Nephrin cellular trafficking and intracellular interactions

Abstract: Nephrin is a 180-kDa transmembrane glycoprotein belonging to the immunoglobulin family of cell adhesion molecules. Nephrin has an extracellular part with eight Ig-like motifs, one fibronectin-like domain, a transmembrane region and a short cytoplasmic tail. In the kidney, nephrin is exclusively expressed by glomerular podocytes, the protein being located in the slit diaphragm (SD) between podocyte foot processes. The nephrin gene is mutated in congenital nephrotic syndrome of the Finnish type (CNF), a rare autosomal recessive disease with an incidence in Finland of approximately 1 in 10,000 newborns. CNF patients exhibit massive proteinuria already in utero. In most cases, nephrin is not detected in the SD. In the first part of the project, we investigated the subcellular localization of 21 missense mutants identified in CNF patients using embryonic kidney cells (HEK293). Immunostaining and Immunoelectron microscopy of stable transfected cells expressing these nephrin mutants demonstrated that most of them were retained in the endoplasmic reticulum (ER) with no detectable cell plasma membrane surface localization. Subcellular fractionation of wild type and a mutant cell line clearly showed an altered subcellular distribution and molecular mobility of the mutant nephrin. Our data suggested the misfolding and defective intracellular transport with consequent absence of the mutant nephrin on the plasma membrane might contribute to the pathomechanism of disease in patients carrying missense mutations resulting in the development of nephrotic syndrome. Nephrin has a typical domain and sequence structure of a cell-cell or cell-matrix adhesion protein with nine intracellular tyrosines, some of which are phosphorylated after podocyte-specific antiganglioside antibody-induced proteinuria. In the second part of the work, we showed that nephrin is a signaling molecule by clustering of nephrin with antibodies on the cell surface, thus mimicking the situation where the interaction between nephrin and its extracellular ligand(s) is altered. We demonstrate that Src family kinase activity is crucial for tyrosine phosphorylation of nephrin, and that apparently several members of the Src family of kinases are able to catalyze nephrin phosphorylation. The aim of the the third paper was to find out whether the disease-causing missense mutations in the podocin gene identified in patients with autosomal recessive steroid-resistant nephrotic syndrome (SRN) might be due to dis- localization of the mutant podocin molecule, similarly to nephrin (first paper). Here, we analyzed 5 different disease-causing missense mutations found in SRN patients and their subcellular localization in human HEK293 cells. Three out of these five mutations, located at the proximal C-terminal part of podocin molecule, failed to localize to the plasma membrane. Interestingly, we also found that the normal localization of wild-type nephrin at the plasma membrane was also altered in cells co-expressing those three podocin mutants. Our date suggest that some disease-causing missense mutations in the podocin gene may abolish proper nephrin localization to the plasma membrane and lead to disruption of normal slit diaphragm structure and function in patients with SRN. Sodium 4-phenylbutyrate (4-PBA) has been shown to function as a chemical chaperone and correct the cellular trafficking of several mislocalized or misfolded mutant plasma membrane proteins. In the last part of study, we explored the potential effect of 4-PBA on the missense mutants found in CNF patients. Immunofluorescence microscopy and cell surface biotinylation showed that treatment with 4-PBA rescued several of the missense mutants from the ER to the cell surface. All the rescued mutants were able to interact with Neph1, another interacting partner of nephrin in the slit diaphragm protein complex. Furthermore, tyrosine phosphorylation of the rescued mutants was rapidly induced by clustering with anti-nephrin antibodies as wild type, implying that the rescued mutants are functionally intact. These results suggest that the use of 4-PBA could become a therapeutic approach for the treatment of CNF or other similar diseases affecting renal filtration.