Project

Discipline

sodium homeostasis; transport proteins; hypertension; ubiquitin; regulation; ion channels; phosphorylation; multimeric membrane protein

Lead
Lay summary
In most developed countries, excess salt intake is a major risk factor of hypertension, one of the most common diseases in the human population, affecting over 1 billion people worldwide. The kidney is able to precisely regulate the balance between uptake and excretion of sodium into and from the blood by regulating sodium transport in the kidney. This transport involves the regulated action of a transport protein called sodium-chloride cotransporter (NCC) that transport sodium and chloride and a channel (called ENaC), which forms a pore across membranes and allows passage of sodium. Because sodium is the most important cation in the blood, changes in its concentration have direct consequences on blood volume and hence on the arterial blood pressure. Both proteins are regulated by hormonal mechanisms, and can be strongly disturbed in diseases such as Familial Hyperkalemic Hypertension (FHHt) or Liddle's syndrome, which are inherited forms of human hypertension We have largely contributed to the understanding of this regulation, by showing that the activity of these proteins is controlled by a mechanism called ubiquitylation, in which a small protein (named ubiquitin) is linked to either the NCC transporter or the ENaC channel, promoting their internalization and degradation. The regulation of sodium reabsorption in the kidney is very complex, and involves a number of hormonal and regulatory pathways that function in concert with each other, assuring a fine tuning of these processes. Aim: In this project we want to study in detail the role of different hormonal and signaling pathways (including ubiquitylation) both in vitro and in vivo, in epithelial cells derived from the kidney, and in mouse models, in which ubiquitylation of NCC or ENaC, or of activity of other potential regulators, has been inactivated. Significance: The process addresses fundamental issues with respect to the regulation of sodium balance (and its consequences for maintenance of blood pressure), and it treats novel biological issues with respect to the regulated ubiquitylation of ion transport proteins and channels.

Direct link to Lay Summary

Last update: 21.02.2013

Number	Title	Start	Funding scheme

Hypertension is the most common disease in the human population, affecting over 1 billion individuals worldwide, and one of the major treatable risk factors for cardiovascular diseases including stroke, myocardial infarctus, heart and kidney failure. Blood pressure is critically dependent on salt handling by the kidney. The mineralocorticoid aldosterone is the main hormone involved in the control of Na+ homeostasis. In this context, the aldosterone-sensitive distal nephron (ASDN) that includes the distal convoluted tubule (DCT), the connecting tubule (CNT), and the collecting duct (CD), precisely tunes the Na+ balance in extracellular fluids (including the blood) by regulating transepithelial Na+ transport via a number of Na+ transporting proteins, including the thiazide-sensitive Na+,Cl--cotransporter (NCC) in the DCT and the epithelial Na+ channel (ENaC) in the DCT/CNT/CD. In recent years it has become evident that aldosterone not only regulates ENaC, but also NCC in a coordinated manner. Remarkably, NCC and ENaC are differentially regulated, a phenomena referred to as the aldosterone paradox. Accordingly, if aldosterone is elevated due to hypovolemia, it stimulates primarily NCC, in order to strongly absorb Na+ without promoting K+ secretion. In contrast, if circulating aldosterone is high due to hyperkalemia, NCC activity is weak, with the purpose of providing large quantities of Na+ to the CNT/CD to stimulate electrogenic Na+ reabsorption via ENaC, serving as driving force for K+ secretion across the same cells. It is not well understood how this differential regulation is achieved. Genetic evidence suggests that a network of kinases, WNK1, WNK4, and SPAK is involved in NCC regulation, but knowledge on the precise molecular mechanisms of their action is sparse. Our recent findings, both in vitro and in vivo, suggest that Sgk1 and Nedd4-2 regulate NCC as well, adding an exciting dimension to these regulatory networks and providing some clues to the unresolved questions in the differential NCC and/or ENaC regulation. Moreover, we have identified a novel aldosterone-induced kinase, Pim3, which may also contribute to this regulatory network. We therefore postulate the following hypothesis:The thiazide-sensitive Na+,Cl--cotransporter NCC and the amiloride-sensitive Na+ channel ENaC are regulated by the ubiquitin-protein ligase Nedd4-2, which is controlled by a complex network of kinases, including the WNK/SPAK, the Sgk1, and the recently identified aldosterone-induced Pim3 kinase.Specific aims:1) To characterize, both in vitro and in vivo the molecular mechanisms of NCC regulation by Nedd4-2 and Sgk1 and their interaction with the WNK1/WNK4 /SPAK signaling pathway.2) To determine the role of the newly identified aldosterone-induced kinase Pim3 in the regulation of NCC and ENaC.Methods: Heterologous expression in Hek293 cells, oocytes and renal epithelial cells. Analysis of various KO mouse modelsExpected value of project:The project will provide novel avenues and a better understanding of the physiological/pathophysiological regulation of the thiazide-sensitive Na+,Cl--cotransporter NCC and the amiloride-sensitive epithelial Na+ channel ENaC, both fundamental for Na+ homeostasis and blood pressure regulation.