Evaluating the role of ubiquitin and endocytosis in Gitelman syndrome

Gitelman’s syndrome (GS) is a renal salt wasting disorder caused by defective reabsortion of NaCl in the kidney. Patients present hypokalemia, renal potassium wasting, activation of the renin-angiotensin-aldosterone axis, and normal blood pressure. GS is transmitted as an autosomal recessive trait. Around 250 distinct mutations have been discovered in the causative gene, the thiazide-sensitive NCC/SLC12A3 transporter. Few of them were functionally characterized. Our goal with this study is to set up assays and tools to examine the molecular regulation of NCC, with the perspective of investigating how ubiquitination and internalization regulate this transporter. To this end we will profit of the knowledge acquired in another well-characterized renal disorder, the Liddle syndrome. Activity of ENaC, the causative factor in Liddle syndrome, is determined by highly regulated phosphorylation and ubiquitination events. Here, Nedd4-2-mediated ubiquitination is followed by endocytic targeting, sorting and degradation of the transporter. This mechanism regulates transporter amount at the cell membrane allowing only the required number of the channel protein to be active at any given time. A disrupted association between Nedd4-2 and ENaC results in a constitutively active channel that allows excessive reabsorption of sodium ions. Recent literature led us to hypothesize a similar regulation exerted by Nedd4-2 on SLC12A3. This hypothesis will be tested using siRNA methods, biochemical assays and cell biological techniques to monitor the traffic of SLC12A3 wt and selected GS mutants. Positive results would provide a framework for exploring the mechanistic basis of the GS mutations. The identification of the endocytic regulatory pathways behind SLC12A3 is predicted to provide a better understanding of the pathophysiology of GS opening a new perspective for the rational discovery of novel treatments.