Nephrin and Neph1 Co-localize at the Podocyte Foot Process Intercellular Junction and Form cis Hetero-oligomers

Abstract

Glomerular visceral epithelial cells (podocytes) appear to play a central role in maintaining the selective filtration barrier of the renal glomerulus. While the immunoglobulin superfamily member Nephrin was proposed to act as a cell adhesion molecule at the podocyte intercellular junction necessary for maintaining glomerular perm selectivity, the Nephrin ligand has not been identified. The existence of a new subfamily of Nephrin-like molecules including Neph1 was recently described. Genetic deletion of Nephrin or Neph1 resulted in similar phenotypes of podocyte foot process effacement and proteinuria. The subcellular localization of Neph1 and the possibility that Nephrin and Neph1 interact was investigated. Polyclonal antiserum for Neph1 was raised and characterized. Neph1 migrated as a 90-kDa protein on SDS-PAGE under reducing conditions. Neph1 was identified in a glomerular and podocyte-specific distribution in adult rat kidney. Like Nephrin and Podocin, Neph1 was enriched in Triton X-100 detergent-resistant membrane fractions. Consistent with this observation, immunogold electron microscopy demonstrated that Neph1 localized exclusively to lateral margins of podocyte foot processes at the insertion of the slit diaphragm. Neph1 and Nephrin participate in a direct cis-interaction involving their cytoplasmic domains. In addition, interactions between the extracellular domain of Nephrin and itself and between the extracellular domain of Nephrin and that of Neph1 were detected. Neph1 did not interact via a homophilic interaction. These observations suggest that Nephrin and Neph1 form a hetero-oligomeric receptor complex in the plane of the membrane that might interact across the foot process intercellular junction through interactions between Nephrin with itself and Neph1.

Nephrin Forms a Complex with Adherens Junction Proteins and CASK in Podocytes and in Madin-Darby Canine Kidney Cells Expressing Nephrin

“…9,10,52 It was suggested that the interaction between nephrin and Neph1 partially stabilizes the interaction between Neph1 and ZO-1. 10 The question of how these two cell adhesion systems contribute to the regulation of slit diaphragms remains to be answered.…”

MAGI-1 is a component of the glomerular slit diaphragm that is tightly associated with nephrin

“…[11][12][13] Neph1 binds to the PDZ domain of ZO-1 via its C-terminal PDZ-binding motif. 13,21 Next, we tested using GST pulldown assay whether nephrin also binds to not only MAGI-1 but also ZO-1.…”

“…11 Neph1 shows high expression in the kidney and is colocalized with nephrin at the podocyte foot process. 12,13 The deletion of Neph1 in the mice results in proteinuria. 11 a-Actinin-4, an actin-binding and crosslinking protein, is highly expressed in podocytes.…”

“…17 Nephrin and Neph1 form heterooligomers. 12 The inhibition of the nephrinNeph1 interaction by antibodies brings in slit diaphragm dysfunction. 13 These reports support the conclusion that intimate interactions among slit diaphragm proteins and slit diaphragm-associated proteins are important for the glomerular filtration barrier.…”

Congenital Nephrotic Syndrome

“…On the other hand, the precise molecular structure of the SD is still unresolved. Recently identified podocyte proteins, such as nephrin, Neph1, Neph2, FAT1, FAT2, and dendrin probably form the backbone of SD [6][7][8]. These proteins associate with each other extracellularly and interact with the adapter proteins, such as podocin, CD2AP, ZO-1, CASK, and MAGI-1, localized in the cytosolic part of the podocyte.…”