Congenital nephrotic syndrome of the Finnish type (NPHS1) is an autosomal-recessive disorder, characterized by massive proteinuria in utero and nephrosis at birth. In this study, the 150 kb critical region of NPHS1 was sequenced, revealing the presence of at least 11 genes, the structures of 5 of which were determined. Four different mutations segregating with the disease were found in one of the genes in NPHS1 patients. The NPHS1 gene product, termed nephrin, is a 1241-residue putative transmembrane protein of the immunoglobulin family of cell adhesion molecules, which is specifically expressed in renal glomeruli. The results demonstrate a crucial role for this protein in the development or function of the kidney filtration barrier (Kestilä, 1998).
Mutation of the protein nephrin, encoded by the NPHS1 gene, singly results in the cellular alterations that result in foot process effacement and nephrotic range proteinuria; the recognition of these results emphasizes the pivotal role that this protein plays in regulating glomerular filter integrity. A cDNA including the full-length mouse nephrin open reading frame has been cloned and sequenced and immuno-affinity purified polyclonal antiserum directed against the cytoplasmic domain of mouse nephrin has been developed. Nephrin identified in mouse glomerular extract is a glycoprotein with an apparent molecular mass of 185 kDa. Nephrin is located only in visceral glomerular epithelial cells, where it was targeted to intercellular junctions of mature podocyte foot processes. In developing glomeruli of newborn mouse, antinephrin immunolocalizes to the earliest slit pore regions between differentiating podocytes, sites where slit diaphragms first become visible. Nephrin likely participates in cell-cell interactions between podocyte foot processes and may represent a component of the slit diaphragm (Holzman, 1999).
The size and location of nephrin, the first protein to be identified at the glomerular podocyte slit diaphragm, is described in this study. In Western blots, nephrin antibodies generated against the two terminal extracellular Ig domains of recombinant human nephrin recognize a 180-kDa protein in lysates of human glomeruli and a 150-kDa protein in transfected COS-7 cell lysates. In immunofluorescence, antibodies to this transmembrane protein reveal reactivity in the glomerular basement membrane region, whereas the podocyte cell bodies remain negative. In immunogold-stained thin sections, nephrin label is found at the slit between podocyte foot processes. The congenital nephrotic syndrome of the Finnish type (NPHS1), a disease in which the nephrin gene is mutated, is characterized by massive proteinuria already in utero and lack of slit diaphragm and foot processes. These features, together with the now demonstrated localization of nephrin to the slit diaphragm area, suggest an essential role for this protein in the normal glomerular filtration barrier. A zipper-like model for nephrin assembly in the slit diaphragm is discussed, based on the present and previous data (Ruotsalainen, 1999).
The developmental expression of nephrin, ZO-1 and P-cadherin was examined in normal fetal kidneys and in NPHS1 kidneys. Nephrin and zonula occludens-1 (ZO-1) are first expressed in late S-shaped bodies. During capillary loop stage, nephrin and ZO-1 localize at the basal margin and in the cell-cell adhesion sites between developing podocytes, especially in junctions with ladder-like structures. In mature glomeruli, nephrin and ZO-1 concentrate at the slit diaphragm area. P-cadherin is first detected in ureteric buds, tubules, and vesicle stage glomeruli. Later, P-cadherin is seen at the basal margin of developing podocytes. Fetal NPHS1 kidneys with Fin-major/Fin-major genotype do not express nephrin, whereas the expression of ZO-1 and P-cadherin is comparable to that of control kidneys. Although early junctional complexes proved structurally normal, junctions with ladder-like structures and slit diaphragms were completely missing. The results indicate that nephrin is dispensable for early development of podocyte junctional complexes. However, nephrin appears to be essential for formation of junctions with ladder-like structures and slit diaphragms (Ruotsalainen, 2000).
Nephrin is a central component of the glomerular podocyte slit diaphragm and is essential for the normal renal filtration process. This study describes the complete structure of the mouse nephrin gene, which was shown to be homologous to the human gene, the major difference being 30 exons in the mouse gene as opposed to 29 in human. The complete primary structure of mouse and rat nephrins was also determined. The sequence identity between the mouse and rat proteins is 93%, while both rodent proteins have only about 83% sequence identity with human nephrin. The availability of the three mammalian sequences is significant for the interpretation of sequence variants and mutations in the nephrin gene in patients with congenital nephrotic syndrome. In situ hybridization analyses of whole mouse embryos and tissues has revealed high expression of nephrin in kidney glomeruli and, surprisingly, an intense and highly restricted expression in a set of cells in hindbrain and spinal cord. No expression is observed elsewhere. This expression pattern may explain occasionally occurring neural symptoms caused by inactivating mutations in the nephrin gene in patients with congenital nephrotic syndrome (Putaala, 2000).
CD2-associated protein (CD2AP) is an 80-kilodalton protein that is critical for stabilizing contacts between T cells and antigen-presenting cells. In CD2AP-deficient mice, immune function was compromised, but the mice died at 6 to 7 weeks of age from renal failure. In the kidney, CD2AP was expressed primarily in glomerular epithelial cells. Knockout mice exhibited defects in epithelial cell foot processes, accompanied by mesangial cell hyperplasia and extracellular matrix deposition. Supporting a role for CD2AP in the specialized cell junction known as the slit diaphragm, CD2AP associates with nephrin, the primary component of the slit diaphragm (Shih, 1999).
CD2-associated protein (CD2AP) is an adapter molecule that can bind to the cytoplasmic domain of nephrin, a component of the glomerular slit diaphragm. Mice lacking CD2AP exhibit a congenital nephrotic syndrome characterized by extensive foot process effacement, suggesting that CD2AP-nephrin interactions are critical to maintaining slit diaphragm function. The patterns of expression of both CD2AP and nephrin have been examined in developing mouse and human kidney. Both proteins are first detected in developing podocytes at the capillary loop stage of glomerulogenesis and eventually became concentrated near the glomerular basement membrane. CD2AP is observed diffusely in collecting duct and apically in many cells of proximal and distal tubule. Kidneys from Cd2ap -/- mice initially exhibit normal nephrin localization, but as the mice age and foot processes become effaced, nephrin disappears. In laminin-beta(2) mutant mice exhibiting nephrotic syndrome, CD2AP in glomeruli is aberrantly localized in a primarily punctate pattern. Extensive extrarenal expression of CD2AP is observed in endothelial and epithelial cells, in many cases with a specific subcellular localization. Together, these results suggest that CD2AP is not only involved in maintaining the slit diaphragm but may also have a general role in maintaining specialized subcellular architecture. The severity of kidney disease in Cd2ap mutant mice may have eclipsed manifestation of defects in other tissues (Li, 2000).
CD2AP, an adapter protein containing multiple SH3 domains, plays a critical role in kidney function. Mice lacking CD2AP die soon after birth because of kidney failure. In the kidney, CD2AP is expressed in glomerular podocytes, which suggests that it may play a role in a specialized adhesion complex known as the slit diaphragm. One of the major components of the slit diaphragm is nephrin, a podocyte-specific protein. CD2AP has been demonstrated to localize to the slit diaphragm in podocytes using immunoelectron microscopy, and nephrin and CD2AP co-immunoprecipitate from a podocyte cell line. The specificity of this interaction was verified by mapping studies, which demonstrated that a novel domain at the C terminus of CD2AP interacts with the C-terminal portion of the nephrin cytoplasmic domain. These studies lend further support to the idea that CD2AP plays a role in the structural integrity of the slit diaphragm (Shih, 2001).
Nephrin is a type-1 transmembrane protein and a key component of the podocyte slit diaphragm, the ultimate glomerular plasma filter. Genetic and acquired diseases affecting expression or function of nephrin lead to severe proteinuria and distortion or absence of the slit diaphragm. Using a surface plasmon resonance biosensor this study shows that soluble recombinant variants of nephrin, containing the extracellular part of the protein, interact with each other in a specific and concentration-dependent manner. This molecular interaction was increased by twofold in the presence of physiological Ca(2+) concentration, indicating that the binding is not dependent on, but rather promoted by Ca(2+). Furthermore, transfected HEK293 cells and an immortalized mouse podocyte cell line overexpressing full-length human nephrin forms cellular aggregates, with cell-cell contacts staining strongly for nephrin. The distance between plasma membranes at the nephrin-containing contact sites was shown by electron microscopy to be 40 to 50 nm, similar to the width of glomerular slit diaphragm. The cell contacts could be dissociated with antibodies reacting with the first two extracellular Ig-like domains of nephrin. Wild-type HEK293 cells express slit diaphragm components CD2AP, P-cadherin, FAT, and NEPH1. The results show that nephrin molecules exhibit homophilic interactions that can promote cellular contacts through direct nephrin-nephrin interactions, and that the other slit diaphragm components expressed could contribute to that interaction (Khoshnoodi, 2003).
Congenital nephrotic syndrome of the Finnish type (NPHS1) is an autosomal recessive disorder that is caused by mutations in the recently discovered nephrin gene, NPHS1 (AF035835). The disease, which belongs to the Finnish disease heritage, exists predominantly in Finland, but many cases have been observed elsewhere in Europe and North America. The nephrin gene consists of 29 exons spanning 26 kb in the chromosomal region 19q13.1. In the present study, the genomic structure of the nephrin gene was analyzed, and 35 NPHS1 patients were screened for the presence of mutations in the gene. A total of 32 novel mutations, including deletions; insertions; nonsense, missense, and splicing mutations; and two common polymorphisms were found. Only two Swedish and four Finnish patients had the typical Finnish mutations: a 2-bp deletion in exon 2 (Finmajor) or a nonsense mutation in exon 26 (Finminor). In seven cases, no mutations were found in the coding region of the NPHS1 gene or in the immediate 5'-flanking region. These patients may have mutations elsewhere in the promoter, in intron areas, or in a gene encoding another protein that interacts with nephrin (Lenkkeri, 1999).
A high-throughput, retrovirus-mediated mutagenesis method based on gene trapping in embryonic stem cells was used to identify a novel mouse gene. The human ortholog encodes a transmembrane protein containing five extracellular immunoglobulin-like domains that is structurally related to human NEPHRIN, a protein associated with congenital nephrotic syndrome. Northern analysis has revealed wide expression in humans and mice, with highest expression in kidney. Based on similarity to NEPHRIN and abundant expression in kidney, this protein was designated NEPH1 and embryonic stem cells containing the retroviral insertion in the Neph1 locus were used to generate mutant mice. Analysis of kidney RNA from Neph1(-/-) mice shows that the retroviral insertion disrupted expression of Neph1 transcripts. Neph1(-/-) pups were represented at the expected normal Mendelian ratios at 1 to 3 days of age but at only 10% of the expected frequency at 10 to 12 days after birth, suggesting an early postnatal lethality. The Neph1(-/-) animals that survived beyond the first week of life were sickly and small but without edema, and all died between 3 and 8 weeks of age. Proteinuria ranging from 300 to 2,000 mg/dl was present in all Neph1(-/-) mice. Electron microscopy has demonstrated NEPH1 expression in glomerular podocytes and revealed effacement of podocyte foot processes in Neph1(-/-) mice. These findings suggest that NEPH1, like NEPHRIN, may play an important role in maintaining the structure of the filtration barrier that prevents proteins from freely entering the glomerular urinary space (Donoviel, 2001).
A mouse model for congenital nephrotic syndrome (NPHS1) was generated by inactivating the nephrin gene (Nphs1) in embryonic stem cells by homologous recombination. The targeting construct contained the Escherichia coli lacZ gene as a reporter for the Nphs1 promoter. Mice homozygous for inactivated Nphs1 were born at an expected frequency of 25%. Although seemingly normal at birth, they immediately developed massive proteinuria and edema and died within 24 h. The kidneys of null mice exhibited enlarged Bowman's spaces, dilated tubuli, effacement of podocyte foot processes and absence of the slit diaphragm, essentially as found in human NPHS1 patients. In addition to expression in glomerular podocytes, the reporter gene is expressed in the brain and pancreas of (+/-) and (-/-) mice. In the brain, the gene is expressed in the ventricular zone of the fourth ventricle, the developing spinal cord, cerebellum, hippocampus and olfactory bulb. In the cerebellum, expression is seen in radial glial cells. Neither anatomical nor morphological abnormalities are observed in the brains of null mice (Putaala, 2001).
Mutations of NPHS1 or NPHS2, the genes encoding for the glomerular podocyte proteins nephrin and podocin, cause steroid-resistant proteinuria. In addition, mice lacking CD2-associated protein (CD2AP) develop a nephrotic syndrome that resembles NPHS mutations suggesting that all three proteins are essential for the integrity of glomerular podocytes. Although the precise glomerular function of either protein remains unknown, it has been suggested that nephrin forms zipper-like interactions to maintain the structure of podocyte foot processes. This study demonstrates that nephrin is a signaling molecule, which stimulates mitogen-activated protein kinases. Nephrin-induced signaling is greatly enhanced by podocin, which binds to the cytoplasmic tail of nephrin. Mutational analysis suggests that abnormal or inefficient signaling through the nephrin-podocin complex contributes to the development of podocyte dysfunction and proteinuria (Huber, 2001).
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. 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. 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. It is concluded that 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 (Ahola, 1999).
Since the discovery of the nephrin gene, many mutations have been reported in the NPHS1 gene in patients from diverse ethnic backgrounds. A surprisingly large number of these mutations are missense mutations resulting in single amino acid substitutions. In order to study the pathomechanism of these missense mutations, the fate of 21 such mutations identified in NPHS1 patients has been examined. Immunostaining of stable transfected cells expressing the nephrin mutants has demonstrated that most of the mutants show only endoplasmic reticulum (ER) staining and no detectable cell surface localization. Immunoelectron microscopy of cells expressing the wild-type and a mutant nephrin further confirmed that the mutant nephrin could be abundantly found in the ER but not on the plasma membrane. Subcellular fractionation of wild-type and a mutant cell line clearly shows an altered subcellular distribution and molecular mobility of the mutant nephrin. In summary, the data indicate that a defective intracellular nephrin transport, most likely due to misfolding, is the most common consequence of missense mutations in NPHS1 (Liu, 2001).
The distribution of nephrin was examined by immunofluorescence microscopy in renal biopsies of patients with nephrotic syndrome: 13 with membranous glomerulonephritis (GN), 10 with minimal change GN, and seven with focal segmental glomerulosclerosis. An extensive loss of staining for nephrin and a shift from a podocyte-staining pattern to a granular pattern was found in patients with nephrotic syndrome, irrespective of the primary disease. In membranous GN, nephrin co-localizes with IgG immune deposits. In the attempt to explain these results, an in vitro investigation was carried out to see whether stimuli acting on the cell cytoskeleton, known to be involved in the pathogenesis of GN, may induce redistribution of nephrin on the surface of human cultured podocytes. Aggregated but not disaggregated human IgG(4), plasmalemmal insertion of membrane attack complex of complement, tumor necrosis factor-alpha, and puromycin, induces the shedding of nephrin with a loss of surface expression. This phenomenon is abrogated by cytochalasin and sodium azide. These results suggest that the activation of cell cytoskeleton may modify surface expression of nephrin allowing a dislocation from plasma membrane to an extracellular site (Doublier, 2001).
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