FC015: High Salt Environment Disrupts the Regulatory Effect of Podocin on the Nephrin-Nephrin Distance

Abstract

Abstract BACKGROUND AND AIMS The podocin encoding NPHS2 is the most frequently mutated gene in steroid-resistant nephrotic syndrome. Podocin homooligomerizes through C-terminal helical regions [1] and binds nephrin in the glomerular slit diaphragm [2]. We formerly showed that podocin regulates the distance between two neighbouring nephrin molecules in cis, i.e. the shortest dimension of the glomerular pore (3.5-4nm) [3, 4]. High sodium diet has been shown to promote glomerular injury and proteinuria [5]. We aimed to assess the effect of high salt environment on the nephrin-nephrin distance in the presence and absence of podocin. METHODS To generate extracellularly tagged nephrin constructs, cDNA sequences encoding YPet or mCherry were inserted in the human wild type nephrin cDNA (YPet-N1 plasmid) replacing the c.3136-3162 nucleotides. Thus, the p.Glu1046-Pro1054 residues of the fibronectin domain, located in the juxtamembraneous extracellular part of nephrin was replaced with either YPet or mCherry. HEK-293 cells were co-transfected with both nephrin constructs with or without wild type HA-tagged podocin construct (pLEX-MCS plasmid). To measure the effect of salt overload, DMEM was supplemented with either 25 mM NaCl or 50 mM mannitol as osmotic control 24-h prior to the FRET measurements. The fluorescence decay curves measured by time-correlated single photon counting (TCSPC) (Chronos BH, ISS Inc.) were decomposed into lifetime components and the longest lifetime population—most suitable for long-range FRET—was used for further calculations. Nonparametric tests were used for statistical analysis. RESULTS High salt environment did not influence the nephrin-nephrin distance in the absence of podocin. In accordance with our previous results, podocin decreased the nephrin-nephrin distance in physiological salt environment, as reflected by an increased FRET efficiency between the extracellular YPet and mCherry tags. High salt environment (+25 mM) completely disrupted this effect. Mannitol of similar osmotic concentration did not modify the effect of podocin (Figure 1). CONCLUSION High salt environment increases the distance between the nephrin molecules in cis by disrupting the organizing effect of podocin. The deleterious effect of high salt is not explained by an increased osmotic pressure.