Involvement of Lipid Rafts in Nephrin Phosphorylation and Organization of the Glomerular Slit Diaphragm

Abstract

NPHS1 has recently been identified as the gene whose mutations cause congenital nephrotic syndrome of the Finnish type. The respective gene product nephrin is a transmembrane protein expressed in glomerular podocytes and primarily localized to the glomerular slit diaphragm. This interpodocyte junction functions in the glomerular filtration by restricting the passage of plasma proteins into the urinary space in a size-selective manner. The functional role of nephrin in this filtration process is so far not very well understood. In this study, we show that nephrin associates in an oligomerized form with signaling microdomains, also known as lipid rafts, and that these localize to the slit diaphragm. We also show that the nephrin-containing rafts can be immunoisolated with the 27A antibody recognizing a podocyte-specific 9-Oacetylated GD3 ganglioside. In a previous study it has been shown that the in vivo injection of this antibody leads to morphological changes of the filtration slits resembling foot process effacement. Here, we report that, in this model of foot process effacement, nephrin dislocates to the apical pole of the narrowed filtration slits and also that it is tyrosine phosphorylated. We suggest that lipid rafts are important in the spatial organization of the glomerular slit diaphragm under physiological and pathological conditions. One of the main functions of the kidney is formation of the primary urine in the glomerulus by plasma ultrafiltration. The structural correlate of ultrafiltration is the glomerular capillary wall with its distinctly layered filtration barrier. It consists of the fenestrated vascular endothelium, the glomerular basement membrane, and a layer of morphologically highly specialized epithelial cells, podocytes.Podocytes form a tight network of multiple interdigitating cellular extensions, called foot processes, which are bridged by so-called "slit diaphragms." 1 The filtration barrier is freely permeable for water, small solutes, and ions but does not allow passage for proteins larger than albumin and other large molecules. The charge selectivity in this process has been attributed to the glomerular basement membrane whereas the slit diaphragm supposedly plays a major role in the size selectivity. 2,3 The importance of the filtration barrier is reflected by a number of human diseases in which the filtration barrier function is disrupted resulting in the loss of plasma proteins into the urine. Patients with persistent proteinuria ultimately develop a nephrotic syndrome with a variety of symptoms including edema, hypoalbuminemia, and hyperlipidemia. 4

Podocyte Biology and Response to Injury

“…Hereby, they provide molecular frameworks for numerous cell biologic processes, such as exocytosis and endocytosis, cell adhesion, and signal transduction events (86 -88). Recent work from our lab established that lipid raft microdomains are critical for the dynamic functional organization of the SD (89). We have shown that nephrin associates with lipid rafts and coimmunoprecipitates with a podocyte-specific 9-O-acetylated ganglioside (89).…”

“…Recent work from our lab established that lipid raft microdomains are critical for the dynamic functional organization of the SD (89). We have shown that nephrin associates with lipid rafts and coimmunoprecipitates with a podocyte-specific 9-O-acetylated ganglioside (89). The in vivo injection of an antibody against this ganglioside causes morphologic changes of the filtration slits resembling FP effacement.…”

“…The in vivo injection of an antibody against this ganglioside causes morphologic changes of the filtration slits resembling FP effacement. In this model, nephrin translocated to the apical pole of the narrowed filtration slits and underwent tyrosine phosphorylation (89).…”

Rostafuroxin Protects from Podocyte Injury and Proteinuria Induced by Adducin Genetic Variants and Ouabain

“…In rat kidneys, preliminary results indicate that ABL1 localizes with nephrin in caveolae subdomains, known to functionally organize signaling pathways (Simons et al, 2001), is present in rat glomerular podocytes, and its expression level is downregulated in MNS and NB glomerular podocytes, as well as nephrin, compared with MHS and NA.…”

Homodimerization and Heterodimerization of the Glomerular Podocyte Proteins Nephrin and NEPH1

“…This interaction was only detectable after solubilization of kidney lysates with 20 mM CHAPS, indicating that a substantial fraction of both proteins is associated with lipid rafts. Addition of 3 mM ATP to the lysis buffer appeared to increase the amount of NEPH1, precipitated with anti-nephrin antiserum versus protein G alone ( Figure 4D, lower panel), indicating that association with both lipid rafts (14) and the cytoskeleton (15) reduces the solubility of NEPH1 and/or nephrin and limits the experimental approaches to verify this interaction in vivo.…”