Recurrence of nephrotic syndrome after transplantation in CNF is due to autoantibodies to nephrin.

The novel gene NPHS1 is defective in the patients with congenital nephrotic syndrome of the Finnish type (CNF) leading to abnormal expression of the respective protein product nephrin in glomerular cells. CNF patients are treated with early nephrectomy and renal transplantation, but about 20% show recurrence of nephrotic syndrome (NS). We used indirect immunofluorescence microscopy and immunoblotting and an ELISA assay to search for circulating autoantibodies to nephrin, the protein defect in CNF patient kidneys. In serial serum samples gathered before and after recurrence of NS, we show an increased antibody titer to nephrin prior to the NS episode and a subsequent drop in antibody level after its successful treatment and reactivity of the high titer sera with glomeruli in indirect immunofluorescence microscopy as well. The results show that the transplantation treatment introduces a neoantigen inducing production of autoantibodies, which may be pathogenic for perturbation of the function of the glomerular filtration barrier.

Lipid peroxidation in human proteinuric disease.

BACKGROUND: While metabolically generated oxidants are produced locally in experimental glomerular diseases, little is still known of their significance and the respective scavenger systems in human glomerular diseases. METHODS: Here we studied kidneys from patients with congenital nephrotic syndrome of the Finnish type (CNF), a human model disease of isolated proteinuria. Expression of specific mRNAs for a major antioxidant system against lipoperoxidation [phospholipid hydroperoxide glutathione peroxidase (PHGPx)] and for mitochondrial proteins were studied in Northern blotting together with analysis of PHGPx in semiquantitative reverse transcription-polymerase chain reaction (RT-PCR). The respective proteins and lipoperoxide (LPO) adducts malonyldialdehyde (MDA) and 4-hydroxynonenal (4-HNE) were analyzed in immunohistochemistry. RESULTS: PHGPx and the mitochondrially encoded subunits of cytochrome-c-oxidase were distinctly down-regulated within the glomeruli of CNF kidneys. These changes were confirmed in semiquantitative RT-PCR. Increases of lipoperoxidation products MDA and 4-HNE were constantly found in the glomeruli of CNF. In agreement with findings in CNF, similar results were obtained in biopsies from other human glomerular diseases. CONCLUSIONS: These findings suggest that local mitochondrial damage initiates LPO, which then causes deposition of the cytotoxic LPO products in glomeruli, as seen especially in CNF kidneys. Together with down-regulation of the local antioxidant protection, these may be important pathophysiologic mechanisms in human glomerular disease.

Nephrin in experimental glomerular disease.

BACKGROUND: The recently identified gene NPHS1 with its mutations causing congenital nephrotic syndrome of the Finnish type (CNF) is highly promising in providing new understanding of pathophysiology of proteinuria. Earlier we cloned a rat NPHS1 homologue, as well as characterized and raised antibodies to the respective protein product nephrin. METHODS: Changes in the expression levels of nephrin-specific mRNA in commonly used experimental models of proteinuria were examined using semiquantitative reverse transcription-polymerase chain reaction, immunofluorescence, and immunoelectron microscopy (IEM) of nephrin. RESULTS: Notably, a 40% down-regulation of the nephrin-specific mRNA of cortical kidney was seen already at day 3 after induction of the puromycin aminonucleoside nephrosis (PAN), while no major elevation of urinary protein secretion was seen at this stage. A further decrease of 80% of nephrin message was seen at the peak of proteinuria at day 10. A similar decrease of up to 70% from the basal levels was seen in mercuric chloride-treated rats. Changes in the protein expression paralleled those of the mRNA in indirect immunofluorescence. Interestingly, a remarkable plasmalemmal dislocation from the normal expression site at the interpodocyte filtration slits could be observed in IEM. CONCLUSIONS: Nephrin appears to be an important causative molecule of proteinuria and shows a remarkable redistribution from the filtration slits to the podocyte plasma membrane, especially in PAN.

Mitochondrial dysfunction in congenital nephrotic syndrome.

The molecular mechanisms maintaining the kidney glomerular filtration barrier remain poorly understood. Recent evidence suggests that mitochondrial dysfunction is a characteristic feature of kidney glomeruli in congenital nephrotic syndrome of the Finnish type (CNF). Here we searched for detailed functional evidence of mitochondrial lesion in CNF kidneys. We used histochemical and immunohistochemical methods, quantitative measurement of mitochondrial DNA, and superoxide production to characterize the mitochondrial function. The results unequivocally show down-regulation of mitochondria-encoded respiratory chain components, whereas the respective nuclearly encoded subunits were close to normal. These results give detailed evidence of distinct mitochondrial dysfunction and of the resulting abnormal production of reactive oxygen species in CNF and suggest a critical role for mitochondria in maintaining the glomerular permeability barrier.

Nephrin localizes at the podocyte filtration slit area and is characteristically spliced in the human kidney.

Defects in the newly reported gene NPHS1 in chromosome 19 cause the massive proteinuria of Finnish type congenital nephrotic syndrome (CNF). Together with its gene product, nephrin, NPHS1 is providing new understanding of the pathophysiological mechanisms of glomerular filtration. Here we show the characteristic splicing of NPHS1 mRNA in the normal and CNF kidneys and localize nephrin exclusively in the glomerulus and to the filtration slit area by light and immunoelectron microscopy. These results indicate that nephrin is a new protein of the interpodocyte filtration slit area with a profound role in the pathophysiology of the filtration barrier.

Cloning and expression of the rat nephrin homolog.

Despite of the increased availability of genetically modified mouse strains, the experimental models in the rat have provided the most widely employed and versatile models for the study of renal pathophysiology and functional genetics. The identification of the human gene mutated in the congenital nephrotic syndrome of the Finnish type (NPHS1) has recently been reported, and its protein product has been termed nephrin. Here we report the molecular cloning and characterization of rat nephrin cDNA. Rat nephrin cDNA has an open reading frame of 3705 bp, shows 82% sequence identity with human nephrin cDNA, and shows characteristic rat-specific splicing variants. The translated nucleotide sequence has 89% sequence identity at the amino acid level. The signal sequence, glycosylation, and cysteine localization patterns are nearly identical to those of human nephrin. As in the human, the rat nephrin transcript is expressed in a tissue-restricted pattern. Antipeptide antibodies raised to the intracellular nephrin-specific domain identified immunoreactivity exclusively within the rat kidney glomerulus by indirect immunofluorescence. Initial results with semiquantitative reverse transcriptase-polymerase chain reaction analysis showed a remarkable down-regulation of nephrin-specific mRNA in the puromycin nephrosis of the rat.

Podocalyxin in rat platelets and megakaryocytes.

Podocalyxin is a membrane protein of rat podocytes and endothelial cells. It has not been described in other cell types, and no amino acid or DNA sequence data are available about it. Here we show that podocalyxin antigens are present in rat platelets and megakaryocytes. In resting platelets, the antigens are mainly intracellular but become surface exposed after thrombin stimulation, as shown by immunofluorescence and flow cytometry. By Western blotting, platelet podocalyxin has an apparent Mr of 140,000. Cytocentrifuge slides of rat bone marrow show that anti-podocalyxin antibodies recognize large polyploid cells also expressing CD62P, indicating that the cells are megakaryocytes. From a rat glomerular cDNA library we isolated a clone covering the carboxyl-terminal nucleotides of rat podocalyxin. Its putative transmembrane or intracellular domains are 100% or >93% identical, respectively, with the human and rabbit podocalyxin-like proteins. The truncated extracellular domain extends to include two of the four conserved cysteines shared by podocalyxin-like proteins. By Northern blotting, a 5.5-kb renal cortical transcript is seen. By in situ hybridization, cRNA probes recognize podocytes, endothelial cells, and megakaryocytes, and by reverse transcription polymerase chain reaction, platelets are shown to contain podocalyxin mRNA. Our results show that rat podocalyxin is a homologue of the previously cloned podocalyxin-like proteins and suggest that also in mammals podocalyxin has a role in hematopoiesis, as previously shown in the chicken.

Altered gene expression and functions of mitochondria in human nephrotic syndrome.

The molecular basis of glomerular permselectivity remains largely unknown. The congenital nephrotic syndrome of the Finnish type (CNF) characterized by massive proteinuria already present but without extrarenal symptoms is a unique human disease model of pure proteinuria. In search of genes and pathophysiologic mechanisms associated with proteinuria, we used differential display-PCR to identify differences in gene expression between glomeruli from CNF and control kidneys. A distinctly underexpressed PCR product of the CNF kidneys showed over 98% identity with a mitochondrially encoded cytochrome c oxidase (COX I). Using a full-length COX I cDNA probe, we verified down-regulation of COX I mRNA to 1/4 of normal kidney values on Northern blots. In addition, transcripts of other mitochondrially encoded respiratory chain complexes showed a similar down-regulation whereas the respective nuclearly encoded complexes were expressed at comparable levels. Additional studies using histochemical, immunohistochemical, in situ hybridization, RT-PCR, and biochemical and electron microscopic methods all showed a mitochondrial involvement in the diseased kidneys but not in extrarenal blood vessels. As a secondary sign of mitochondrial dysfunction, excess lipid peroxidation products were found in glomerular structures in CNF samples. Our data suggest that mitochondrial dysfunction occurs in the kidneys of patients with CNF, with subsequent lipid peroxidation at the glomerular basement membrane. Our additional studies have revealed similar down-regulation of mitochondrial functions in experimental models of proteinuria. Thus, mitochondrial dysfunction may be a crucial pathophysiologic factor in this symptom.

Morphologic changes suggesting abnormal renal differentiation in congenital nephrotic syndrome.

Retrograde differentiation (or dedifferentiation) has recently been proposed as a pathogenetic mechanism involved also in various renal diseases. Here we studied whether evidence of these mechanisms can be found in the kidneys of patients with congenital nephrotic syndrome of the Finnish type (CNF). These patients show isolated massive proteinuria but no primary symptoms from any other organ systems. For the analysis we used antibody markers of early (fibronectin, stem cell factor, Wilms' tumor gene product, cytokeratin) and later (laminin, midgestation and kidney, heparin binding growth-associated molecule) stages of nephron differentiation as well as for apoptosis (acridine orange staining), rescue from apoptosis (anti-Bcl-2 antibodies) and cell proliferation (antibodies to proliferating cell nuclear antigen). In the peritubular spaces atypically organized areas were found which appeared positive with markers of low stages of differentiation, but neither abnormal cell proliferation nor activation of the apoptotic pathway could be detected. As morphologic signs of abnormal tissue organization, we found clusters of tightly compacted and large glomeruli corresponding to the size of two to three normal glomeruli. However, all individual glomerular cell compartments (mesangial, endothelial, visceral epithelial cells) appeared balanced in relative cell numbers. Together these results may indicate abnormal early mesenchymoepithelial tissue interaction leading to excessive and poorly organized formation of glomeruli. This could be causally related also to the serious functional immaturity of CNF kidneys presented as isolated proteinuria.

Expression of nine developmental stage-specific genes in human kidney and cultured renal cells.

BACKGROUND: Several genes transiently expressed during the maturation of the metanephrogenic mesenchyme have been reported in recent years while there is accumulating evidence of a reverted developmental pathway during tissue damage and loss of function. METHODS: Here we studied the expression of nine genes associated with kidney maturation from samples of normal human fetal, juvenile and adult kidneys and cultured glomerular cells using Northern blotting analysis. Subsequently, kidneys from patients with congenital nephrotic syndrome of the Finnish type (CNF), presenting with heavy proteinuria, and Wilms' tumor tissue were studied for the corresponding expression pattern for evidence of dedifferentiation/persistence of a fetal expression pattern. RESULTS: All the genes studied had their distinct expression pattern within the tissues and cells tested. No conclusive evidence of dedifferentiation could be obtained in the samples from CNF kidneys, whereas, as expected, the gene expression pattern of Wilms' tumor tissue was highly similar to that of fetal kidney tissue. CONCLUSION: Some genes thought to be only transiently expressed during kidney maturation were, however, constantly found in the samples from fetal to newborn and adult kidneys.

Mechanisms of proteinuria: vascular permeability factor in congenital nephrotic syndrome of the Finnish type.

Vascular permeability factor (VPF) is the most potent known mediator of vessel wall permeability. In the kidney, it is expressed preferentially in the glomerular visceral epithelial cells. The present study was designed to clarify the proposed role of VPF in diseases with increased glomerular permeability as here exemplified by the congenital nephrotic syndrome of the Finnish type (CNF). For this, we studied the expression levels and the sites of synthesis of VPF and its kinase-insert domain receptor (KDR) in kidneys of patients with CNF using Northern and in situ hybridization techniques and immunohistologic staining with anti-VPF antibody. In addition, we extended the study to include analysis of fetal kidney tissue and cultured glomerular cells of normal and CNF kidneys. In CNF and in normal kidneys VPF was localized in the visceral epithelial aspect of the glomeruli and in the collecting ducts, as also earlier described. A new finding was its localization also in the juxtaglomerular area. The VPF receptor KDR was found in glomeruli in the endothelial cells, but it was not detected in the peritubular capillaries. no consistent differences in the levels of VPF or KDR mRNAs or in their sites of production were seen in CNF and control samples. Also the distribution of VPF antigen in the CNF kidneys and normal kidneys was similar. Thus, we propose that VPF and KDR are not directly involved in the pathogenesis of the proteinuria in CNF.

Sphingolipid activator proteins in a human hereditary renal disease with deposition of disialogangliosides.

Congenital nephrotic syndrome of the Finnish type is a recessively inherited renal disease with glomerular deposits of the disialoganglioside O-acetyl-GD3. Sphingolipid activator proteins (saposins) stimulate the degradation of glycosphingolipids by lysosomal enzymes, and defects in saposins cause accumulation of substrate lipids in the affected tissues in lysosomal storage disease. Here we report a study of the role of saposins in the accumulation of O-acetyl-GD3 in kidneys of congenital nephrotic syndrome patients. At the mRNA level, the expression of saposin precursor in diseased kidneys appeared normal, and the nucleotide sequence analysis of cDNA clones did not reveal abnormalities in the prosaposin gene. Immunohistologically, saposins were localized mainly to the epithelial cells of the distal renal tubules or to the parietal epithelial cells of glomeruli. In the nephrotic syndrome kidneys, the staining pattern was highly granular and appeared mostly in the apical part of the epithelial lining, unlike the control kidneys. These results show that a major site of ganglioside metabolism is located in the distal nephron. Furthermore, these results suggest that saposins are not directly involved in the metabolism of the terminal sialic acids of disialogangliosides in the nephrotic syndrome kidneys.

Decrease of glomerular disialogangliosides in puromycin nephrosis of the rat.

Puromycin aminonucleoside nephrosis (PAN) is a model for human minimal change nephropathy induced in rats by injection of puromycin. In PAN, defective sialylation of a major sialoprotein of podocytes, podocalyxin, has been demonstrated and the consequent decrease of anionic charge suggested as a causative factor for increased glomerular permeability and proteinuria. Whether defective sialylation is a general feature of PAN affecting also glomerular glycosphingolipids is not known. We have shown that rat glomeruli are rich in disialogangliosides GD3 and O-acetyl GD3, the functions of which are not known. Here, we made a sequential analysis of the glomerular gangliosides, especially of GD3 and its O-acetyl derivative in acute PAN using immunohistochemical and biochemical techniques and compared the results with another rat model of glomerular disease, Heymann nephritis. The prominent immunohistochemical finding was the almost total disappearance of glomerular O-acetyl GD3 and a substantial decrease of its precursor GD3 peaking at 10 days after injection of puromycin. Segmental areas lacking these gangliosides remained in glomeruli still at 30 days after injection. The response was dose dependent. Semiquantitative analysis by thin layer chromatograms showed that O-acetyl GD3 was decreased by 41% already at 3 days and by 60% at 10 days after injection of puromycin. Also GD3, the immediate precursor of O-acetyl GD3, was decreased by 20 and 19%, respectively, at 3 and 10 days after injection. At 3 days after injection, overt proteinuria had not started. At these times, no other changes were observed in the glomerular gangliosides. The decrease of glomerular GD3 and O-acetyl GD3 indicates a decrease of GD3 synthase activity and perhaps of O-acetyltransferase activity in PAN nephrosis. As these changes preceded the overt proteinuria, they may have a causal relationship to it. In the glomeruli of Heymann nephritic rats, no similar changes were seen, suggesting that the sialylation defect is not due to proteinuria but is a consequence of targeted puromycin action on cells.

Glomerular antigens in severe hereditary nephrosis.

In search of the basic defect and cell type responsible for the massive treatment-resistant proteinuria of congenital nephrotic syndrome of the Finnish type (CNF), we examined tissue samples of CNF kidneys using established antibody and lectin markers of various glomerular cell types. Markers of vascular endothelium (antibodies to factor VIII and a human homologue of podocalyxin (anti-PHM5) and UEA I lectin) showed no qualitative changes in the endothelial cells of glomeruli or peritubular areas in CNF as compared with controls. Markers of glomerular mesangial cells (antibodies to desmin, smooth muscle actin, RCA I lectin) revealed a secondary increase in mesangial reactivity reflecting the sclerosis and expansion of the mesangial areas in CNF. Markers of visceral epithelial cells (antibodies to a human homologue of podocalyxin, C3b receptor, vimentin, common lymphocytic leukemia antigen, gp44, and the WGA, LFA and, after neuraminidase treatment, PNA lectin) failed to show appreciable qualitative changes in CNF kidney samples. Interestingly, the alpha 2 beta 1 integrins appeared greatly reduced in all CNF samples studied, possibly explaining the mechanisms of CNF-associated proteinuria.

Major and minor breakpoint sites of chromosomal translocation t(14;18) in subtypes of non-Hodgkin's lymphomas.

The frequencies of major breakpoint region (MBR) and minor cluster region (MCR) breakpoint sites of t(14;18) were examined by polymerase chain reaction and Southern blotting in 50 non-Hodgkin's lymphomas with cytogenetic evidence of t(14;18). A translocation breakpoint was detected in 41 cases (82%). The MBR was involved in 66%, and the MCR in 16% of the cases. Most cases in the present series were lymphomas with a follicular or diffuse growth pattern, 38 being of the centroblastic/centrocytic type and eight of the centroblastic type. The series also included four lymphomas of probable non-follicular center cell origin. MBR and/or MCR breakpoints were found in all studied lymphoma subtypes and in the majority of these most of the breakpoints were in the MBR and a minor portion of the breakpoints in the MCR. Our results suggest that a breakpoint site is not related to growth pattern or neoplastic cell type in follicular center cell lymphomas with t(14;18).

Immunoglobulin constant kappa gene alleles in twelve strains of mice.

Genomic DNA for the immunoglobulin (Ig) constant kappa Igk-C gene region was amplified by the polymerase chain reaction (PCR) method and sequenced from twelve commonly used inbred mouse strains. PCR products were used directly as templates in dideoxy-DNA-sequencing, a method which avoids the sequencing errors caused by Taq polymerase, since no cloning step is required. In restriction fragment length polymorphism (RFLP) studies the SJL mouse strain has been shown to belong to a Igk-C allogroup different from other common inbred mouse strains. The BALB/c Igk-C region was sequenced earlier, but our Igk-C sequences clarify the situation and confirm the existence of three Igk-C alleles in inbred mice (Mus musculus domesticus). Mice belonging to the kappa (Igk) haplotype e (SJL) have allele c of the Igk-C gene. The strains belonging to the kappa haplotype [a albino strain, K subline (AKR), PL and d (C58)] have allele a, and all other eight strains belonging to three different Igk haplotypes (b, c, and f) use allele b of the gene. Allele b has at least one (possibly two) nucleotide differences from allele a in the Igk-C region, but five compared to allele c. The allelic sequences also predict two allotypic kappa polypeptide chains among twelve inbred strains. Alleles a and b encode identical polypeptides, but allele c (SJL) has a conserved lysine to arginine substitution in residue 142.

The same few V genes account for a majority of oxazolone antibodies in most mouse strains.

The early primary anti-phenyloxazolone antibodies of 12 mouse strains were studied by determining proportions of two defined subsets id495 (the classical phOx idiotype) and id350. Id495-positive antibodies bear an H chain encoded by VHOx1 gene (family Q52) and an L chain usually coded for by VKOx1 but occasionally by other VK genes. Id350-positive antibodies are encoded by a VK gene VK45.1, and usually by a VH gene of the S107 family. All 12 strains (representing nine H-chain and four kappa-chain haplotypes) produced id350-positive anti-phOx antibodies. While id495 is the predominant major subset in the BALB/c response (originally studied), id350 seems to be the predominant subset of early anti-phOx antibodies in the mouse species. The combined proportion of the two subsets varied from ca. 50 to almost 100% of the total in all strains except C57BL.

Allelic polymorphism of mouse Igh-J locus, which encodes immunoglobulin heavy chain joining (JH) segments.

The mouse genome contains four functional JH genes, which encode immunoglobulin heavy chain joining segments. The JH gene cluster is located a few kilobases 5' from the constant region genes (C genes) on chromosome 12. The polymerase chain reaction (PCR)-technique was used to amplify DNA stretches from mouse genome of approximately 1,340 nucleotides in length containing all four JH genes (Igh-J locus). PCR products were directly used as templates in Sanger's dideoxy-sequencing, and sequences were determined. Twelve inbred mouse strains belonging to ten different Igh-C haplotypes were studied. The strains were: BALB/c, C58/J, RIII, DBA/2, CE, RF, CBA, NZB/J, AKR, C57BL/10, SJL, and A/J. Five allelic forms of the Igh-J locus were found among these strains. The A/J mouse has an allele (e) which differs from the BALB/c allele (a) by 15 nucleotides. C57BL and SJL have the allele (b) with eight differences from BALB/c. The CBA allele (j) has two differences, and the CE allele (f) has a single nucleotide difference compared with the BALB/c sequence. Based on the JH, variable (V) and constant (C) region sequences we conclude that independent reshuffling of VH, JH, and CH gene clusters occurred during the evolution of Mus musculus.

V genes of oxazolone antibodies in 10 strains of mice.

One pair of V genes (V kappa 45.1 and V11) code for a great portion of phenyloxazolone (anti-phOx) antibodies in 10 strains of mice. This combination replaces the first-known major combination VHOx1-V kappa Ox1 in some strains, and is important in most strains. C57BL/10 and SJL mice have an additional subset of antibodies encoded by genes V kappa 45.1 and V13 (a relative of V11). All three genes involved (V kappa 45.1, V11 and V13) have "allelic" variation. Four alleles of V11 were found, one in Igh haplotypes a, c and g, the second in haplotypes d, j and n, the third in b, and the fourth in f. The most distant alleles d, j, n and f had 10 nucleotide differences out of 429 determined (97.7% homology). Only one allele of the V13 gene was found from anti-phOx hybridomas but two others have been published. Three alleles of the V kappa 45.1 gene were found; one in NZB mice (Ig kappa haplotype b) another in CE (haplotype f), and the third in eight strains including representatives of three Ig kappa haplotypes (a, c and e). The three alleles had greater than 99.0% homology. The V11 and V13 genes that code for anti-phOx antibodies in C57BL/10 and SJL mice were different from the related genes found from the C57BL/10 germ line. C57BL/10 mice must have a chromosome bearing two V11 and two V13 genes. RF mice were found to have two V11 genes, and both code for anti-phOx antibodies. Our data show that the majority of antibodies in the anti-phOx response are encoded by the same restricted collection of V genes in most mouse strains. Antibody responses appear to be no less heritable than other functions of the body.