Podocin Localizes in the Kidney to the Slit Diaphragm Area

Abstract

We recently cloned a novel gene, NPHS2, involved in autosomal recessive steroid-resistant nephrotic syndrome. This gene encodes a novel podocyte protein, podocin. Given its similarity with the stomatin family proteins, podocin is predicted to be an integral membrane protein with a single membrane domain forming a hairpin-like structure placing both N- and C-termini in the cytosol. Here, we show by in situ hybridization, that during development, the NPHS2 transcript is first expressed in mesonephric podocytes from the S-shaped body and, later, in the metanephric kidney, in the future podocytes at the late S-shaped body stage. In the mature kidney, NPHS2 is exclusively expressed in the podocytes of mature glomeruli. We generated rabbit polyclonal antibodies against fusion proteins derived from the N- and the C-terminal regions of podocin which detected a single band of 49-kd in transfected HEK293 cell lysates by immunoprecipitation and Western blotting. By immunohistology, podocin was detected in podocytes from the early capillary loop stage in the developing nephrons, and at the basal pole, along the GBM, in mature glomeruli. By electron microscopy, we demonstrate that podocin is facing the slit diaphragm with its two ends in the cytoplasm of the foot processes, in agreement with its predicted structure. Our results suggest that podocin could serve to anchor directly or indirectly components of the slit diaphragm to the cytoskeleton.

Functional and molecular alterations of the glomerular barrier in long-term diabetes in mice

“…For a long time the glomerular basement membrane (GBM) has been considered to be the main barrier for filtration. More recently, focus has shifted to the podocytes, since several novel proteins in the podocyte slit diaphragm have been found to be important for the barrier function [1][2][3]. In addition, we have suggested a role for the glomerular endothelial cell glycocalyx [4][5][6][7][8][9][10], an idea supported also by others [11,12].…”

“…Under normal conditions, the glomerular barrier restricts the passage of macromolecules such as albumin, while it is highly permeable to water and small solutes. The underlying mechanisms leading to proteinuria are still poorly understood, although the importance of the podocyte for glomerular integrity has been clearly documented [1][2][3]. Consequently, there is no specific therapy for diabetic nephropathy.…”

Ancient origin of reggie (flotillin), reggie-like, and other lipid-raft proteins: convergent evolution of the SPFH domain

“…While their SPFH domains are predicted to be globular, the C termini seem more likely to be unfolded and lack multiple EA motifs. Finally, podocin is an integral membrane protein exclusively expressed in podocytes of the kidney, and plays a key role in the stability of glomerular slit diaphragm [45]. Podocin contains an SPFH domain covering about 40% of the polypeptide (human position 123-279) and a transmembrane domain (positions 102-128).…”

Podocyte Biology and Response to Injury

“…Stomatin is present as high-order oligomers in erythrocyte lipid rafts, where it has a scaffolding function (83). Podocin localizes to the SD (16,21), accumulates there in an oligomeric form in lipid rafts and associates via its C-terminus with CD2AP and nephrin (16). Further studies revealed direct interaction of podocin and CD2AP (16).…”

“…Neighboring FP are connected by a cell-cell junction, the glomerular SD, which represents the main size selective filter barrier in the kidney (8 -10). The SD is thought to be a modified adherens junction (11) that is composed of a growing number of proteins, including nephrin (12)(13)(14), P-cadherin, CD2AP (15)(16)(17)(18), ZO-1 (19), FAT (20), podocin (16,21), and possibly Neph1 (see reference 22 and below). In addition to the contractile proteins described above, we have reported the association of synaptopodin with the actin filaments in FP (23).…”