Juan Pablo Arroyo

Nedd4-2 Modulates Renal Na+-Cl− Cotransporter via the Aldosterone-SGK1-Nedd4-2 Pathway

Regulation of renal Na(+) transport is essential for controlling blood pressure, as well as Na(+) and K(+) homeostasis. Aldosterone stimulates Na(+) reabsorption by the Na(+)-Cl(-) cotransporter (NCC) in the distal convoluted tubule (DCT) and by the epithelial Na(+) channel (ENaC) in the late DCT, connecting tubule, and collecting duct. Aldosterone increases ENaC expression by inhibiting the channels ubiquitylation and degradation; aldosterone promotes serum-glucocorticoid-regulated kinase SGK1-mediated phosphorylation of the ubiquitin-protein ligase Nedd4-2 on serine 328, which prevents the Nedd4-2/ENaC interaction. It is important to note that aldosterone increases NCC protein expression by an unknown post-translational mechanism. Here, we present evidence that Nedd4-2 coimmunoprecipitated with NCC and stimulated NCC ubiquitylation at the surface of transfected HEK293 cells. In Xenopus laevis oocytes, coexpression of NCC with wild-type Nedd4-2, but not its catalytically inactive mutant, strongly decreased NCC activity and surface expression. SGK1 prevented this inhibition in a kinase-dependent manner. Furthermore, deficiency of Nedd4-2 in the renal tubules of mice and in cultured mDCT(15) cells upregulated NCC. In contrast to ENaC, Nedd4-2-mediated inhibition of NCC did not require the PY-like motif of NCC. Moreover, the mutation of Nedd4-2 at either serine 328 or 222 did not affect SGK1 action, and mutation at both sites enhanced Nedd4-2 activity and abolished SGK1-dependent inhibition. Taken together, these results suggest that aldosterone modulates NCC protein expression via a pathway involving SGK1 and Nedd4-2 and provides an explanation for the well-known aldosterone-induced increase in NCC protein expression.

Aldosterone Paradox: Differential Regulation of Ion Transport in Distal Nephron

The mechanisms through which aldosterone promotes apparently opposite effects like salt reabsorption and K(+) secretion remain poorly understood. The identification, localization, and physiological analysis of ion transport systems in distal nephron have revealed an intricate network of interactions between several players, revealing the complex mechanism behind the aldosterone paradox. We review the mechanisms involved in differential regulation of ion transport that allow the fine tuning of salt and K(+) balance.

The SLC12 family of electroneutral cation-coupled chloride cotransporters ☆

The SLC12 family encodes electroneutral cation-coupled chloride cotransporters that are critical for several physiological processes including cell volume regulation, modulation of intraneuronal chloride concentration, transepithelial ion movement, and blood pressure regulation. Members of this family are the targets of the most commonly used diuretic drugs, have been shown to be the causative genes for inherited disease such as Gitelman, Bartter and Andermann syndromes, and potentially play a role in polygenic complex diseases like arterial hypertension, epilepsy, osteoporosis, and cancer.

Insulin Increases the Functional Activity of the Renal NaCl cotransporter

Objectives: Insulin is recognized to increase renal salt reabsorption in the distal nephron and hyperinsulinemic states have been shown to be associated with increased expression of the renal NaCl cotransporter (NCC). However, the effect of insulin on NCC functional activity has not been reported. Methods: Using a heterologous expression system of Xenopus laevis oocytes, a mouse distal convoluted cell line, mDCT15 cells, endogenously expressing NCC, and an ex-vivo kidney perfusion technique, we assessed the effect of insulin on the activity and phosphorylation of NCC. The signaling pathway involved was analyzed. Results: In Xenopus oocytes insulin increases the activity of NCC together with its phosphorylation at threonine residue 58. Activation of NCC by insulin was also observed in mDCT15 cells. Additionally, insulin increased the NCC phosphorylation in kidney under the ex-vivo perfusion technique. In oocytes and mDCT15 cells, insulin effect on NCC was prevented with inhibitors of phosphatidylinositol 3-kinase (PI3K), mTORC2, and AKT1 kinases, but not by inhibitors of MAP or mTORC1 kinases, suggesting that PI3K-mTORC2-AKT1 is the intracellular pathway required. Additionally, activation of NCC by insulin was not affected by wild-type or mutant versions of with no lysine kinase 1, with no lysine kinase 4, or serum glucocorticoid kinase 1, but it was no longer observed in the presence of wild-type or the dominant negative, catalytically inactive with no lysine kinase 3, implicating this kinase in the process. Conclusion: Insulin induces activation and phosphorylation of NCC. This effect could play an important role in arterial hypertension associated with hyperinsulinemic states, such as obesity, metabolic syndrome, or type 2 diabetes mellitus.

WNK3 abrogates the NEDD4-2-mediated inhibition of the renal Na+-Cl− cotransporter

The serine/threonine kinase WNK3 and the ubiquitin-protein ligase NEDD4-2 are key regulators of the thiazide-sensitive Na+-Cl- cotransporter (NCC), WNK3 as an activator and NEDD2-4 as an inhibitor. Nedd4-2 was identified as an interacting partner of WNK3 through a glutathione-S-transferase pull-down assay using the N-terminal domain of WNK3, combined with LC-MS/MS analysis. This was validated by coimmunoprecipitation of WNK3 and NEDD4-2 expressed in HEK293 cells. Our data also revealed that the interaction between Nedd4-2 and WNK3 does not involve the PY-like motif found in WNK3. The level of WNK3 ubiquitylation did not change when NEDD4-2 was expressed in HEK293 cells. Moreover, in contrast to SGK1, WNK3 did not phosphorylate NEDD4-2 on S222 or S328. Coimmunoprecipitation assays showed that WNK3 does not regulate the interaction between NCC and NEDD4-2. Interestingly, in Xenopus laevis oocytes, WNK3 was able to recover the SGK1-resistant NEDD4-2 S222A/S328A-mediated inhibition of NCC and further activate NCC. Furthermore, elimination of the SPAK binding site in the kinase domain of WNK3 (WNK3-F242A, which lacks the capacity to bind the serine/threonine kinase SPAK) prevented the WNK3 NCC-activating effect, but not the Nedd4-2-inhibitory effect. Together, these results suggest that a novel role for WNK3 on NCC expression at the plasma membrane, an effect apparently independent of the SPAK kinase and the aldosterone-SGK1 pathway.

Advances in WNK Signaling of Salt and Potassium Metabolism: Clinical Implications

Recent evidence due to the discovery of a family of kinases implicated in arterial hypertension now points to the underlying molecular mechanisms that dictate Na+, K+ and water handling in the nephron. These new key players need to be understood in order to fully comprehend the pathophysiology, manifestations, and treatment of common clinical entities such as hypovolemic shock, congestive heart failure, primary hyperaldosteronism, nephrotic syndrome and hypertension. It is through the analysis of the volume status and electrolyte abnormalities that commonly present with these diseases that we can begin to create a link between the abstract concept of a kinase regulation and how a patient will respond to a particular treatment. This review is an attempt to bridge that gap.

Independent regulation of Na+ and K+ balance by the kidney.

The understanding of the independent regulation of sodium and potassium by the kidney has remained elusive. Recent evidence now points to dissimilar regulatory mechanisms in ion handling, dependent on the presence of either aldosterone alone or angiotensin II with aldosterone among other factors. This review summarizes past and present information in an attempt to reconcile the current concepts of differential regulation of sodium and potassium balance through the with-no-lysine (K) kinase (WNK) system and the previous knowledge regarding ion transport mechanisms in the distal nephron.