Author: stefania bellini
Date: 06/05/2009



Nephrin is a kidney glomerular filtration barrier protein that is an essential component of the interpodocyte-spanning slit diaphragm that constitute the size-selective filter of the kidney. Mutations in the Nephrin gene (NPHS1) are associated with congenital nephrotic syndrome, a disease characterized by massive proteinuria and edema.

NPHS1 is located on the chromosome 19 (location: 19q13.1) , in a region of 25,883 bases on the minus strand and contains 29 exons.

Protein names:
1. Raccomanded name: Nephrin
2. Alternative name: NPHN, renal glomerulus-specific cell adhesion receptor

Gene names:
1. Official symbol: HPHS1
2. Aliases: CNF, NPHN

Tissue specificity:
Nephrin is specifically expressed by the kidney. Within the kidney Nephrin is exclusively expressed by podocytes and predominantly localized to the slit diaphragm. In addition to its expression in podocytes, Nephrin was also detected in different regions of the brain and in the pancreas β-cells.

Nephrin linkage to the membrane is stabilized by podocin

β-Arrestin2 mediates nephrin endocytosis and impairs slit diaphragm integrity 2006 Movie

Aminoacid percentage

Alternative products of the gene: two isoforms produced by alternative splicing are described:

  • ISOFORM 1: predict an amino acid sequence of 1241 residues with a calculate molecular mass of 134,742Da without post-translational modifications. This isoform has been chosen as the “canonical” sequence (all information refers to it).
  • ISOFORM 2 (Alpha): amino acid sequence of 1201 residues with molecular mass of 130,546 Da. Its sequence differs from the canonical one because of the missing of residues from 1056 to 1095

Regulation: Little is understood about pathophysiologic substances that regulate nephrin expression.

BIOACTIVE SUBSTANCES: three physiological ligands of nuclear receptors including all-trans-retinoic acid , 1,25-dihydroxyvitamin D3 , and dexamethasone significantly activated the nephrin gene promoter in a dose-dependent manner. The effects of these substances were synergistic, and the maximum effect was observed by combination of three agents. The retinoids, biologically active metabolites of vitamin A , play a very important role in nefrogenesis, as well as their receptors. Deficiency of this vitamin causes severe renal malformations, such as renal hypoplasia, or in serious cases the renal genesi. In contrast, inflammatory cytokines IL-1β and TNFα significantly downregulated the activity of the nephrin promoter as well as nephrin gene expression.

TRANSCRITION FACTORS: WT1 ( Wilms’ tumor gene ) is a transcription factor specifically expressed in developing and differentiated podocytes. WT1 (upregulated by HIF) encodes a zinc finger protein that can bind and activate the Nephrin promoter; this binding is essential for podocyte-specific expression in vivo. Consistently, nephrin expression is reduced in mouse models that lack WT1.
Upon WT1 induction, endogenous nephrin mRNA becomes highly upregulated 10-fold. A WT1 responsive element in the nephrin promoter is required for the binding of WT1 protein. Mutations or deletion of this WT1 responsive element completely abolished transactivation of the Nephrin promoter by WT1.
Expression of WT1 continues in podocytes of adult kidneys, suggesting that this gene is also required during glomerular function. Consistent with this observation, we recently found that the two major podocyte proteins nephrin and podocalyxin were lowered in the kidneys of mice with reduced expression of WT1, suggesting co-regulation of nephrin and WT1.

External links:
HUGO 7908
Entrez Gene 4868
OMIM 602716
Ensembl GeneView
Refseq-mRNA NM_004646.2
Refseq-Protein NP_004637.1

Chemical structures:

Nephrin is a single-pass type I membrane glycoprotein and a member of the immunoglobulin superfamily (IgCAM/NCAM). The extracellular portion containing 8 Ig motifs and one type III-fibronectin-like domain. The cytosolic domain contains 9 tyrosine residues, some of which could become phosphorylated during ligand binding of Nephrin.
Nephrin molecules extending toward each other from two adjacent foot processes are likely to interact with each other in the slit diaphragm through homophilic interactions. Such homophilic head-to-head assembly of Nephrin molecules in the slit have a zipperlike arrangement. The amino terminal extracellular domain of Nephrin contain 6 consecutive Ig repeats, followed by a spacer domain, 2 additional Ig repeats and one type III-fibronectin domain. Each Ig motif contains 2 cysteine residues that similarly to corresponding motifs in other proteins, can be assumed to form a disulfide bridge within the repeat structure (Ig motifs have been shown to adopt a globular or ellipsoid structure with an axis length of between 24 and 47 Å). The Ig repeats form a chain-like structure, all 8 motifs would contribute to a length of about 28 nm. The region between Ig repeats 6 and 7 (spacer domain) and fibronectine type III domain would added more length to the protein; consequently, a single Nephrin molecule can extend through most of the width of the slit diaphragm. In addition to the 2 cysteine residues in each Ig motif, Nephrin contains 3 free cysteines: one in the Ig motif 1, one in the spacer domain and one in the fibronectin domain close to the plasma membrane. The 3 free cysteines have a function in forming intermolecular disulfide bridges that provide strength to the slit diaphragm (they are important because their absence results in proteinuria and congenital nephrotic syndrome). In this model, the free cysteine of the Ig motif 1 in one molecule interacts with the cysteine residue of the spacer in another Nephrin molecule. Such disulfide bonds could “lock” the homophilic unit of 6 Ig repeats of one Nephrin molecule to similar units of two adjacent Nephrin molecule.

A centrally located aggregate of numerous Nephrin molecules along the slit diaphragm between two foot processes could constitute the central filament visualized by electron microscopy.

Nephrin has 10 potential N-glycosylation sites in its extracellular domain: the serine-glycine doublets (SG) are potential heparan sulfate attachment sites. Heparan sulfate is a glycosaminoglycan or GAG (an unbranched polysaccharide chain composed of repeating disaccharide units of d-glucosamine or l-iduronic acid and N-acetyl-d-glucosamine).

Synthesis and turnover:

Northern hybridization analysis carried out with poly(A) mRNA from human embryonic and adult tissues revealed a high level of NPHS1 transcript in the kidney, but not notably in other tissues. In humans, Nephrin mRNA was first detected in 23 weeks-old embrio kidneys with intense expression signals in the glomeruli.
Nephrin mRNA is composed by 1241 amino acidic residues. Note that the region by 1 to 22 aa is a SP motif or Signal Peptide : a peptide present on proteins that are destined either to be secreted or to be membrane components.

Amino acids modifications: N-linked glycosylation, disulfide bounds and phosphorylation.
The polypeptide chain of Nephrin, or core protein, is made on membrane-bound ribosomes and threaded into the lumen of the endoplasmic reticulum. The heparan sulfate polysaccharide chains are assembled on this core protein in the Golgi apparatus. First, a special link tetrasaccharide is attached to a serine side chain on the core protein to serve as a primer for polysaccharide growth; then, one sugar at a time is added by specific glycosyl transferases. While still in the Golgi apparatus, many of the polymerized sugars are covalently modified by a sequential and coordinated series of reactions. Epimerizations alter the configuration of the substituents around individual carbon atoms in the sugar molecule; sulfations increase the negative charge. The addition of the polysaccharide chains at the sites of glycosylation, increases the weight of the primary sequence of about 35 kDa, the weight of the modified protein is approximately 180 kDa. It was demonstrated that pharmacological inhibition of the process of glycosylation leads to retention of nonglycosylated Nephrin molecules in the endoplasmic reticulum. It was concluded that N-glycosylation of Nephrin is crucial for its proper folding and thereby plasma membrane localization; therefore, inhibition of this process might be an important factor in the onset of pathogenesis of some acquired glomerular diseases.

Cellular functions:

Based on its primary structure, Nephrin seems to has three main functions: it is an adhesion, filtering and signaling molecule. Inactivation of NPHS1 in mice leads to an immediate massive proteinuria and edema after birth, causing death within 1 day. The kidney of NPHS1 deficient mice showed effacement of podocytes foot processes and the absence of the slit diaphragm.

ADHESION AND FILTERING ROLE: Nephrin has a role in the development or function of the kidney glomerular filtration barrier and may anchor the podocytes slit diaphragm to the actin cytoskeleton. Nephrin Ig extracellular domains, through intermolecular homophilic interactions, form the ladderlike structure typical of the slit diaphragm (the size-selective filter of the kidney); heparan sulfate chains linked to the extracellular domain, because of their sulfate groups, are highly negatively charged. The whole structure has the role of filter molecules passing into the urine from the bloodstream, forming a selective sieve that regulate this traffic according to both size and charge of the molecules themselves.

SIGNALING ROLE: Nephrin is also a signaling molecule: the Ig-like modules of Nephrin are all of type C2, which is particularly found in proteins involved in modulating cell-cell and cell-matrix (ECM) interactions, inducinducing signal transduction events, which are crucial for cell adhesion, motility, cell growth and survival. Phosphorylated tyrosine residues in the intracellular domain may provide a binding site for tyrosine kinases of the Src family. The phosphorylation could have a role in the activation of signal transduction pathway upon binding of the extracellular ligands (other proteins interact with Nephrin both on the podocyte surface and with the cytosolic domain: they could change variably the function of the podocyte adhesion system). The evidence on interaction of the cytosolic CD2AP with Nephrin provides information on potential linkage mechanisms between Nephrin and the actin cytoskeleton of podocyte foot processes. The deletion of most of the cytoplasmatic domain results in foot process effacement and proteinuria, indicating the importance of this domain.

Diagnostic use:

MUTATIONS: NPHS1 has been identified as the gene whose mutations cause congenital nephrotic syndrome of the Finnish type/CNF (an autosomal recessive disease affecting ~ 1:10000 newborns in Finland, characterized by massive proteinuria and edema); a total of about 50 mutations have been reported so far (deletions and insertions, splice-site and nonsense mutations, missense mutations). The identification of the gene will immediately find application for diagnosis of the disease. The two most important mutation causing the disease are Finmajor and Finminor . In Finmajor (the most diffused) there is a 2-bp deletion in exon 2 that causes a frameshift and a translation stop at the end of exon 2. In Finminor there is a nonsense mutation in the exon 26 that introduced a premature stop codon.

DISEASES: The link between permutations in Nephrin expression and proteinuria has been confirmed in animal studies; in humans it was shown a reduced expression of Nephrin in patients with primary or acquired nephropatic syndrome, independent from the initial pathogenic mechanism. Proteinuria is also the clinical hallmark of Diabetic Nephropathy (DN) and recent studies have assessed the role of Nephrin in the loss of glomerular permselectivity in DN. In humans both type 1 and type 2 diabetic patients with nephrotic syndrome have an extensive reduction in glomerular Nephrin expression.
Urinary mRNA expression of podocyte markers, such as Nephrin and are significantly different between proteinuric disease categories. Further, NephRNA correlated with the rate of decline in renal function: this suggests that urinary podocyte gene expression may be a useful non-invasive tool which provides additional information for the management of proteinuric diseases.

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