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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.

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.

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.

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 V H Oxl gene (family Q52) and an L chain usually coded for by V K Oxl but occasionally by other V K genes. Id350-positive antibodies are encoded by a V K gene V K 45.1, and usually by a V H 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.

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.

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.

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.

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 mouse genome contains four functional J H genes, which encode immunoglobulin heavy chain joining segments. The J H 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 J H 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 J H , variable (V) and constant (C) region sequences we conclude that independent reshuffling of V H ,J H , and C H gene clusters occurred during the evolution of Mus musculus.

A VH gene (Ox1) has a major role in the early antibody response of several mouse strains to hapten phenyloxazolone (phOx). Antibodies that are coded by this gene are positive for idiotype 495. Idiotype-positive monoclonal antibodies originating from the early primary response of nine strains were partially sequenced (mRNA). All 21 antibodies were coded by this gene, most of them also by one VL gene, VKOx1(H3). Very few somatic mutations were found, and the germ-line sequence of the two genes in several strains can be predicted. Four 'alleles' of the VHOx1 gene have 99-99.7% sequence homology to each other. One allele was found in Igh allotype j strains CBA and C3H, another in allotype c strains DBA/2 and RF, the third in allotype f strain CE and the fourth in BALB/c, 129, A/J and RIII mice (allotypes a, e or g). The VKOx1(H3) gene has the same sequence in eight strains. RF mice do not use this gene for the anti-phOx response. Our data suggest that antibody responses are inherited to a considerable extent and that immunoglobulin V genes are as stable as other genes in evolution.

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.