Over the past few years, considerable advances have been made in our understanding of the molecul... more 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 ...
Background: Membranous nephropathy (MN) is the most common histological diagnosis in adults with ... more Background: Membranous nephropathy (MN) is the most common histological diagnosis in adults with nephrotic syndrome and a leading cause of end-stage kidney failure from glomerulonephritis. Little is known about the underlying aetiology, although anti-glomerular antibodies have been implicated. No specific underlying genetic defect has yet been identified. Methods: In a family with four members in three generations affected by primary MN, the serum of affected members and their mothers were assessed for anti-glomerular antibodies. Results: All four affected are male and connected through the maternal line, indicative of X-linked inheritance. Age of onset of nephrotic syndrome varied between 1 and 67 years of age, suggesting that a potential underlying gene may confer a genetic predisposition to MN, but other factors, genetic or environmental, are necessary to trigger the disease. Serologic studies revealed antibodies against glomerular and peritubular endothelial cells in the mother ...
Patients with hereditary hemorrhagic telangiectasia have increased plasma levels of vascular endo... more Patients with hereditary hemorrhagic telangiectasia have increased plasma levels of vascular endothelial growth factor and transforming growth factor-beta1 as well as high ALK1 tissue expression. Haematologica 2005;90:818-28.
L1, a member of the immunoglobulin superfamily, is a cell adhesion and signal transducing molecul... more L1, a member of the immunoglobulin superfamily, is a cell adhesion and signal transducing molecule. In the kidney, L1 is expressed in the mesonephric duct and the metanephros throughout collecting duct development. We show that mice with a targeted deletion of the L1 gene display diverse renal malformations including (i) a duplex kidney with two ureters partially or totally separated, accompanied by hydronephrosis; and (ii) an enlarged elongated kidney with a malformed or incorrectly positioned inner medulla. The type, penetrance and severity of these phenotypes are influenced by the genetic background. The development of a duplex kidney is initiated by double ureteral budding from the Wolffian duct or by an accessory budding from the main ureter, whereas medullary malformation is due to an improper growth and branching pattern of ureteral branches. Multiple developmental defects in formation of the collecting system promote subsequent renal damage and progression to renal insufficiency. Various features of mouse ureteral duplication resemble the human congenital anomalies of the kidney and urinary tract (CAKUT) although disturbances of medulla development have not yet been reported in men.
A 62-year-old woman presented with nephrotic syndrome, monoclonal gammopathy, and membranouslike ... more A 62-year-old woman presented with nephrotic syndrome, monoclonal gammopathy, and membranouslike nephropathy with nonorganized deposits composed of monoisotypic immunoglobulin G1 protein. Nephrotic syndrome remitted after a brief course of treatment with melphalan despite ongoing production of the monoclonal protein. The circulating monoclonal immunoglobulin G1 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.
Introduction and Aims: Hypertriglyceridemia is a cardinal feature of nephrotic syndrome. A recent... more Introduction and Aims: Hypertriglyceridemia is a cardinal feature of nephrotic syndrome. A recent study (Clement Nature Medicine 2011) shows that podocyte secreted, but not circulating Angiopoietin-like-4 (Angptl4) also noted in minimal change disease, induces proteinuria. Here, we investigated the role of circulating Angptl4 in nephrotic syndrome. Methods: Models of proteinuria were induced in Wild-Type and KnockOut Angptl4 mice, and in Wild-Type and generated Angptl4 transgenic rats. Proteinuria, albuminuria and hypertriglyceridemia were studied in those animals. Stable transfection was realized in HEK293 cells in order to produce recombinant Angptl4. Recombinant Angptl4 was injected into animals and incubated with rat glomerular endothelial cells. Immunogold electron microscopy was used to determine glomerular localization of Angptl4. Results: Circulating Angptl4 is known to inhibit endothelium bound lipoprotein lipase, which reduces tissue uptake of triglycerides from circulation, resulting in hypertriglyceridemia. We induced the gamma2-NTS (gamma2-Nephrotoxic Serum) and LPS (Lipopolysaccharides) models of proteinuria in Angptl4 −/− and + / + mice. Angptl4 −/− mice did not develop hypertriglyceridemia despite significant residual proteinuria, suggesting that Angptl4 is the key determinant of hypertriglyceridemia in nephrotic syndrome. Next, we studied albuminuria in adipose tissue specific Angptl4 transgenic rats (aP2-Angptl4), that have hypertriglyceridemia due to increased circulating Angptl4, and noted reduced albuminuria in transgenic compared to wild type rats (P < 0.05). Induction of PAN (Puromycin Aminonucleoside) in these rats caused lower levels of proteinuria (Day 8 wild type 315 + 2 mg/18 hours, transgenic 194 + 25 mg/18 hours, mean + SE, P < 0.05). Immunogold EM showed binding of Angptl4-V5 to glomerular endothelial surface. Cultured rat glomerular endothelial cells were protected from oxidative stress by recombinant neutral pI Angptl4 similar to that present in the circulation (P < 0.001). By inducing puromycin nephrosis in podocyte specific Angptl4 transgenic rats (NPHS2-Angptl4), we determined that beyond the initial stages, the bulk of the circulating Angptl4 is contributed to by peripheral tissues, predominantly adipose tissue. In addition, injection of recombinant neutral pI Angptl4 reduces proteinuria in multiple animal models of proteinuria. Conclusions: Our studies suggest that in later stages of nephrotic syndrome, adipose tissue secretes Angptl4 into the circulation to reduce proteinuria by glomerular endothelial stabilization, but this increase in circulating Angptl4 also results in hypertriglyceridemia.
Pediatric steroid-sensitive nephrotic syndrome (pSSNS) is the most common childhood glomerular di... more Pediatric steroid-sensitive nephrotic syndrome (pSSNS) is the most common childhood glomerular disease. Previous genome-wide association studies (GWAS) identified a risk locus in the HLA Class II region and three additional independent risk loci. But the genetic architecture of pSSNS, and its genetically driven pathobiology, is largely unknown. Here, we conduct a multi-population GWAS meta-analysis in 38,463 participants (2440 cases). We then conduct conditional analyses and population specific GWAS. We discover twelve significant associations-eight from the multi-population meta-analysis (four novel), two from the multi-population conditional analysis (one novel), and two additional novel loci from the European meta-analysis. Fine-mapping implicates specific amino acid haplotypes in HLA-DQA1 and HLA-DQB1 driving the HLA Class II risk locus. Non-HLA loci colocalize with eQTLs of monocytes and numerous T-cell subsets in independent datasets. Colocalization with kidney eQTLs is lacking but overlap with kidney cell open chromatin suggests an uncharacterized disease mechanism in kidney cells. A polygenic risk score (PRS) associates with earlier disease onset. Altogether, these discoveries expand our knowledge of pSSNS genetic architecture across populations and provide cell-specific insights into its molecular drivers. Evaluating these associations in additional cohorts will refine our understanding of population specificity, heterogeneity, and clinical and molecular associations. Pediatric steroid-sensitive nephrotic syndrome (pSSNS) is a rare disease of the glomerular filtration barrier. Its incidence ranges from 1.15-16.9 cases in every 100,000 children, occurring most frequently in South Asian populations 1. pSSNS causes massive proteinuria and increases the risk of thromboembolism, sepsis, and progression to chronic kidney disease (CKD)/end-stage kidney disease (ESKD) 2-7. And those progressing to ESKD have increased odds of recurrent NS in their allograft 8. pSSNS is impactful across the lifespan-31-50% of those affected have relapses in adulthood 9. Much of pSSNS's morbidity is related to side effects of the non-specific immunosuppressants which allow some to achieve remission of their proteinuria 7, 10-17. Despite intensive investigation, there are no known monogenic forms of pSSNS to illuminate its pathobiology. However, we know that immune dysregulation is a major contributor 18,19. But determining causal immune factors via case-control studies of cytokines profiles, cell types, and transcriptomic signatures is challenging. The dynamic responses of the immune system at different disease stages and to various stimuli make it difficult to determine whether observed differences are causal, correlated, or due to independent biological/
medRxiv (Cold Spring Harbor Laboratory), Sep 14, 2022
doi: medRxiv preprint NOTE: This preprint reports new research that has not been certified by pee... more doi: medRxiv preprint NOTE: This preprint reports new research that has not been certified by peer review and should not be used to guide clinical practice.
Journal of Histochemistry and Cytochemistry, Jan 6, 2006
The HNK-1 carbohydrate epitope is a 3-sulfo-glucuronyl residue attached to lactosamine structures... more 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.
Journal of The American Society of Nephrology, Jun 14, 2018
Background Steroid-sensitive nephrotic syndrome (SSNS) is a childhood disease with unclear pathop... more Background Steroid-sensitive nephrotic syndrome (SSNS) is a childhood disease with unclear pathophysiology and genetic architecture. We investigated the genomic basis of SSNS in children recruited in Europe and the biopsy-based North American NEPTUNE cohort. Methods We performed three ancestry-matched, genome-wide association studies (GWAS) in 273 children with NS (Children Cohort Nephrosis and Virus [NEPHROVIR] cohort: 132 European, 56 African, and 85 Maghrebian) followed by independent replication in 112 European children, transethnic meta-analysis, and conditional analysis. GWAS alleles were used to perform glomerular cis-expression quantitative trait loci studies in 39 children in the NEPTUNE cohort and epidemiologic studies in GWAS and NEPTUNE (97 children) cohorts. Results Transethnic meta-analysis identified one SSNS-associated single-nucleotide polymorphism (SNP) rs1063348 in the 39 untranslated region of HLA-DQB1 (P59.3310 223). Conditional analysis identified two additional independent risk alleles upstream of HLA-DRB1 (rs28366266, P53.7310 211) and in the 39 untranslated region of BTNL2 (rs9348883, P59.4310 27) within introns of HCG23 and LOC101929163. These three risk alleles were independent of the risk haplotype DRB1*07:01-DQA1*02:01-DQB1*02:02 identified in European patients. Increased burden of risk alleles across independent loci was associated with higher odds of SSNS. Increased burden of risk alleles across independent loci was associated with higher odds of SSNS, with younger age of onset across all cohorts, and with increased odds of complete remission across histologies in NEPTUNE children. rs1063348 associated with decreased glomerular expression of HLA-DRB1, HLA-DRB5, and HLA-DQB1. Conclusions Transethnic GWAS empowered discovery of three independent risk SNPs for pediatric SSNS. Characterization of these SNPs provide an entry for understanding immune dysregulation in NS and introducing a genomically defined classification.
Journal of The American Society of Nephrology, Feb 21, 2013
The M-type receptor for phospholipase A2 (PLA2R1) is the major target antigen in idiopathic membr... more 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 (,1%) in the Single Nucleotide Polymorphism Database or the 1000 Genomes project, and 9 known to be common polymorphisms. Although we confirmed significant associations among 6 of the identified common variants and iMN, only 9 patients had the private or rare variants, and only 4 of these patients were among the 60 who were PLA2R positive. In conclusion, rare variants in the coding sequence of PLA2R1, including splice sites, are unlikely to explain the pathogenesis of iMN.
We immunopurified a surface antigen specific for the collecting duct (CD) epithelium. Microsequen... more We immunopurified a surface antigen specific for the collecting duct (CD) epithelium. Microsequencing of three polypeptides identified the antigen as the neuronal cell adhesion molecule L1, a member of the immunoglobulin superfamily. The kidney isoform showed a deletion of exon 3. L1 was expressed in the mesonephric duct and the metanephros throughout CD development. In the adult CD examined by electron microscopy, L1 was not expressed on intercalated cells but was restricted to CD principal cells and to the papilla tall cells. By contrast, L1 appeared late in the distal portion of the elongating nephron in the mesenchymally derived epithelium and decreased during postnatal development. Immunoblot analysis showed that expression, proteolytic cleavage, and the glycosylation pattern of L1 protein were regulated during renal development. L1 was not detected in epithelia of other organs developing by branching morphogenesis. Addition of anti-L1 antibody to kidney or lung organotypic cultures induced dysmorphogenesis of the ureteric bud epithelium but not of the lung. These results suggest a functional role for L1 in CD development in vitro. We further postulate that L1 may be involved in the guidance of developing distal tubule and in generation and maintenance of specialized cell phenotypes in CD.
Idiopathic membranous nephropathy (MN) is the most frequent cause of the nephrotic syndrome in ad... more Idiopathic membranous nephropathy (MN) is the most frequent cause of the nephrotic syndrome in adults. The diagnosis is based on typical findings observed via electron microscopy (EM) and immunofluorescence (IF) studies. On some occasions, tissues are only available for analysis using an optical microscope (OM); in these cases, it can be difficult to differentiatebetween MN and minimal change disease (MCD). Recent advances have shown that MN is a kidney specific autoimmune disease induced by antibodies specific for podocyte antigens. Complement plays an important role, even if mechanisms of activation have not been clarified yet. C4d is a fragment of C4 that is produced during activation of the classical or lectin complement pathway. We might therefore expect to find C4d deposition as a marker of complement activation in MN. The aim of our study was to determine whether immunohistochemical detection of C4d in patients with MN could be useful as a diagnostic tool. METHODS: All adult patients diagnosed with idiopathic minimal change disease (MCD) and MN biopsied in our unit between January 2001-December 2016 were considered for inclusion in the study. Diagnoses of MCD and MN were based on histological assessment of renal biopsy tissue with LM, IF and EM studies. In October 2008, 3lm sections of renal tissue fixed in formaldehyde were removed from paraffin and rehydrated. The samples were then stained for C4d immunohistochemical analysis using anti-human polyclonal antibodies obtained from rabbits. 51 patients with MCD and 91 with MN were finally included. RESULTS: Our study included a final sample of 91 patients with MCD and 51 with MN. No C4d deposition was observed in any of the glomeruli of patients with MCD, and 100% of these patients were classified as "negative". C4d was detected in 100% of patients with NM in the form of a uniform granular distribution that outlined all the capillary loops and spared the mesangium. Detectable C1q deposits by IF were detected in only two patients with MN and was classified as negative in all patients diagnosed of MCD. CONCLUSIONS: The demonstration of C4d by means of immunohistochemicaltechniques can thus be used as a tool for the differential diagnosis of MN andMCD. The deposit of C4d and no C1q deposit suggest that the alternative and/or lectin pathways might be predominantly involved in complement activation and formation of the C5b-9 complex in MN.
A protein with Na+/P, co-transporter activity has been extracted from rabbit brush-border membran... more A protein with Na+/P, co-transporter activity has been extracted from rabbit brush-border membranes with chloroform/methanol and purified by hydroxyapatite chromatography. The protein has been incorporated by the dilution method into liposomes formed from different types and ratios of lipids. The greatest reconstitution has been achieved into liposomes prepared from cholesterol (20 %), phosphatidylcholine (20 %), phosphatidylethanolamine (30 %) and phosphatidylserine (30 %) (CH/PC/PE/PS). Pi uptake by these proteoliposomes had the following characteristics: (i) the initial rate was markedly greater in the presence of an inwardly directed Na+ gradient (600 pmol/l0 s per mg) than with a K+ gradient (65 pmol/ I0 s per mg); (ii) maximal uptake was increased 8-fold above the equilibrium value ('overshoot') when a Na+ gradient was applied; (iii) Pi was not merely bound to proteoliposomes but was transported intravesicularly; and (iv) Na+-dependent Pi uptake was sensitive to the known phosphate transport inhibitors. This first successful attempt of reconstitution of Na+/Pi transport activity into proteoliposomes led us to isolate and characterize physico-chemically the protein responsible. Its isoelectric point was about 5.8, and urea/SDS gel electrophoresis revealed a broad band of molecular mass ranging from 63 to 66 kDa under both reducing and non-reducing conditions. In the native form, the molecular mass analysed by gel filtration was estimated to be 170+10 kDa, suggesting that the protein is a polymer, probably stabilized by hydrophobic bonds. Endoglycosidase F treatment decreased the molecular mass to approx. 50 kDa. It is postulated that this acidic glycoprotein might represent a subunit of the intact Na+/P1 co-transporter from rabbit kidney brush-border membranes.
Purpose: The L1 cell adhesion molecule is overexpressed in many human carcinomas. The objectives ... more Purpose: The L1 cell adhesion molecule is overexpressed in many human carcinomas. The objectives of the study were to provide a comprehensive description of L1 distribution in human kidney and to establish the prognostic relevance of L1 expression in renal cell carcinomas (RCC).Experimental Design: Using two antibodies to the extracellular part and the cytoplasmic domain, respectively, we first compared L1 expression in normal kidney and renal tumors of diverse histopathologic origin, then we studied L1 expression together with tumor stage, grade, molecular prognostic biomarkers, and metastatic behavior.Results: In normal kidney, L1 immunoreactive with both antibodies was expressed in all epithelial cells originating from the ureteric bud except for intercalated cells. In renal tumors, L1 was mainly detected in those originating from cells that do not express L1 in the normal kidney [i.e., 33 of 72 clear cell RCC (ccRCC) and 25 of 88 papillary RCC (papRCC)]. Both in ccRCC and papRCC...
ABSTRACT Membranous nephropathy (MN) is characterized by an accumulation of immune deposits on th... more ABSTRACT 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 antigen is neutral endopeptidase (NEP), the alloantigen involved in neonatal cases of MN that occur in newborns from NEP-deficient mothers. The second antigen, which is also the first autoantigen identified in idiopathic MN in the adult, is the M-type phospholipase A(2) receptor (PLA(2)R). Both NEP and PLA(2)R are present on the podocyte surface where they can serve as targets for circulating antibodies. Antibodies against cytoplasmic podocytic antigens, such as aldose reductase, superoxide dismutase-2 and alpha-enolase, have also been reported although their role in the pathogenesis remains controversial because they are not present at the podocyte membrane in normal conditions. In addition to podocyte antigens, a circulating antigen, cationic bovine serum albumin, was shown to be implicated in cases of early childhood MN. This suggests that dietary antigens may be involved through charge-dependent binding to anionic glomerular capillary wall and in situ formation of immune complexes. This review will be focused on non-PLA(2)R antigens. (C) 2014 S. Karger AG, Basel
Over the past few years, considerable advances have been made in our understanding of the molecul... more 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 ...
Background: Membranous nephropathy (MN) is the most common histological diagnosis in adults with ... more Background: Membranous nephropathy (MN) is the most common histological diagnosis in adults with nephrotic syndrome and a leading cause of end-stage kidney failure from glomerulonephritis. Little is known about the underlying aetiology, although anti-glomerular antibodies have been implicated. No specific underlying genetic defect has yet been identified. Methods: In a family with four members in three generations affected by primary MN, the serum of affected members and their mothers were assessed for anti-glomerular antibodies. Results: All four affected are male and connected through the maternal line, indicative of X-linked inheritance. Age of onset of nephrotic syndrome varied between 1 and 67 years of age, suggesting that a potential underlying gene may confer a genetic predisposition to MN, but other factors, genetic or environmental, are necessary to trigger the disease. Serologic studies revealed antibodies against glomerular and peritubular endothelial cells in the mother ...
Patients with hereditary hemorrhagic telangiectasia have increased plasma levels of vascular endo... more Patients with hereditary hemorrhagic telangiectasia have increased plasma levels of vascular endothelial growth factor and transforming growth factor-beta1 as well as high ALK1 tissue expression. Haematologica 2005;90:818-28.
L1, a member of the immunoglobulin superfamily, is a cell adhesion and signal transducing molecul... more L1, a member of the immunoglobulin superfamily, is a cell adhesion and signal transducing molecule. In the kidney, L1 is expressed in the mesonephric duct and the metanephros throughout collecting duct development. We show that mice with a targeted deletion of the L1 gene display diverse renal malformations including (i) a duplex kidney with two ureters partially or totally separated, accompanied by hydronephrosis; and (ii) an enlarged elongated kidney with a malformed or incorrectly positioned inner medulla. The type, penetrance and severity of these phenotypes are influenced by the genetic background. The development of a duplex kidney is initiated by double ureteral budding from the Wolffian duct or by an accessory budding from the main ureter, whereas medullary malformation is due to an improper growth and branching pattern of ureteral branches. Multiple developmental defects in formation of the collecting system promote subsequent renal damage and progression to renal insufficiency. Various features of mouse ureteral duplication resemble the human congenital anomalies of the kidney and urinary tract (CAKUT) although disturbances of medulla development have not yet been reported in men.
A 62-year-old woman presented with nephrotic syndrome, monoclonal gammopathy, and membranouslike ... more A 62-year-old woman presented with nephrotic syndrome, monoclonal gammopathy, and membranouslike nephropathy with nonorganized deposits composed of monoisotypic immunoglobulin G1 protein. Nephrotic syndrome remitted after a brief course of treatment with melphalan despite ongoing production of the monoclonal protein. The circulating monoclonal immunoglobulin G1 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.
Introduction and Aims: Hypertriglyceridemia is a cardinal feature of nephrotic syndrome. A recent... more Introduction and Aims: Hypertriglyceridemia is a cardinal feature of nephrotic syndrome. A recent study (Clement Nature Medicine 2011) shows that podocyte secreted, but not circulating Angiopoietin-like-4 (Angptl4) also noted in minimal change disease, induces proteinuria. Here, we investigated the role of circulating Angptl4 in nephrotic syndrome. Methods: Models of proteinuria were induced in Wild-Type and KnockOut Angptl4 mice, and in Wild-Type and generated Angptl4 transgenic rats. Proteinuria, albuminuria and hypertriglyceridemia were studied in those animals. Stable transfection was realized in HEK293 cells in order to produce recombinant Angptl4. Recombinant Angptl4 was injected into animals and incubated with rat glomerular endothelial cells. Immunogold electron microscopy was used to determine glomerular localization of Angptl4. Results: Circulating Angptl4 is known to inhibit endothelium bound lipoprotein lipase, which reduces tissue uptake of triglycerides from circulation, resulting in hypertriglyceridemia. We induced the gamma2-NTS (gamma2-Nephrotoxic Serum) and LPS (Lipopolysaccharides) models of proteinuria in Angptl4 −/− and + / + mice. Angptl4 −/− mice did not develop hypertriglyceridemia despite significant residual proteinuria, suggesting that Angptl4 is the key determinant of hypertriglyceridemia in nephrotic syndrome. Next, we studied albuminuria in adipose tissue specific Angptl4 transgenic rats (aP2-Angptl4), that have hypertriglyceridemia due to increased circulating Angptl4, and noted reduced albuminuria in transgenic compared to wild type rats (P < 0.05). Induction of PAN (Puromycin Aminonucleoside) in these rats caused lower levels of proteinuria (Day 8 wild type 315 + 2 mg/18 hours, transgenic 194 + 25 mg/18 hours, mean + SE, P < 0.05). Immunogold EM showed binding of Angptl4-V5 to glomerular endothelial surface. Cultured rat glomerular endothelial cells were protected from oxidative stress by recombinant neutral pI Angptl4 similar to that present in the circulation (P < 0.001). By inducing puromycin nephrosis in podocyte specific Angptl4 transgenic rats (NPHS2-Angptl4), we determined that beyond the initial stages, the bulk of the circulating Angptl4 is contributed to by peripheral tissues, predominantly adipose tissue. In addition, injection of recombinant neutral pI Angptl4 reduces proteinuria in multiple animal models of proteinuria. Conclusions: Our studies suggest that in later stages of nephrotic syndrome, adipose tissue secretes Angptl4 into the circulation to reduce proteinuria by glomerular endothelial stabilization, but this increase in circulating Angptl4 also results in hypertriglyceridemia.
Pediatric steroid-sensitive nephrotic syndrome (pSSNS) is the most common childhood glomerular di... more Pediatric steroid-sensitive nephrotic syndrome (pSSNS) is the most common childhood glomerular disease. Previous genome-wide association studies (GWAS) identified a risk locus in the HLA Class II region and three additional independent risk loci. But the genetic architecture of pSSNS, and its genetically driven pathobiology, is largely unknown. Here, we conduct a multi-population GWAS meta-analysis in 38,463 participants (2440 cases). We then conduct conditional analyses and population specific GWAS. We discover twelve significant associations-eight from the multi-population meta-analysis (four novel), two from the multi-population conditional analysis (one novel), and two additional novel loci from the European meta-analysis. Fine-mapping implicates specific amino acid haplotypes in HLA-DQA1 and HLA-DQB1 driving the HLA Class II risk locus. Non-HLA loci colocalize with eQTLs of monocytes and numerous T-cell subsets in independent datasets. Colocalization with kidney eQTLs is lacking but overlap with kidney cell open chromatin suggests an uncharacterized disease mechanism in kidney cells. A polygenic risk score (PRS) associates with earlier disease onset. Altogether, these discoveries expand our knowledge of pSSNS genetic architecture across populations and provide cell-specific insights into its molecular drivers. Evaluating these associations in additional cohorts will refine our understanding of population specificity, heterogeneity, and clinical and molecular associations. Pediatric steroid-sensitive nephrotic syndrome (pSSNS) is a rare disease of the glomerular filtration barrier. Its incidence ranges from 1.15-16.9 cases in every 100,000 children, occurring most frequently in South Asian populations 1. pSSNS causes massive proteinuria and increases the risk of thromboembolism, sepsis, and progression to chronic kidney disease (CKD)/end-stage kidney disease (ESKD) 2-7. And those progressing to ESKD have increased odds of recurrent NS in their allograft 8. pSSNS is impactful across the lifespan-31-50% of those affected have relapses in adulthood 9. Much of pSSNS's morbidity is related to side effects of the non-specific immunosuppressants which allow some to achieve remission of their proteinuria 7, 10-17. Despite intensive investigation, there are no known monogenic forms of pSSNS to illuminate its pathobiology. However, we know that immune dysregulation is a major contributor 18,19. But determining causal immune factors via case-control studies of cytokines profiles, cell types, and transcriptomic signatures is challenging. The dynamic responses of the immune system at different disease stages and to various stimuli make it difficult to determine whether observed differences are causal, correlated, or due to independent biological/
medRxiv (Cold Spring Harbor Laboratory), Sep 14, 2022
doi: medRxiv preprint NOTE: This preprint reports new research that has not been certified by pee... more doi: medRxiv preprint NOTE: This preprint reports new research that has not been certified by peer review and should not be used to guide clinical practice.
Journal of Histochemistry and Cytochemistry, Jan 6, 2006
The HNK-1 carbohydrate epitope is a 3-sulfo-glucuronyl residue attached to lactosamine structures... more 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.
Journal of The American Society of Nephrology, Jun 14, 2018
Background Steroid-sensitive nephrotic syndrome (SSNS) is a childhood disease with unclear pathop... more Background Steroid-sensitive nephrotic syndrome (SSNS) is a childhood disease with unclear pathophysiology and genetic architecture. We investigated the genomic basis of SSNS in children recruited in Europe and the biopsy-based North American NEPTUNE cohort. Methods We performed three ancestry-matched, genome-wide association studies (GWAS) in 273 children with NS (Children Cohort Nephrosis and Virus [NEPHROVIR] cohort: 132 European, 56 African, and 85 Maghrebian) followed by independent replication in 112 European children, transethnic meta-analysis, and conditional analysis. GWAS alleles were used to perform glomerular cis-expression quantitative trait loci studies in 39 children in the NEPTUNE cohort and epidemiologic studies in GWAS and NEPTUNE (97 children) cohorts. Results Transethnic meta-analysis identified one SSNS-associated single-nucleotide polymorphism (SNP) rs1063348 in the 39 untranslated region of HLA-DQB1 (P59.3310 223). Conditional analysis identified two additional independent risk alleles upstream of HLA-DRB1 (rs28366266, P53.7310 211) and in the 39 untranslated region of BTNL2 (rs9348883, P59.4310 27) within introns of HCG23 and LOC101929163. These three risk alleles were independent of the risk haplotype DRB1*07:01-DQA1*02:01-DQB1*02:02 identified in European patients. Increased burden of risk alleles across independent loci was associated with higher odds of SSNS. Increased burden of risk alleles across independent loci was associated with higher odds of SSNS, with younger age of onset across all cohorts, and with increased odds of complete remission across histologies in NEPTUNE children. rs1063348 associated with decreased glomerular expression of HLA-DRB1, HLA-DRB5, and HLA-DQB1. Conclusions Transethnic GWAS empowered discovery of three independent risk SNPs for pediatric SSNS. Characterization of these SNPs provide an entry for understanding immune dysregulation in NS and introducing a genomically defined classification.
Journal of The American Society of Nephrology, Feb 21, 2013
The M-type receptor for phospholipase A2 (PLA2R1) is the major target antigen in idiopathic membr... more 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 (,1%) in the Single Nucleotide Polymorphism Database or the 1000 Genomes project, and 9 known to be common polymorphisms. Although we confirmed significant associations among 6 of the identified common variants and iMN, only 9 patients had the private or rare variants, and only 4 of these patients were among the 60 who were PLA2R positive. In conclusion, rare variants in the coding sequence of PLA2R1, including splice sites, are unlikely to explain the pathogenesis of iMN.
We immunopurified a surface antigen specific for the collecting duct (CD) epithelium. Microsequen... more We immunopurified a surface antigen specific for the collecting duct (CD) epithelium. Microsequencing of three polypeptides identified the antigen as the neuronal cell adhesion molecule L1, a member of the immunoglobulin superfamily. The kidney isoform showed a deletion of exon 3. L1 was expressed in the mesonephric duct and the metanephros throughout CD development. In the adult CD examined by electron microscopy, L1 was not expressed on intercalated cells but was restricted to CD principal cells and to the papilla tall cells. By contrast, L1 appeared late in the distal portion of the elongating nephron in the mesenchymally derived epithelium and decreased during postnatal development. Immunoblot analysis showed that expression, proteolytic cleavage, and the glycosylation pattern of L1 protein were regulated during renal development. L1 was not detected in epithelia of other organs developing by branching morphogenesis. Addition of anti-L1 antibody to kidney or lung organotypic cultures induced dysmorphogenesis of the ureteric bud epithelium but not of the lung. These results suggest a functional role for L1 in CD development in vitro. We further postulate that L1 may be involved in the guidance of developing distal tubule and in generation and maintenance of specialized cell phenotypes in CD.
Idiopathic membranous nephropathy (MN) is the most frequent cause of the nephrotic syndrome in ad... more Idiopathic membranous nephropathy (MN) is the most frequent cause of the nephrotic syndrome in adults. The diagnosis is based on typical findings observed via electron microscopy (EM) and immunofluorescence (IF) studies. On some occasions, tissues are only available for analysis using an optical microscope (OM); in these cases, it can be difficult to differentiatebetween MN and minimal change disease (MCD). Recent advances have shown that MN is a kidney specific autoimmune disease induced by antibodies specific for podocyte antigens. Complement plays an important role, even if mechanisms of activation have not been clarified yet. C4d is a fragment of C4 that is produced during activation of the classical or lectin complement pathway. We might therefore expect to find C4d deposition as a marker of complement activation in MN. The aim of our study was to determine whether immunohistochemical detection of C4d in patients with MN could be useful as a diagnostic tool. METHODS: All adult patients diagnosed with idiopathic minimal change disease (MCD) and MN biopsied in our unit between January 2001-December 2016 were considered for inclusion in the study. Diagnoses of MCD and MN were based on histological assessment of renal biopsy tissue with LM, IF and EM studies. In October 2008, 3lm sections of renal tissue fixed in formaldehyde were removed from paraffin and rehydrated. The samples were then stained for C4d immunohistochemical analysis using anti-human polyclonal antibodies obtained from rabbits. 51 patients with MCD and 91 with MN were finally included. RESULTS: Our study included a final sample of 91 patients with MCD and 51 with MN. No C4d deposition was observed in any of the glomeruli of patients with MCD, and 100% of these patients were classified as "negative". C4d was detected in 100% of patients with NM in the form of a uniform granular distribution that outlined all the capillary loops and spared the mesangium. Detectable C1q deposits by IF were detected in only two patients with MN and was classified as negative in all patients diagnosed of MCD. CONCLUSIONS: The demonstration of C4d by means of immunohistochemicaltechniques can thus be used as a tool for the differential diagnosis of MN andMCD. The deposit of C4d and no C1q deposit suggest that the alternative and/or lectin pathways might be predominantly involved in complement activation and formation of the C5b-9 complex in MN.
A protein with Na+/P, co-transporter activity has been extracted from rabbit brush-border membran... more A protein with Na+/P, co-transporter activity has been extracted from rabbit brush-border membranes with chloroform/methanol and purified by hydroxyapatite chromatography. The protein has been incorporated by the dilution method into liposomes formed from different types and ratios of lipids. The greatest reconstitution has been achieved into liposomes prepared from cholesterol (20 %), phosphatidylcholine (20 %), phosphatidylethanolamine (30 %) and phosphatidylserine (30 %) (CH/PC/PE/PS). Pi uptake by these proteoliposomes had the following characteristics: (i) the initial rate was markedly greater in the presence of an inwardly directed Na+ gradient (600 pmol/l0 s per mg) than with a K+ gradient (65 pmol/ I0 s per mg); (ii) maximal uptake was increased 8-fold above the equilibrium value ('overshoot') when a Na+ gradient was applied; (iii) Pi was not merely bound to proteoliposomes but was transported intravesicularly; and (iv) Na+-dependent Pi uptake was sensitive to the known phosphate transport inhibitors. This first successful attempt of reconstitution of Na+/Pi transport activity into proteoliposomes led us to isolate and characterize physico-chemically the protein responsible. Its isoelectric point was about 5.8, and urea/SDS gel electrophoresis revealed a broad band of molecular mass ranging from 63 to 66 kDa under both reducing and non-reducing conditions. In the native form, the molecular mass analysed by gel filtration was estimated to be 170+10 kDa, suggesting that the protein is a polymer, probably stabilized by hydrophobic bonds. Endoglycosidase F treatment decreased the molecular mass to approx. 50 kDa. It is postulated that this acidic glycoprotein might represent a subunit of the intact Na+/P1 co-transporter from rabbit kidney brush-border membranes.
Purpose: The L1 cell adhesion molecule is overexpressed in many human carcinomas. The objectives ... more Purpose: The L1 cell adhesion molecule is overexpressed in many human carcinomas. The objectives of the study were to provide a comprehensive description of L1 distribution in human kidney and to establish the prognostic relevance of L1 expression in renal cell carcinomas (RCC).Experimental Design: Using two antibodies to the extracellular part and the cytoplasmic domain, respectively, we first compared L1 expression in normal kidney and renal tumors of diverse histopathologic origin, then we studied L1 expression together with tumor stage, grade, molecular prognostic biomarkers, and metastatic behavior.Results: In normal kidney, L1 immunoreactive with both antibodies was expressed in all epithelial cells originating from the ureteric bud except for intercalated cells. In renal tumors, L1 was mainly detected in those originating from cells that do not express L1 in the normal kidney [i.e., 33 of 72 clear cell RCC (ccRCC) and 25 of 88 papillary RCC (papRCC)]. Both in ccRCC and papRCC...
ABSTRACT Membranous nephropathy (MN) is characterized by an accumulation of immune deposits on th... more ABSTRACT 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 antigen is neutral endopeptidase (NEP), the alloantigen involved in neonatal cases of MN that occur in newborns from NEP-deficient mothers. The second antigen, which is also the first autoantigen identified in idiopathic MN in the adult, is the M-type phospholipase A(2) receptor (PLA(2)R). Both NEP and PLA(2)R are present on the podocyte surface where they can serve as targets for circulating antibodies. Antibodies against cytoplasmic podocytic antigens, such as aldose reductase, superoxide dismutase-2 and alpha-enolase, have also been reported although their role in the pathogenesis remains controversial because they are not present at the podocyte membrane in normal conditions. In addition to podocyte antigens, a circulating antigen, cationic bovine serum albumin, was shown to be implicated in cases of early childhood MN. This suggests that dietary antigens may be involved through charge-dependent binding to anionic glomerular capillary wall and in situ formation of immune complexes. This review will be focused on non-PLA(2)R antigens. (C) 2014 S. Karger AG, Basel
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Papers by Hanna Debiec