. Next, in vitro experiments using Tcf21-expressing murine podocyte cell line, we could observe some Tcf21-dependent effects, related with actin cytoskeleton dysregulation and apoptosis. Our study illustrated TCF21 expression changes in vivo and its in vitro-functional significance injured podocytes.

To understand the genetics of steroid-sensitive nephrotic syndrome (SSNS), we conducted a genome-wide association study in 987 childhood SSNS patients and 3,206 healthy controls with Japanese ancestry. Beyond known associations in the HLA-DR/DQ region, common variants in NPHS1-KIRREL2 (rs56117924, P=4.94E-20, odds ratio (OR) =1.90) and TNFSF15 (rs6478109, P=2.54E-8, OR=0.72) regions achieved genome-wide significance and were replicated in Korean, South Asian and African populations. Trans-ethnic meta-analyses including Japanese, Korean, South Asian, African, European, Hispanic and Maghrebian populations confirmed the significant associations of variants in NPHS1-KIRREL2 (P(meta)=6.71E-28, OR=1.88) and TNFSF15 (P(meta)=5.40E-11, OR=1.33) loci. Analysis of the NPHS1 risk alleles with glomerular NPHS1 mRNA expression from the same person revealed allele specific expression with significantly lower expression of the transcript derived from the risk haplotype (Wilcox test p=9.3E-4). Because rare pathogenic variants in NPHS1 cause congenital nephrotic syndrome of the Finnish type (CNSF), the present study provides further evidence that variation along the allele frequency spectrum in the same gene can cause or contribute to both a rare monogenic disease (CNSF) and a more complex, polygenic disease (SSNS).

International audience

Membranous nephropathy is a noninflammatory autoimmune disease of the kidney glomerulus, characterized by the formation of immune deposits, complement-mediated proteinuria, and risk of renal failure. Considerable advances in understanding the molecular pathogenesis have occurred with the identification of several antigens [neutral endopeptidase, phospholipase A2 receptor (PLA2R), thrombospondin domain-containing 7A (THSD7A)] in cases arising from the neonatal period to adulthood and the characterization of antibody-binding domains (that is, epitopes). Immunization against PLA2R occurs in 70% to 80% of adult cases. The development of highly specific and sensitive assays of circulating antibodies has induced a paradigm shift in diagnosis and treatment monitoring. In addition, several interacting loci in HLA-DQ, HLA-DR, and PLA2R1, as well as classical human leukocyte antigen (HLA)-D alleles have been identified as being risk factors, depending on a patient's ethnicity. Additionally, mechanisms of antibody pathogenicity and pathways of complement activation are now better understood. Further research is mandatory for designing new therapeutic strategies, including the identifying triggering events, the molecular bases of remission and progression, and the T cell epitopes involved.

In this edition of Kidney International, we publish 2 series of patients who had undergone kidney transplantation during the coronavirus disease 2019 (COVID-19) pandemic that have taken different approaches to transplant immunosuppressive therapy and antiviral treatment in the early days of the developing pandemic in the United Kingdom and Italy. The first series of 7 cases of COVID-19 infection in patients who had undergone kidney transplantation comes from St. George's, St. Helier’s, and King’s College Hospitals in south London, United Kingdom,1 and the second series of 20 cases originates from Spedali Civili University Hospital in Brescia, Italy. [...]

International audience; Primary membranous nephropathy (PMN) is characterized by antibodies to the podocyte, but little is known about B- and T-cell populations and their response to rituximab is controversial. To help resolve this we compared 33 lymphocyte subpopulations and 27 cytokines/chemokines in 25 patients with severe PMN and 27 age-matched healthy individuals. At baseline, patients had a significantly increased percentage of naive B-cells with significantly decreased switched and non-switched memory B-cells. There was a significantly decreased percentage of natural killer (NK) cells with an increase in the CD56brightCD16-/lo NK subset. There were a significantly decreased percentage of regulatory T cells, together with an increased plasma concentration of TNF-alpha, IL-5 and IL-2RA. We then investigated 16 patients at eight days and three and six months after treatment with rituximab added to supportive therapy compared to nine patients with supportive therapy alone. After rituximab, B-cell recovery was still incomplete at six months, with persistent alterations of B-cell subsets, a significant increase of both T-regulatory (Treg) cells and NK cells, and a significant decrease of both the CD56brightCD16-/lo NK subset and TNF-alpha levels. The patients who clinically responded to rituximab had a significantly lower percentage of Tregs at baseline compared to non-responders and a significantly increased percentage at day eight. Tregs remained unchanged in non-responders and in patients treated with supportive therapy alone. Thus, evaluation of Tregs might be useful for predicting early response to rituximab.

Collagen IV is a major component of basement membranes (BMs). The α1(IV) chain, encoded by the COL4A1 gene, is expressed ubiquitously and associates with the α2(IV) chain to form the α1α1α2(IV) heterotrimer. Several COL4A1 mutations affecting a conformational domain containing integrin-binding sites are responsible for the systemic syndrome of hereditary angiopathy, nephropathy, aneurysms, and cramps (HANAC). To analyze the pathophysiology of HANAC, Col4a1 mutant mice bearing the p.Gly498Val mutation were generated. Analysis of the skeletal muscles of Col4a1G498V mutant animals showed morphologic characteristics of a muscular dystrophy phenotype with myofiber atrophy, centronucleation, focal inflammatory infiltrates, and fibrosis. Abnormal ultrastructural aspects of muscle BMs was associated with reduced extracellular secretion of the mutant α1α1α2(IV) trimer. In addition to muscular dystrophic features, endothelial cell defects of the muscle capillaries were observed, with intracytoplasmic accumulation of the mutant α1α1α2(IV) molecules, endoplasmic reticulum cisternae dilation, and up-regulation of endoplasmic reticulum stress markers. Induction of the unfolded protein response in Col4a1 mutant muscle tissue resulted in an excess of apoptosis in endothelial cells. HANAC mutant animals also presented with a muscular functional impairment and increased serum creatine kinase levels reflecting altered muscle fiber sarcolemma. This extensive description of the muscular phenotype of the Col4a1 HANAC murine model suggests a potential contribution of primary endothelial cell defects, together with muscle BM alterations, to the development of COL4A1-related myopathy.

The podocyte is a key component of the glomerular filtration barrier. Podocyte dysfunction is central to the underlying pathophysiology of many common glomerular diseases, including diabetic nephropathy, glomerulonephritis and genetic forms of nephrotic syndrome. Collectively, these conditions affect millions of people worldwide, and account for the majority of kidney diseases requiring dialysis and transplantation. The 12th International Podocyte Conference was held in Montreal, Canada from May 30 to June 2, 2018. The primary aim of this conference was to bring together nephrologists, clinician scientists, basic scientists and their trainees from all over the world to present their research and to establish networks with the common goal of developing new therapies for glomerular diseases based on the latest advances in podocyte biology. This review briefly highlights recent advances made in understanding podocyte structure and metabolism, experimental systems in which to study podocytes and glomerular disease, disease mediators, genetic and immune origins of glomerulopathies, and the development of novel therapeutic agents to protect podocyte and glomerular injury.

International audience; Membranous nephropathy is characterized by deposition of immune complexes along the glomerular basement membrane. PLA2R and THSD7A are target antigens in 70% and 1-5% of primary membranous nephropathy cases, respectively. In the remaining cases, the target antigen is unknown. Here, laser microdissection of glomeruli followed by mass spectrometry was used to identify novel antigen(s) in PLA2R-negative membranous nephropathy. An initial pilot mass spectrometry study in 35 cases of PLA2R-negative membranous nephropathy showed high spectral counts for neural tissue encoding protein with EGF-like repeats, NELL-1, in six cases. Mass spectrometry failed to detect NELL-1 in 23 PLA2R-associated membranous nephropathy and 88 controls. NELL-1 was localized by immunohistochemistry, which showed bright granular glomerular basement membrane staining for NELL-1 in all six cases. Next, an additional 23 NELL-1 positive cases of membranous nephropathy were identified by immunohistochemistry in a discovery cohort of 91 PLA2R-negative membranous nephropathy cases, 14 were confirmed by mass spectrometry. Thus, 29 of 126 PLA2R-negative cases were positive for NELL-1. PLA2R-associated membranous nephropathy and controls stained negative for NELL-1. We then identified five NELL-1 positive cases of membranous nephropathy out of 84 PLA2R and THSD7A-negative cases in two validation cohorts from France and Belgium. By confocal microscopy, both IgG and NELL-1 co-localized to the glomerular basement membrane. Western blot analysis showed reactivity to NELL-1 in five available sera, but no reactivity in control sera. Clinical and biopsy findings of NELL-1 positive membranous nephropathy showed features of primary membranous nephropathy. Thus, a subset of membranous nephropathy is associated with accumulation and co-localization of NELL-1 and IgG along the glomerular basement membrane, and with anti-NELL-1 antibodies in the serum. Hence, NELL-1 defines a distinct type of primary membranous nephropathy.

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Hepatitis B virus (HVB) infection is a worldwide epidemic that, in addition to the liver, causes damage in other organs. HBV-membranous nephropathy (HBV-MN) is the most common renal manifestation in patients with HBV infection,1, 2, 3 possibly linked to subepithelial deposits of viral immune complexes.4, 5 HBV RNA and DNA were detected in glomerular and tubular cells, although a pathogenetic role for these viral nucleic acids remains to be confirmed.6 The effects of vaccination programs on the decline of HBV-associated MN, particularly in Asian and African children,7, 8 further support a possible relationship between HBV infection and MN.9 Identification in 2009 of PLA2R as the major antigen associated with idiopathic membranous nephropathy provided the first diagnostic biomarker.10 The 2 available assays (immunofluorescence test assay [IFTA] and enzyme-linked immunosorbent assay) for the detection of circulating anti-PLA2R antibodies (PLA2R-Abs) have shown that PLA2R-Abs are a specific and sensitive biomarker of MN, being detected in 70% to 80% of patients with primary MN worldwide in early serum samples, and consistently absent in healthy individuals and in patients with other nephropathies or autoimmune diseases.11 Because anti-PLAR antibody levels fluctuate and can rapidly decrease under immunosuppressive treatment, detection of PLA2R antigen in subepithelial immune deposits of patients with MN is even more sensitive than serology.12, 13 Although Hoxha et al. suggested that the diagnosis between primary MN and secondary MN could rely on the presence or absence of PLA2R antigen in the immune deposits,14 other investigators showed that the antigen could be detected in cases of active sarcoidosis,15, 16 hepatitis C virus,17 and HBV infection–associated MN.13, 18 In the Chinese population, 25 of 39 patients (64%) with HBV-MN had deposited subepithelial PLA2R antigen, and all tested sera contained anti-PLA2R antibody.18 These findings suggest that HBV infection could be the trigger of autoimmunity anti-PLA2R. Whatever the mechanism of autoimmunity, we are facing the conundrum of antiviral versus immunosuppressive therapy with the risk of aggravating the viral infection. Most data have been obtained in small-scale case series published before the availability of anti-PLA2R antibody assays, and a standard of care has not yet been established.19, 20 Here, we report the first 2 cases in which rituximab was used after controlling viral replication.

Familial steroid-sensitive nephrotic syndrome (SSNS) is a rare condition. The disease pathophysiology remains elusive. However, bi-allelic mutations in the EMP2 gene were identified, and specific variations in HLA-DQA1 were linked to a high risk of developing the disease. Clinical data were analyzed in 59 SSNS families. EMP2 gene was sequenced in families with a potential autosomal recessive (AR) inheritance. Exome sequencing was performed in a subset of 13 families with potential AR inheritance. Two variations in HLA-DQA1 were genotyped in the whole cohort. Transmission was compatible with an AR (n = 33) or autosomal dominant (AD, n = 26) inheritance, assuming that familial SSNS is a monogenic trait. Clinical features did not differ between AR and AD groups. All patients, including primary (n = 7) and secondary steroid resistant nephrotic syndrone (SRNS), (n = 13) were sensitive to additional immunosuppressive therapy. Both HLA-DQA1 variations were found to be highly linked to the disease (OR = 4.34 and OR = 4.89; p

G.L. and P.R. contributed equally to this work.; International audience; The phospholipase A2 receptor (PLA2R1) is the major autoantigen in primary membranous nephropathy. Several PLA2R1 epitopes have been characterized, and a retrospective study identified PLA2R1 epitope spreading as a potential indicator of poor prognosis. Here, we analyzed the predictive value of anti-PLA2R1 antibody (PLA2R1-Ab) titers and epitope spreading in a prospective cohort of 58 patients positive for PLA2R1-Ab randomly allocated to rituximab (n=29) or antiproteinuric therapy alone (n=29). At baseline, the epitope profile (CysR, CysRC1, CysRC7, or CysRC1C7) did not correlate with age, sex, time from diagnosis, proteinuria, or serum albumin, but epitope spreading strongly correlated with PLA2R1-Ab titer (P

Of the glomerulonephritis associated with sarcoidosis, membranous nephropathy (MN) is the most prevalent. Coincidence or a causal relationship between these two diseases is unclear. Here, we present for the first time a high prevalence of PLA2R-related MN among patients with MN associated with active sarcoidosis. Our results suggest some causal link between sarcoidosis and PLA2R-related MN. Detection of anti-PLA2R antibodies in serum or PLA2R antigen in biopsy should not be taken as evidence against a secondary cause, particularly sarcoidosis. This important observation can affect treatment decision in these patients.

International audience; BACKGROUND AND OBJECTIVES:Thrombospondin type-I domain-containing 7A (THSD7A) was recently identified as the target antigen in about 10% of patients with M-type phospholipase A2 receptor (PLA2R)-negative membranous nephropathy in European and North American populations. The prevalence of THSD7A in other populations and their clinical associations deserve further clarification.DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS:Immunofluorescence assay was performed to investigate anti-THSD7A antibodies in 578 consecutive patients with biopsy-proven idiopathic membranous nephropathy, 114 patients with secondary membranous nephropathy, 64 disease controls, and 20 healthy controls. Glomerular expression of THSD7A antigen was examined by immunohistochemistry. Anti-PLA2R antibodies and glomerular PLA2R expression were also screened.RESULTS:Among the 578 patients with idiopathic membranous nephropathy, 12 (2%) patients were identified as THSD7A-positive: ten patients were THSD7A-positive alone, which accounted for 16% (ten of 64) of PLA2R-negative patients; two patients were dual-positive for both anti-THSD7A and anti-PLA2R antibodies and showed enhanced expression of both antigens colocalized in glomeruli. Among the 114 patients with secondary membranous nephropathy, one among 44 (2%) patients with cancer had anti-THSD7A antibodies, whereas 18 of 44 (41%) had anti-PLA2R antibodies. No anti-THSD7A antibody was detected in other disease controls or healthy individuals. Clinical features were comparable between the patients with and without THSD7A. During follow-up, two patients who achieved remission had a clearance of circulating antibodies against THSD7A, whereas antibodies increased in parallel with proteinuria in a patient with a relapse.CONCLUSIONS:THSD7A-associated membranous nephropathy has a low prevalence in Chinese patients. The double-positive patients suggest dual autoimmune responses.

International audience; By use of immunodepletion studies, we characterized four monoclonal antibodies reactive with rabbit brush-border (BB) as specific for aminopeptidase N (AP), dipeptidylpeptidase IV (DPPIV), neutral endopeptidase (EP), and angiotensin-converting enzyme (ACE), and we used these antibodies for immunohistochemical detection of these four hydrolases. Expression within the kidney was studied by light and electron microscopy. All four hydrolases are expressed on the various segments of the proximal tubule. In addition, EP and DPPIV are detectable on visceral epithelial cells of the glomerulus and AP on the cells of Bowman's capsule. Outside the kidney, the four hydrolases are expressed within the digestive and genital tracts, where AP, EP, and DPPIV predominate on epithelial structures, whereas ACE is essentially located in vascular structures. The latter localization is also characteristic of ACE in the other organs studied, where clear-cut systematic distribution of the other hydrolases was often difficult to demonstrate. In addition, AP, DPPIV, and EP were detected on lymphoid cells. As compared to reports of data obtained essentially by enzymatic or immunoradiometric assays, these observations suggest considerable interspecies variations of extrarenal expression of the major BB hydrolases. This should be taken into account in attempting to define a general physiological role for a given enzyme.

International audience; Epitopes of phospholipase A2 receptor (PLA2R), the target antigen in idiopathic membranous nephropathy (iMN), must be presented by the HLA-encoded MHC class II molecules to stimulate autoantibody production. A genome-wide association study identified risk alleles at HLA and PLA2R loci, with the top variant rs2187668 within HLA-DQA1 showing a risk effect greater than that of the top variant rs4664308 within PLA2R1. How the HLA risk alleles affect epitope presentation by MHC class II molecules in iMN is unknown. Here, we genotyped 261 patients with iMN and 599 healthy controls at the HLA-DRB1, HLA-DQA1, HLA-DQB1, and HLA-DPB1 loci with four-digit resolution and extracted the encoded amino acid sequences from the IMGT/HLA database. We predicted T cell epitopes of PLA2R and constructed MHC-DR molecule-PLA2R peptide-T cell receptor structures using Modeler. We identified DRB1*1501 (odds ratio, 4.65; 95% confidence interval [95% CI], 3.39 to 6.41; P

The HANAC syndrome is caused by mutations in the gene coding for collagen4a1, a major component of blood vessel basement membranes. Ocular symptoms include an increase in blood vessel tortuosity and occasional hemorrhages. To examine how vascular defects can affect neuronal function, we analyzed the retinal phenotype of a HANAC mouse model. Heterozygous mutant mice displayed both a thinning of the basement membrane in retinal blood vessels and in Bruch's membrane resulting in vascular leakage. Homozygous mice had additional vascular changes, including greater vessel coverage and tortuosity. This greater tortuosity was associated to higher expression levels of vascular endothelial growth factor (VEGF). These major changes to the blood vessels were correlated with photoreceptor dysfunction and degeneration. The neuronal damage was associated with reactive gliosis in astrocytes and Müller glial cells, and by the migration of microglial cells into the outer retina. This study illustrates how vascular changes can trigger neuronal degeneration in a new model of HANAC syndrome that can be used to further study dysfunctions of neurovascular coupling. Summary statement: This study provides a phenotypic analysis of a novel mouse model of HANAC syndrome focusing on the retinal aspect. It recapitulates most of the aspects of the human disease and is therefore a great tool to study and to address this condition.

International audience; Membranous nephropathy (MN) is characterized by an accumulation of immune deposits on the subepithelial side of the glomerular basement membrane, which results in complement activation and proteinuria. Since 2002, several major antigens of the podocyte have been identified in human MN, the first one being neutral endopeptidase (NEP), the alloantigen involved in neonatal cases of MN that occur in newborns from NEP-deficient mothers. This discovery opened the field to the major advances that have occurred since then in the pathophysiology and treatment of MN. It is remarkable that experimental models such as Heymann nephritis and cationic bovine serum albumin-induced MN in the rabbit predicted the pathomechanisms of the human glomerulopathy. The podocyte is at the center of the pathogenesis of MN either by providing a source of endogenous antigens or by creating an environment favorable to deposition and accumulation of immune complexes containing exogenous (non-podocyte) antigens. The podocyte is also a victim of complement activation and antibody blocking activity against enzymes or receptors. A search for innovative drugs aimed at protecting this cell against complement activation and the effects of prolonged ER stress has become a priority.

International audience; Objective. Renal involvement is a rare event during primary SS (pSS). We aimed to describe the clinico-biological and histopathological characteristics of pSS-related nephropathy and its response to treatment. Methods. We conducted a French nationwide, retrospective, multicentre study including pSS patients fulfilling American–European Consensus Group criteria or enlarged American–European Consensus Group criteria, and with biopsy-proven renal involvement. Results. A total of 95 patients were included (median age 49 years). An estimated glomerular filtration rate (eGFR) of

International audience; The M-type receptor for phospholipase A2 (PLA2R1) is the major target antigen in idiopathic membranous nephropathy (iMN). Our recent genome-wide association study showed that genetic variants in an HLA-DQA1 and phospholipase A2 receptor (PLA2R1) allele associate most significantly with biopsy-proven iMN, suggesting that rare genetic variants within the coding region of the PLA2R1 gene may contribute to antibody formation. Here, we sequenced PLA2R1 in a cohort of 95 white patients with biopsy-proven iMN and assessed all 30 exons of PLA2R1, including canonical (GT-AG) splice sites, by Sanger sequencing. Sixty patients had anti-PLA2R1 in serum or detectable PLA2R1 antigen in kidney tissue. We identified 18 sequence variants, comprising 2 not previously described, 7 reported as rare variants (

Maternal-fetal alloimmunisation with antenatal glomerulopathies (FMAIG) is a recently described allo-immune disorder that results from the production of maternal antibodies which cross the placenta, bind to fetal glomerular podocytes and thereby cause renal dysfunction. The pathogenic antibodies are directed against CD10/neutral endopeptidase (NEP). The infant's mother is apparently healthy but is genetically NEP-deficient, and thus becomes immunized against CD10/NEP expressed by placental cells during her first pregnancy. This disease, that we have now diagnosed in five families, is the first described organ-specific disorder due to maternal-fetal allo-immunisation. Because future pregnancies in CD10/NEP-immunized mothers are at high risk for the fetus, antigen-driven therapies aimed at eliminating pathogenic antibodies are urgently needed. This will require identification of the pathogenic epitopes born by the antigen.

Key points Antibodies to neutral endopeptidase, a podocyte protein, are responsible for rare alloimmune neonatal membranous nephropathy that develops in children from neutral endopeptidase-deficient mothers. Neutral endopeptidase was the first podocyte antigen described in human membranous nephropathy. PLA2R1, the type-M receptor of soluble phospholipase A2, is a major target antigen in so-called idiopathic membranous nephropathy in adults. Antibodies to PLA2R1 are detected in 60 to 80% of patients before immunosuppressive treatment, and are only occasionally found in secondary membranous nephropathy. To date, they have not been detected in other pathological conditions and in healthy individuals. PLA2R1 and HLA-DQA1 gene variants defined by single nucleotide polymorphisms are strongly associated with idiopathic membranous nephropathy in patients of white ancestry, and can thus be considered as predisposing genes. In addition to their diagnostic value, anti-PLA2R1 antibodies can be used to monitor treatment. Immunization against cationic bovine serum albumin is a cause of early childhood membranous nephropathy. This finding points to a possible role of food and environmental antigens in membranous nephropathy. The newly identified antigen-antibody systems should be considered as molecular signatures challenging the uniform histological definition and having a major impact on patient care in a near future.

International audience; Over the past few years, considerable advances have been made in our understanding of the molecular pathomechanisms of human membranous nephropathy, inspired by studies of Heymann nephritis, a faithful experimental model of this disease. This research led to the identification of neutral endopeptidase, the M-type receptor for secretory phospholipase A(2) (PLA(2)R1) and cationic bovine serum albumin as target antigens of circulating and deposited antibodies in alloimmune neonatal, adult 'idiopathic' and early-childhood membranous nephropathy, respectively. A genome-wide association study has provided further evidence for a highly significant association between PLA2R1 and HLA-DQA1 loci and idiopathic membranous nephropathy in patients of white ancestry. Additional antibody specificities for cytoplasmic antigens have also been identified, but their pathogenic role is uncertain. The time has come to revisit the spectrum of membranous nephropathies based on the newly identified antigen-antibody systems that should be considered as molecular signatures of the disease and that challenge the uniform histological definition. These signatures will soon have a major impact on patient care.

Summary 1. embranous nephropathy is characterized by an accumulation of immune deposits on the outer aspect of the glomerular basement membrane. 2. In the rat model described by Heymann in 1959, the target antigen of antibodies is megalin, a multiligand receptor expressed in the rat glomerulus but absent from the human glomerulus. 3. In recent years, two major antigens have been identified in human membranous nephropathy (MN). The first is neutral endopeptidase (NEP), the alloantigen involved in neonatal cases of MN that occur in newborns from NEP-deficient mothers. The second is the M-type phospholipase A2 receptor (PLA2R), the first autoantigen identified in idiopathic MN in the adult. Megalin, NEP and PLA2R are all expressed on the podocyte surface, where they can serve as targets for circulating antibodies, leading to in situ immune complex formation, complement activation and proteinuria. 4. In addition to podocyte antigens, we recently showed that some patients with childhood MN had both circulating cationic bovine serum albumin (BSA) and anti-BSA antibodies, with BSA being present in immune deposits. This suggests that food antigens may be involved in MN through charge-dependent binding to the anionic glomerular capillary wall and in situ formation of immune complexes.

The M-type phospholipase A(2) receptor (PLA(2)R) was recently identified as a candidate antigen in 70% of cases of idiopathic membranous nephropathy, a common form of the nephrotic syndrome. The nature of antigens involved in other idiopathic and secondary membranous nephropathies remains unclear.We searched for antibodies against bovine serum albumin and circulating bovine serum albumin by means of enzyme-linked immunosorbent assay and Western blotting in serum specimens obtained from 50 patients with membranous nephropathy and 172 controls. The properties of immunopurified circulating bovine serum albumin obtained from serum specimens were analyzed with the use of two-dimensional sodium dodecyl sulfate-polyacrylamide-gel electrophoresis. We detected bovine serum albumin in glomerular deposits and analyzed the reactivity of eluted IgG.Eleven patients, including four children, had high levels of circulating anti-bovine serum albumin antibodies, of both the IgG1 and IgG4 subclasses. These patients also had elevated levels of circulating bovine serum albumin, without an increase in circulating immune complex levels. Bovine serum albumin immunopurified from the serum specimens of these four children migrated in the basic range of pH, whereas the bovine serum albumin from adult patients migrated in neutral regions as native bovine serum albumin. Bovine serum albumin was detected in subepithelial immune deposits only in the children with both high levels of cationic circulating bovine serum albumin and bovine serum albumin-specific antibodies, and it colocalized with IgG in the absence of PLA(2)R. IgG eluted from such deposits was specific for bovine serum albumin.Some patients with childhood membranous nephropathy have both circulating cationic bovine serum albumin and anti-bovine serum albumin antibodies. Bovine serum albumin is present in immune deposits, suggesting that cationic bovine serum albumin is pathogenic through binding to the anionic glomerular capillary wall and in situ formation of immune complexes, as shown in experimental models.

The COL4A1 gene encodes the α1-chain of type IV collagen, which is ubiquitously expressed in basement membranes. Mutations in COL4A1 have been reported in autosomal-dominant porencephaly and in patients with symptomatic small vessel brain disease, inconstantly associated with eye defects. We have previously reported three COL4A1 mutations associated with a systemic phenotype that we called HANAC (Hereditary Angiopathy, Nephropathy, Aneurysms, and Cramps). We carried out a clinical and genetic study of three families presenting with characteristic features of HANAC syndrome. Common systemic signs included arterial retinal tortuosity and muscle cramps, with a variable combination of small vessel brain disease, Raynaud phenomena, and kidney defects. Three novel COL4A1 missense substitutions are described, which affect highly conserved glycine residues within the collagenous domain of the protein. All six known mutations associated with the HANAC phenotype are localized within the CB3[IV] fragment of COL4A1, which encompasses major integrin-binding sites. Our results confirm that HANAC syndrome is a distinct clinical entity within the COL4A1-related disorders, which is characterized by systemic involvement and usually asymptomatic brain disease. The restricted distribution of COL4A1 mutations within the CB3[IV] region is a characteristic of the reports of patients with HANAC, which suggests that abnormal cell-type IV collagen interactions may underlie the systemic defects observed in this syndrome.

Renal involvement is frequent in patients suffering from primary Sjögren’s syndrome (pSS). Tubulointerstitial infiltration is the most common renal lesion, while glomerular involvement is rare. We report the case of a 50-year-old woman with pSS who developed renal failure due to an unusual proliferative glomerulonephritis with humps and monotypic IgG1-kappa deposits. Searches for cryoglobulinaemia, anti-double-stranded DNA and anti-neutrophil cytoplasmic antibodies were negative. Serum protein electrophoresis and immunofixation revealed no monoclonal immunoglobulin. Extensive work-up excluded associated infectious, collagen or lymphoproliferative disease. This case adds to the spectrum of pSS-related glomerular disease which is reviewed in depth.

Purpose of review Membranous nephropathy is one of the most common glomerulopathies. Current treatments are entirely empirical, and pathophysiology-driven therapies are dramatically lacking. This review focuses on new pathophysiologic aspects of the disease, with special emphasis on the antigenic targets of pathogenic antibodies. Recent findings In the past few years, two major antigens have been identified in human membranous nephropathy. The first is neutral endopeptidase (NEP), the alloantigen involved in neonatal cases of membranous nephropathy that occur in newborns from NEP-deficient mothers. The second is the M-type phospholipase A2 receptor (PLA2R), the first autoantigen identified in idiopathic membranous nephropathy in the adult. Summary A common denominator shared by most cases of membranous nephropathy is the expression by podocytes of antigenic targets for in-situ formation of glomerular immune deposits. The recent discovery of neutral endopeptidase and PLA2R provides new tools for the monitoring of disease activity and should be of value in designing new antigen-driven therapeutic strategies.

A 62-year-old woman presented with nephrotic syndrome, monoclonal gammopathy, and membranous-like nephropathy with nonorganized deposits composed of monoisotypic immunoglobulin G1 lambda protein. Nephrotic syndrome remitted after a brief course of treatment with melphalan despite ongoing production of the monoclonal protein. The circulating monoclonal immunoglobulin G1 lambda showed unusual in vitro aggregation properties, including dependence on low ionic strength and neutral pH, suggesting that electrostatic interactions had a role in the precipitation process. This case illustrates the importance of looking for monoclonal immunoglobulin deposits when kidney biopsy findings are suggestive of membranous nephropathy. In addition, our in vitro demonstrations of the role of physicochemical factors in immunoglobulin precipitation help elucidate the pathogenesis of immunoglobulin deposition disorders. Although binding to podocyte antigens is a well-recognized determinant of subepithelial immunoglobulin deposition, proneness to aggregation as described in this case also might be nephritogenic.

Early events in kidney organogenesis involve reciprocal interactions between the ureteric bud and the metanephric mesenchyme, which lead to remodeling of the extracellular matrix. This remodeling involves matrix metalloproteases (MMPs), but the specific roles of individual MMPs in kidney development are not completely understood. Here, we analyzed MMP9-deficient mice at the first step of kidney development and found that MMP9 deficiency delayed embryonic kidney maturation and increased apoptosis ex vivo by 2.5-fold. These early defects resulted in a 30% decrease in nephron number, a 20% decrease in adult kidney weight, and altered kidney function and morphology at 12 mo. The membrane form of stem cell factor (SCF) increased, whereas the activated form of the SCF receptor, c-kit, decreased in MMP9-deficient embryonic kidneys. In organotypic culture, MMP9-deficient kidneys failed to secrete SCF, and addition of recombinant SCF partially rescued both apoptosis and the branching defect. In conclusion, these data show that MMP9 protects mesenchymal cells from apoptosis during kidney development and stimulates ureteric bud branching morphogenesis, most likely by releasing the soluble form of SCF, suggesting that normal renal development requires MMP9.

Fetomaternal alloimmunization with antenatal glomerulopathies (FMAIG) is a recently described alloimmune disorder, which results from the production of maternal antibodies that cross the placenta, bind to fetal glomerular podocytes, and mediate renal disease. The pathogenic antibodies are directed against CD10/neutral endopeptidase (NEP). The infant's mother is NEP-deficient and thus she becomes immunized during the first pregnancy against CD10/NEP expressed by placental cells. Because future pregnancies in CD10/NEP-immunized mothers are at high risk for the fetus, detection of anti-NEP antibodies in pregnant mothers and antigen-driven therapies including induction of tolerance, are urgently needed. This ideally requires identification of the pathogenic epitopes born by the antigen and specifically recognized by B- and T-cells. We have recently characterized such epitopes that will be used in diagnostic tests (ELISA) and for new therapeutic approaches based on peptide-specific immune intervention. For this purpose, we have developed an experimental model by crossing NEP/CD10-deficient female mice to wild-type males. The females develop an alloimmune reaction against NEP, which is a prerequisite for tolerance induction experiments. Although NEP/CD10 does not seem to be involved in common idiopathic forms of membranous nephropathy in the adult, alloimmune antibodies may be implicated in de novo membranous nephropathy that develop in the kidney graft and after alloimmune bone marrow transplantation.

Remodeling of extracellular matrix (ECM) is a key event in progression and reversal of kidney disease. This process results from synthesis of ECM components and their degradation, mostly by matrix metalloproteinases (MMPs). However, because of both the multiplicity of their targets that include non-matrix substrates, and the complexity of their regulation, MMPs may exert different, and even opposite, effects during the different phases of the evolution of kidney diseases. The major challenge with future therapeutic interventions will be to accomplish temporal control of MMP activity. In addition to MMPs, enzymes that stabilize ECM (transglutaminase) and cell receptors for ECM components including integrins and discoidin domain receptor-1 (DDR1), play an important role in the cell response to matrix remodeling. Novel therapeutic approaches aimed at targeting transglutaminase and ECM receptors, particularly DDR1, are a promising option provided that specific and safe pharmacological inhibitors be developed. These therapies together with pharmacological agents controlling MMP activity, given in appropriate windows of the course of kidney diseases may represent a useful adjunct to blockers of the renin–angiotensin system.

The deterioration of renal function in chronic kidney disease is related to the progression of renal fibrosis, which was long considered unavoidable. Today, the reversibility of renal fibrotic lesions is a reality, although still clinically rare. Because angiotensin II is highly profibrotic, blocking its action effectively protects the kidney, as numerous clinical trials have shown. The development of interstitial fibrosis is secondary to the epithelial-to-mesenchymal transition induced by transforming growth factor (TGF)-beta. Bone morphogenic protein-7 (BMP-7) and hepatocyte growth factor (HGF) induce the reverse transition and thus open up perspectives for treatment. Degradation of the extracellular matrix by matrix metalloproteinases or other enzymes is another therapeutic pathway. Renal regeneration may be promoted by modulation of hypoxia-inducible factor-1 (HIF-1) and vascular endothelial growth factor (VEGF).

Kidney podocytes and their foot processes maintain the ultrafiltration barrier and prevent urinary protein loss (proteinuria). Here we show that the GTPase dynamin is essential for podocyte function. During proteinuric kidney disease, induction of cytoplasmic cathepsin L leads to cleavage of dynamin at an evolutionary conserved site, resulting in reorganization of the podocyte actin cytoskeleton and proteinuria. Dynamin mutants that lack the cathepsin L site, or render the cathepsin L site inaccessible through dynamin self-assembly, are resistant to cathepsin L cleavage. When delivered into mice, these mutants restored podocyte function and resolve proteinuria. Our study identifies dynamin as a critical regulator of renal permselectivity that is specifically targeted by proteolysis under pathological conditions.

International audience; Most patients with idiopathic membranous nephropathy (IMN) have IgG4 autoantibodies against phospholipase A2 receptor (PLA2R). C3 and C5b-9 are found in immune deposits of IMN kidney biopsy specimens, but the pathway of complement activation in IMN remains elusive. We report the case of a patient who developed IMN with intense staining for PLA2R, IgG4, C3, C5b-9, factor B, and properdin and very weak staining for C1q, C4d, and IgG1. Measurement of mannan binding lectin (MBL) antigenic level and activity revealed MBL deficiency. Genotyping revealed a heterozygous (A/C) polymorphism in codon 57 of MBL2 exon 1 associated with homozygous and heterozygous variations in the promoter region at -550 (L/L) and -221 (X/Y), respectively, suggesting that the patient harbored the LXA/LYC haplotypes linked to MBL deficiency. Genetic sequencing in 77 consecutive patients with IMN identified four patients with MBL2 promoter and coding region variations associated with MBL deficiency and the same complement pattern in immune deposits as the index patient. In contrast, patients with wild-type MBL2 had immune deposits with intense Cd4 staining. Thus, IMN can develop in patients with complete MBL deficiency, with complement activated mainly by the alternative pathway, whereas the lectin pathway is also activated in those with wild-type MBL2.

International audience; Rituximab induces nephrotic syndrome (NS) remission in two-thirds of patients with primary membranous nephropathy (MN), even after other treatments have failed. To assess the relationships among treatment effect, circulating nephritogenic anti-phospholipase A2 receptor (anti-PLA2R) autoantibodies and genetic polymorphisms predisposing to antibody production we serially monitored 24-hour proteinuria and antibody titer in patients with primary MN and long-lasting NS consenting to rituximab (375 mg/m(2)) therapy and genetic analyses. Over a median (range) follow-up of 30.8 (6.0-145.4) months, 84 of 132 rituximab-treated patients achieved complete or partial NS remission (primary end point), and 25 relapsed after remission. Outcomes of patients with or without detectable anti-PLA2R antibodies at baseline were similar. Among the 81 patients with antibodies, lower anti-PLA2R antibody titer at baseline (P=0.001) and full antibody depletion 6 months post-rituximab (hazard ratio [HR], 7.90; 95% confidence interval [95% CI], 2.54 to 24.60; P

8 pages; Light-, light- and heavy-, and heavy-chain deposition diseases belong to a family of diseases that include light-chain (AL)-amyloid, nonamyloid fibrillary and immunotactoid glomerulonephritis, and cryoglobulinemic glomerulonephritis, in which monoclonal Ig or their subunits become deposited in kidney. In clinical and pathologic terms, light-, light- and heavy-, and heavy-chain deposition diseases essentially are similar and are characterized by prominent renal involvement with severe renal failure; extrarenal manifestations; diabetes-like nodular glomerulosclerosis; marked thickening of tubular basement membranes; and monotypic deposits of light chain, mostly kappa, and/or heavy chain that feature a nonorganized granular, electron-dense appearance by electron microscopy. The most common cause is myeloma. Recent progress has been made in the understanding of the molecular pathomechanisms of Ig-chain deposition and extracellular matrix accumulation, which opens up new therapeutic avenues in addition to eradication of the Ig-secreting plasma cell clone. Because these diseases represent a model of glomerular and interstitial fibrosis that is induced by a single molecule species, a better understanding of their pathomechanisms may help to unravel the pathophysiology of kidney fibrosis and renal disease progression.

Membranous nephropathy is one of the most common glomerulopathies. Current treatments are entirely empirical, and concept-driven therapies are dramatically lacking. This review focuses on new pathophysiologic aspects of the disease, with special emphasis on the antigenic targets of pathogenic antibodies.Neutral endopeptidase - a podocyte antigen that can digest biologically active peptides - was recently identified as the target antigen of antibodies deposited in the subepithelial space of glomeruli in a subset of patients with antenatal membranous nephropathy. The mothers became immunized because they are deficient in neutral endopeptidase due to truncating mutations in the gene. Membranous nephropathy could be transferred to the rabbit by injection of mothers' immunoglobulin. Development of the renal disease was associated with anti-neutral endopeptidase IgG1.Membranous nephropathy most likely is a heterogeneous disease, although a common denominator may be that podocytes provide antigenic targets for in-situ formation of glomerular immune deposits. Identification of neutral endopeptidase and additional (podocyte) antigens and characterization of their epitopes should make it possible to design more effective and better tolerated therapies. Fetomaternal alloimmunization is a novel mechanism of renal disease that may apply to other organs as well.

The HNK-1 carbohydrate epitope is a 3-sulfo-glucuronyl residue attached to lactosamine structures on glycoproteins, proteoglycans, or glycolipids mostly expressed in the nervous system. Here, using monoclonal antibodies against the sulfated HNK-1 carbohydrate epitope, we first examined its distribution in developing and adult kidneys, then its expression in kidneys with tubular necrosis and renal neoplasms. This HNK-1 epitope was expressed in the human, rabbit, and rat, but not mouse kidney. It was detected within a subset of epithelial cells in the renal vesicle and in comma- and S-shaped bodies during early stages of nephrogenesis. In ureteral bud derivatives, the epitope was present transiently in the area where the collecting duct fused with the nephron. In the adult kidney, expression of the HNK-1 epitope became mainly restricted to the thin ascending loop of Henle where this epitope was carried by heparan- and chondro-proteoglycan. In pathological conditions, HNK-1 epitope expression increased dramatically in proximal epithelial tubule cells in kidneys with acute tubular necrosis. In tumors, the HNK-1 epitope was expressed in the epithelial component of nephroblastomas and in a subgroup of papillary renal cell carcinomas. These data suggest that molecules carrying the sulfated HNK-1 carbohydrate epitope may play an important role in critical stages of renal development and in the physiology of thin ascending loop of Henle. (J Histochem Cytochem 54:575-584, 2006)

The myelin protein 0 (MPZ or P0) is a transmembrane glycoprotein that represents the most abundant myelin component. Mutations in the P0 gene are associated with one form of autosomal dominant demyelinating peripheral neuropathy, Charcot-Marie-Tooth disease type 1B (CMT1B). Because CMT1 may be associated with renal involvement, mostly focal segmental glomerulosclerosis, we hypothesized that P0 could be expressed in the kidney. P0 mRNA was detected by reverse transcriptase–PCR in the human and mouse renal cortex. P0 transcripts were identified by in situ hybridization at different stages of the mouse kidney development, especially in embryonic structures that give rise to the glomerulus. P0 protein was also detected by Western blot in human and rat glomerular extracts and in a human podocyte cell line using a monoclonal anti-P0 antibody. Immunofluorescence studies on human kidney sections showed that the podocytes were intensely labeled. Immunogold electron microscopy disclosed a predominant staining of the membranes of intracellular vesicles in podocytes. P0 was also detected in the podocyte cell membrane, including at the foot processes. P0 −/− mice exhibited mild growth retardation and demyelinating neuropathy similar to the one observed in patients with CMT1B. They also presented mild albuminuria, without significant ultrastructural change of the glomerular basement membrane or the podocytes. These results demonstrate that P0, the major myelin protein, is also expressed during nephrogenesis and in mature kidney, mostly in podocytes. They suggest that P0 gene mutations might be involved in renal diseases.