Proteinuria and Perinatal Lethality in Mice Lacking NEPH1, a Novel Protein with Homology to NEPHRIN

Abstract

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 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 showed 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 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.NEPHRIN is a transmembrane protein of the immunoglobulin (Ig) superfamily that is expressed by epithelial podocytes of developing glomeruli (13, 17). Congenital nephrotic syndrome of the Finnish type results from mutations in NPHS1, the human gene encoding NEPHRIN, indicating a role for NEPHRIN in maintaining the filtration barrier that prevents proteins from freely entering the glomerular urinary space (6,17). Recent studies localized NEPHRIN to the slit diaphragms that form the junctions between podocyte foot processes interdigitating along the glomerular basement membrane. This and other studies suggest that NEPHRIN proteins extending toward each other from adjacent podocyte foot processes may interdigitate in a zipper-like structure to form the crucial filtration barrier in the slit diaphragm. The eight Ig-like domains of each NEPHRIN protein, which are of the C2 type of Ig domain known to be involved in cell-cell interactions, are thought to provide the homophilic interactions that bind these NEPHRIN proteins together (13, 17).We use a high-throughput mutagenesis method based on gene trapping in embryonic stem (ES) cells that allows automated production of sequence tags from the trapped and mutated genes (20). These ES cell clones are stored in a library called Omnibank, and the sequence tag from the gene trapped in each clone, referred to as the Omnibank sequence tag or OST, is entered into a searchable database. A protein with Ig domains was identified with...

Focal and Segmental Glomerulosclerosis in Mice with Podocyte-Specific Expression of Mutant α-Actinin-4

“…Nephrin, a key player in the formation of the slit diaphragm, possesses extracellular IgGlike repeats, a single membrane-spanning region, and a cytoplasmic tail that interacts with intracellular molecules including CD2AP, which may anchor it to the actin cytoskeleton (17,18). The importance of this molecule in maintaining the glomerular barrier to protein is illustrated by the fact that humans with mutations in the NPHS1 gene and NPHS1-null mice are nephrotic and fail to develop normal podocyte foot processes (19,34). Many recent studies have associated reduced nephrin mRNA or protein expression with the onset of proteinuria in a number of nephropathies including diabetic nephropathy (35,36), minimal change disease (37), passive Heymann nephritis (38 -40), and membranous proliferative glomerulonephritis (37).…”

Inherited Podocytopathies

“…Mice lacking NEPH1, a nephrin homolog sharing structural features as well as high renal expression with nephrin, develop severe nephrosis and die perinatally (75). Electron microscopy studies showed podocyte expression of NEPH1, and, in NEPH1-deficient mice, diffusely effaced foot processes.…”

Association analysis of podocyte slit diaphragm genes as candidates for diabetic nephropathy

“…Defects in the podocin gene (NPHS2) manifest soon after birth and are characterised by uncontrolled, steroid-resistant proteinuria [3]. Mice lacking the gene encoding the nephrinlike protein NEPH1 (Kirrel) develop massive proteinuria and foot process effacement [4]. Mutations in the ACTN4 gene, which encodes alpha actinin 4, account for the familial form of focal segmental glomerulosclerosis with later onset of the disease [5].…”