Gamma-melanocyte stimulating hormone regulates the expression and cellular localization of epithelial sodium channel in inner medullary collecting duct cells.

Gamma(2)-melanocyte-stimulating hormone (γ2MSH) is a peptide hormone released by the pituitary gland which is thought to act directly on the renal inner medulla to promote increased sodium excretion into urine (natriuresis). The aim of this study was to determine if a stable analog, [Nle(3), D-Phe(6)]-γ2MSH (NDP-γ2MSH), of the native peptide regulated the activity, expression and cellular localization of epithelial sodium channel (ENaC) in a murine inner medullary collecting duct (mIMCD-3) cell line. Our results indicate that expression of the γ2MSH receptor, melanocortin receptor 3 receptor (MC3R), is up-regulated by culturing the cells in media with an increased osmolality (∼400mOsm/kg). Furthermore, stimulation of cAMP signaling and sodium transport by 1nM NDP-γ2MSH occurs only in cells cultured in the high osmolality media. Finally, treatment of mIMCD-3 cells cultured in high osmolality medium for 1h with 1nM NDP-γ2MSH causes a reduction in expression of serum- and glucocorticoid-induced kinase (sgk1) and a reduction in expression and cell surface abundance of the alpha subunit of ENaC. Collectively, this data suggest that γ2MSH directly regulates both ENaC expression and cellular localization in the inner medulla to exert its natriuretic effect.

[Nle3,d-Phe6 ]-γ2 -melanocyte-stimulating hormone possesses the renal excretory but not the cardiovascular actions of the native γ2 -melanocyte-stimulating hormone in anaesthetized rats.

The present study compared the cardiovascular and renal actions of γ(2) -melanocyte-stimulating hormone (γ(2) MSH) with those of the synthetic analogue [Nle(3) ,d-Phe(6) ]-γ(2) MSH (NDP-γ(2) MSH) and explored the effects of high dietary salt intake on the renal actions of NDP-γ(2) MSH. Both peptides were infused systemically (3-1000 nmol/kg) and intrarenally (500 fmol/min) into innervated and renally denervated rats fed either a normal (0.4% NaCl) or high-salt (4% NaCl; HS) diet. Mean arterial pressure (MAP), glomerular filtration rate (GFR), urinary sodium excretion (U(N) (a) V), urinary output (UV) and fractional sodium excretion were determined, as was expression of the melanocortin MC(3) receptor in inner medullary collecting duct (IMCD) epithelial cells. Both renal and systemic infusion of γ(2) MSH increased MAP by 23 ± 2% and 54 ± 4%, respectively, but equivalent doses of NDP-γ(2) MSH had no significant pressor effects. Both peptides had similar natriuretic and diuretic effects in rats fed a normal salt diet. However, NDP-γ(2) MSH increased U(N) (a) V and UV by two- to threefold in rats fed the normal salt diet and by six- to sevenfold in rats fed the HS diet. Furthermore, NDP-γ(2) MSH induced a 3.5-fold increase in GFR only in rats fed the HS diet. These renal effects of NDP-γ(2) MSH were not abolished by prior renal denervation. Rats fed the HS diet also exhibited a 4.5-fold increase in MC(3) receptor expression in IMCD epithelial cells. Intrarenal infusion of NDP-γ(2) MSH induced the natriuretic but not the cardiovascular effects exhibited by γ(2) MSH. The renal activities may be attributed to a direct binding of NDP-γ(2) MSH to MC(3) receptors expressed in IMCD cells, leading to a potent natriuretic effect that is independent of renal innervation.

Expression of the epithelial Na(+) channel and other components of an aldosterone response pathway in human adrenocortical cells.

We have unexpectedly found expression of the epithelial Na(+) channel (ENaC) in human adrenocortical cells and tested the hypothesis that these cells contain the components of an aldosterone response pathway. Tissue was obtained from patients undergoing adrenalectomy and mRNA and protein expression of recognised components of an aldosterone-response pathway were determined by RT-PCR and Western blotting. The effects of mineralocorticoid receptor agonists and antagonists, amiloride analogues, and extracellular Na(+) on basal and stimulated aldosterone release from immortalised (H295R) cells were determined by radioimmunoassay. Expression of mRNA for alpha-, beta- and gamma-subunits of ENaC, the mineralocorticoid receptor, Nedd4L, Sgk1 and 11beta hydroxysteroid dehydrogenase type II was confirmed in human adrenal cortex. Using Western blotting alpha-, beta- and gamma-ENaC expression was demonstrated in adrenocortical cells. Measurements of 24 h aldosterone release from H295R cells showed stimulation by K(+) and angiotensin II, suppression by both Na(+) and high-concentration 5-(N-ethyl-N-isopropyl) amiloride (EIPA, blocker of Na(+)-H(+) exchange) and no change with benzamil (ENaC blocker). (22)Na-uptake into H295R cells was inhibited by EIPA, but not by benzamil. Our experiments suggest that the components of an aldosterone response pathway are present in human adrenal cortex. Studies in H295R cells, however, suggest that ENaC is not an important mediator of (22)Na-uptake or aldosterone production. Further studies are required to determine the importance of an adrenal aldosterone response pathway.

The acidic motif of WNK4 is crucial for its interaction with the K channel ROMK.

WNK kinases have rapidly emerged as important regulators of Na+ and K+ homoeostasis in the mammalian kidney where they regulate the trafficking of proteins such as the NaCl-cotransporter (NCCT) and K+ channel, ROMK. However, an increasing number of WNK effects are kinase-independent, including their interaction with ROMK, and involve instead protein-protein interactions. Outside of their kinase domain all WNKs contain a unique run of predominantly negatively charged amino acids dubbed the acidic motif, where the WNK4 disease mutations causing Gordon's syndrome also cluster. To look further at the role of this motif we studied the effects of WNK4 fragments, including one with a deleted acidic motif (DeltaAM) and a 10-mer acidic motif peptide on ROMK expression in Xenopus oocytes. We found that an N-terminal fragment of WNK4 (1-620 WNK4) containing the acidic motif retains full activity in inhibiting ROMK currents. However, DeltaAM WNK4 is completely inactive and the effect of WNK4 or 1-620 WNK4 can be completely blocked by co-injection of the 10-mer acidic motif peptide. The blocking action of the peptide was sequence specific as a peptide with a randomised sequence was inactive. These results on ROMK currents were paralleled by changes in membrane expression of fluorescent EGFP-ROMK. Finally, we show that 1-620 WNK4 can pull down ROMK and this interaction can be blocked with the acidic motif peptide. These results confirm the important role of the acidic motif of WNK4 in its protein-protein interaction with the ROMK channel.

Regulation of the expression of the Na/Cl cotransporter by WNK4 and WNK1: evidence that accelerated dynamin-dependent endocytosis is not involved.

The novel serine/threonine kinases (with no lysine kinases or WNKs), WNK1 and WNK4, are encoded by the disease genes for Gordon syndrome (PRKWNK1 and PRKWNK4), a rare monogenic syndrome of hypertension and hyperkalemia. These proteins alter the expression of the thiazide-sensitive Na/Cl cotransporter (NCCT) in Xenopus laevis oocytes, although the details are controversial. We describe here our own experience and confirm that kinase-dead WNK4 (318D>A) is unable to affect Na+ fluxes through the thiazide-sensitive Na/Cl transporter (NCCT) or its membrane expression as an ECFP-NCCT fusion protein. However, the kinase domain is not sufficient for a functional WNK4 since deletion of the acidic motif (a motif unique to WNK family members) completely abolishes functional activity. Indeed, the NH2 terminal of WNK4 (1-620) containing the kinase domain and acidic motif retains full activity, but does not interact directly with NCCT in pull-down assays. Coexpression of WNK1 antagonizes the action of WNK4, and kinase-dead WNK1 (368D>A) or WNK1 carrying a WNK4 disease mutation (565Q>E) behaves in the same way as wild-type WNK1. This suggests kinase activity and charge conservation within the acidic motif are not essential for the WNK1-WNK4 interaction. We also report that WNK4 probably reduces surface expression largely through an effect on forward trafficking. Hence, the effect of WNK4 on NCCT expression is mimicked by dynamin, but the dominant-negative K44A dynamin mutant does not block the action of WNK4 itself. These results further highlight important differences in the mechanism by which WNK kinases affect expression of NCCT vs. other membrane proteins such as ROMK.

WNK1 affects surface expression of the ROMK potassium channel independent of WNK4.

The WNK (with no lysine kinase) kinases are a novel class of serine/threonine kinases that lack a characteristic lysine residue for ATP docking. Both WNK1 and WNK4 are expressed in the mammalian kidney, and mutations in either can cause the rare familial syndrome of hypertension and hyperkalemia (Gordon syndrome, or pseudohypoaldosteronism type 2). The molecular basis for the action of WNK4 is through alteration in the membrane expression of the NaCl co-transporter (NCCT) and the renal outer-medullary K channel KCNJ1 (ROMK). The actions of WNK1 are less well defined, and evidence to date suggests that it can affect NCCT expression but only in the presence of WNK4. The results of co-expressing WNK1 with ROMK in Xenopus oocytes are reported for the first time. These studies show that WNK1 is able to suppress total current directly through ROMK by causing a marked reduction in its surface expression. The effect is mimicked by a kinase-dead mutant of WNK1 (368D > A), suggesting that it is not dependent on its catalytic activity. Study of the time course of ROMK expression further suggests that WNK1 accelerates trafficking of ROMK from the membrane, and this effect seems to be dynamin dependent. Using fragments of full-length WNK1, it also is shown that the effect depends on residues in the middle section of the protein (502 to 1100 WNK1) that contains the acidic motif. Together, these findings emphasize that the molecular mechanisms that underpin WNK1 regulation of ROMK expression are distinct from those that affect NCCT expression.

A new kindred with pseudohypoaldosteronism type II and a novel mutation (564D>H) in the acidic motif of the WNK4 gene.

We identified a new kindred with the familial syndrome of hypertension and hyperkalemia (pseudohypoaldosteronism type II or Gordon's syndrome) containing an affected father and son. Mutation analysis confirmed a single heterozygous G to C substitution within exon 7 (1690G>C) that causes a missense mutation within the acidic motif of WNK4 (564D>H). We confirmed the function of this novel mutation by coexpressing it in Xenopus oocytes with either the NaCl cotransporter (NCCT) or the inwardly rectifying K-channel (ROMK). Wild-type WNK4 inhibits 22Na+ flux in Xenopus oocytes expressing NCCT by approximately 90% (P<0.001), whereas the 564D>H mutant had no significantly inhibitory effect on flux through NCCT. In oocytes expressing ROMK, wild-type WNK4 produced >50% inhibition of steady-state current through ROMK at a +20-mV holding potential (P<0.001). The 564D>H mutant produced further inhibition with steady-state currents to some 60% to 70% of those seen with the wild-type WNK4. Using fluorescent-tagged NCCT (enhanced cyan fluorescent protein-NCCT) and ROMK (enhanced green fluorescent protein-ROMK) to quantify the expression of the proteins in the oocyte membrane, it appears that the functional effects of the 564D>H mutation can be explained by alteration in the surface expression of NCCT and ROMK. Compared with wild-type WNK4, WNK4 564D>H causes increased cell surface expression of NCCT but reduced expression of ROMK. This work confirms that the novel missense mutation in WNK4, 564D>H, is functionally active and highlights further how switching charge on a single residue in the acid motif of WNK4 affects its interaction with the thiazide-sensitive target NCCT and the potassium channel ROMK.

WNK kinases and the control of blood pressure.

The WNK kinases are a small group of serine/threonine kinases with unique catalytic domains that lack the lysine residue used in other kinases to co-ordinate ATP (hence, With No K [WNK]). Their closest homologues are found within the mitogen-activated protein kinase (MAPK) pathway suggesting a role in signalling. Two WNK isoforms, WNK1 and WNK4, have been identified as the disease genes for a rare monogenic hypertension syndrome (Gordon's syndrome or pseudohypoaldosteronism type 2 [PHA2]) implicating them in salt homeostasis by the kidney. This is supported by recent data showing widespread expression of WNK1 and WNK4 in mammalian transporting epithelia. Within the kidney, WNKs probably regulate the surface expression of several proteins involved in ion transport, including the sodium-chloride cotransporter (NCCT) and the potassium channel renal outer medullary potassium channel (ROMK), based on co-expression studies in Xenopus oocytes. WNKs, especially WNK4, have been suggested as candidate genes for essential hypertension itself, but evidence for this is lacking. Some of the effects of the WNKs are independent of their kinase function, suggesting that they are dependent on specific protein-protein interactions. It seems likely that the WNKs are part of much larger protein scaffolds in cells and have effects in cells beyond ion transport. However, because of their effect on expression of the NCCT they are attractive drug targets for the development of novel antihypertensive agents. These agents could potentially offer the efficacy of a thiazide diuretic, but without the metabolic side effects usually seen with this class of antihypertensive therapy.