Randomized Trial on the Effect of Oral Potassium Chloride Supplementation on the Thiazide-Sensitive Sodium Chloride Cotransporter in Healthy Adults.

Introduction: The putative "renal-K switch" mechanism links dietary potassium intake with sodium retention and involves activation of the sodium chloride (NaCl) cotransporter (NCC) in the distal convoluted tubule in response to low potassium intake, and suppression in response to high potassium intake. This study examined NCC abundance and phosphorylation (phosphorylated NCC [pNCC]) in urinary extracellular vesicles (uEVs) isolated from healthy adults on a high sodium diet to determine tubular responses to alteration in potassium chloride (KCl) intake. Methods: Healthy adults maintained on a high sodium (∼4.5 g [200 mmol]/d) low potassium (∼2.3 g [60 mmol]/d) diet underwent a 5-day run-in period followed by a crossover study, with 5-day supplementary KCl (active phase, Span-K 3 tablets (potassium 24 mmol) thrice daily) or 5-day placebo administrated in random order and separated by 2-day washout. Ambulatory blood pressure (BP) and biochemistries were assessed, and uEVs were analyzed by western blotting. Results: Among the 18 participants who met analysis criteria, supplementary KCl administration (vs. placebo) was associated with markedly higher levels of plasma potassium and 24-hour urine excretion of potassium, chloride, and aldosterone. KCl supplementation was associated with lower uEV levels of NCC (median fold change (KCl/Placebo) = 0.74 [0.30-1.69], P < 0.01) and pNCC (fold change (KCl/Placebo) = 0.81 [0.19-1.75], P < 0.05). Plasma potassium inversely correlated with uEV NCC (R2 = 0.11, P = 0.05). Conclusions: The lower NCC and pNCC in uEVs in response to oral KCl supplementation provide evidence to support the hypothesis of a functional "renal-K switch" in healthy human subjects.

Dissociation of sodium-chloride cotransporter expression and blood pressure during chronic high dietary potassium supplementation.

Dietary potassium (K+) supplementation is associated with a lowering effect in blood pressure (BP), but not all studies agree. Here, we examined the effects of short- and long-term K+ supplementation on BP in mice, whether differences depend on the accompanying anion or the sodium (Na+) intake and molecular alterations in the kidney that may underlie BP changes. Relative to the control diet, BP was higher in mice fed a high NaCl (1.57% Na+) diet for 7 weeks or fed a K+-free diet for 2 weeks. BP was highest on a K+-free/high NaCl diet. Commensurate with increased abundance and phosphorylation of the thiazide sensitive sodium-chloride-cotransporter (NCC) on the K+-free/high NaCl diet, BP returned to normal with thiazides. Three weeks of a high K+ diet (5% K+) increased BP (predominantly during the night) independently of dietary Na+ or anion intake. Conversely, 4 days of KCl feeding reduced BP. Both feeding periods resulted in lower NCC levels but in increased levels of cleaved (active) α and γ subunits of the epithelial Na+ channel ENaC. The elevated BP after chronic K+ feeding was reduced by amiloride but not thiazide. Our results suggest that dietary K+ has an optimal threshold where it may be most effective for cardiovascular health.

In Primary Aldosteronism Acute Potassium Chloride Supplementation Suppresses Abundance and Phosphorylation of the Sodium-Chloride Cotransporter.

Background: Elevated abundance of sodium-chloride cotransporter (NCC) and phosphorylated NCC (pNCC) are potential markers of primary aldosteronism (PA), but these effects may be driven by hypokalemia. Methods: We measured plasma potassium in patients with PA. If potassium was <4.0 mmol/L, patients were given sufficient oral potassium chloride (KCl) over 24 hours to achieve as close to 4.0 mmol/L as possible. Clinical chemistries were assessed, and urinary extracellular vesicles (uEVs) were examined to investigate effects on NCC. Results: Among 21 patients with PA who received a median total dose of 6.0 g (2.4-16.8 g) of KCl, increases were observed in plasma potassium (from 3.4 to 4.0 mmol/L; P<0.001), aldosterone (from 305 to 558 pmol/L; P=0.01), and renin (from 1.2 to 2.5 mIU/L; P<0.001), whereas decreases were detected in uEV levels of NCC (median fold change(post/basal) [FC]=0.71 [0.09-1.99]; P=0.02), pT60-NCC (FC=0.84 [0.06-1.66]; P=0.05), and pT55/60-NCC (FC=0.67 [0.08-2.42]; P=0.02). By contrast, in 10 patients with PA who did not receive KCl, there were no apparent changes in plasma potassium, NCC abundance, and phosphorylation status, but increases were observed in plasma aldosterone (from 178 to 418 pmol/L; P=0.006) and renin (from 2.0 to 3.0 mU/L; P=0.009). Plasma potassium correlated inversely with uEV levels of NCC (R 2=0.11; P=0.01), pT60-NCC (R 2=0.11; P=0.01), and pT55/60-NCC (R 2=0.11; P=0.01). Conclusions: Acute oral KCl loading replenished plasma potassium in patients with PA and suppressed NCC abundance and phosphorylation, despite a significant rise in plasma aldosterone. This supports the view that potassium supplementation in humans with PA overrides the aldosterone stimulatory effect on NCC. The increased plasma aldosterone in patients with PA without KCl supplementation may be due to aldosterone response to posture challenge.

Potassium Effects on NCC Are Attenuated during Inhibition of Cullin E3-Ubiquitin Ligases.

The thiazide-sensitive sodium chloride cotransporter (NCC) plays a vital role in maintaining sodium (Na+) and potassium (K+) homeostasis. NCC activity is modulated by with-no-lysine kinases 1 and 4 (WNK1 and WNK4), the abundance of which is controlled by the RING-type E3 ligase Cullin 3 (Cul3) and its substrate adapter Kelch-like protein 3. Dietary K+ intake has an inverse correlation with NCC activity, but the mechanism underlying this phenomenon remains to be fully elucidated. Here, we investigated the involvement of other members of the cullin family in mediating K+ effects on NCC phosphorylation (active form) and abundance. In kidneys from mice fed diets varying in K+ content, there were negative correlations between NCC (phosphorylated and total) and active (neddylated) forms of cullins (Cul1, 3, 4, and 5). High dietary K+ effects on phosphorylated NCC were attenuated in Cul3 mutant mice (CUL3-Het/Δ9). Short-term (30 min) and long-term (24 h) alterations in the extracellular K+ concentration did not affect cullin neddylation levels in ex vivo renal tubules. In the short term, the ability of high extracellular K+ to decrease NCC phosphorylation was preserved in the presence of MLN4924 (pan-cullin inhibitor), but the response to low extracellular K+ was absent. In the long term, MLN4924 attenuated the effects of high extracellular K+ on NCC phosphorylation, and responses to low extracellular K+ were absent. Our data suggest that in addition to Cul3, other cullins are involved in mediating the effects of K+ on NCC phosphorylation and abundance.

The Cl-/HCO3- exchanger pendrin is downregulated during oral co-administration of exogenous mineralocorticoid and KCl in patients with primary aldosteronism.

In primary aldosteronism (PA), the occurrence of K+ loss and hypertension suggest alterations in renal tubular transport, but the molecular basis of these alterations in humans is unclear. In this study, urinary extracellular vesicles (uEVs) isolated from patients undergoing fludrocortisone suppression testing (FST, as a means of confirming or excluding PA) were analyzed using mass spectrometry-based proteomics to determine the combined effects of an aldosterone analogue, NaCl and KCl supplementation on renal tubular protein abundance. Of quantified proteins, the Cl-/HCO3- exchanger pendrin decreased by a median 37% [-15, 57] (P < 0.01) and the potassium channel ROMK increased by a median 31% [-10, 85] (P < 0.01) during FST among 10 PA subjects. The trends remained, but to a lesser degree, in two subjects cured of PA by unilateral adrenalectomy. In PA subjects, plasma K+ increased from median 3.6 to 4.2 mM (P < 0.01) and 24 h urine K+ from 101 to 202 mmol (P < 0.01), while 24 h urine Na+/K+ decreased from 2.3 to 0.8 (P < 0.01). At baseline, pendrin negatively correlated with plasma K+ (P < 0.05) and positively correlated with plasma aldosterone (P < 0.01). There were no clear correlations between Δ pendrin (Δ = D4-D0) and changes in blood or urine variables, and no correlations between ROMK in any of the blood or urine variables either at baseline or during FST. We conclude that oral co-administration of mineralocorticoid and KCl in PA patients is associated with reduced pendrin and enhanced ROMK in uEVs. Pendrin reduction during FST suggests that the suppressive effects of oral KCl may outweigh pendrin upregulation by mineralocorticoids.

K+ and the renin-angiotensin-aldosterone system: new insights into their role in blood pressure control and hypertension treatment.

Approximately 25% of the adult population is diagnosed with hypertension and it is therefore one of the biggest challenges for the health sector. The renin-angiotensin-aldosterone system (RAAS) adjusts effective circulating volume and ultimately blood pressure (BP). Accordingly, antihypertensive drugs targeting the RAAS have been a major focus in modern medical treatment. Low and high dietary K+ intakes are associated with increased or decreased BP and risk of cardiac failure, respectively, suggesting that dietary K+ augmentation has the potential to supplement or replace conventional anti-hypertensive drugs. Animal studies have indicated that the beneficial effects of high dietary K+ may be linked to a dominant regulatory role of plasma K+ on key renal transport proteins controlled by the RAAS. However, only a limited number of studies have investigated whether the reported mechanisms in animal models apply to humans. Furthermore, hypertension is often treated with so-called 'K+ sparing' drugs, thus complicating co-treatment with K+ supplementation. In this review, we revisit old concepts of RAAS effects in the kidney, relate them to effects of dietary K+ manipulation, and finally consider the clinical potential of treating hypertension with K+ supplementation alone or in combination with RAAS inhibitors. Collectively, a wealth of data suggest that increased dietary K+ intake may have beneficial effects on BP in the general population, but underlying medical conditions or current treatment regimens need to be carefully considered before implementing K+ supplementation in patients.

Changes in urinary excretion of water and sodium transporters during amiloride and bendroflumethiazide treatment.

AIM: To quantify changes in urinary excretion of aquaporin2 water channels (u-AQP2), the sodium-potassium-chloride co-transporter (u-NKCC2) and the epithelial sodium channels (u-ENaC) during treatment with bendroflumethiazide (BFTZ), amiloride and placebo. METHODS: In a randomized, double-blinded, placebo-controlled, 3-way crossover study we examined 23 healthy subjects on a standardized diet and fluid intake. The subjects were treated with amiloride 5 mg, BFTZ 1.25 mg or placebo twice a day for 4.5 d before each examination day. On the examination day, glomerular filtration rate was measured by the constant infusion clearance technique with (51)Cr-EDTA as reference substance. To estimate the changes in water transport via AQP2 and sodium transport via NKCC2 and ENaC, u-NKCC2, the gamma fraction of ENaC (u-ENaCγ), and u-AQP2 were measured at baseline and after infusion with 3% hypertonic saline. U-NKCC2, u-ENaCγ, u-AQP2 and plasma concentrations of vasopressin (p-AVP), renin (PRC), angiotensin II (p-ANG II) and aldosterone (p-Aldo) were measured, by radioimmunoassay. Central blood pressure was estimated by applanation tonometry and body fluid volumes were estimated by bio-impedance spectroscopy. General linear model with repeated measures or related samples Friedman's two-way analysis was used to compare differences. Post hoc Bonferroni correction was used for multiple comparisons of post infusion periods to baseline within each treatment group. RESULTS: At baseline there were no differences in u-NKCC2, u-ENaCγ and u-AQP2. PRC, p-Ang II and p-Aldo were increased during active treatments (P < 0.001). After hypertonic saline, u-NKCC2 increased during amiloride (6% ± 34%; P = 0.081) and increased significantly during placebo (17% ± 24%; P = 0.010). U-AQP2 increased significantly during amiloride (31% ± 22%; P < 0.001) and placebo (34% ± 27%; P < 0.001), while u-NKCC2 and u-AQP2 did not change significantly during BFTZ (-7% ± 28%; P = 0.257 and 5% ± 16%; P = 0.261). U- ENaCγ increased in all three groups (P < 0.050). PRC, AngII and p-Aldo decreased to the same extent, while AVP increased, but to a smaller degree during BFTZ (P = 0.048). cDBP decreased significantly during BFTZ (P < 0.001), but not during amiloride or placebo. There were no significant differences in body fluid volumes. CONCLUSION: After hypertonic saline, u-NKCC2 and u-AQP2 increased during amiloride, but not during BFTZ. Lower p-AVP during BFTZ potentially caused less stimulation of NKCC2 and AQP2 and subsequent lower reabsorption of water and sodium.

Quantitative apical membrane proteomics reveals vasopressin-induced actin dynamics in collecting duct cells.

In kidney collecting duct cells, filamentous actin (F-actin) depolymerization is a critical step in vasopressin-induced trafficking of aquaporin-2 to the apical plasma membrane. However, the molecular components of this response are largely unknown. Using stable isotope-based quantitative protein mass spectrometry and surface biotinylation, we identified 100 proteins that showed significant abundance changes in the apical plasma membrane of mouse cortical collecting duct cells in response to vasopressin. Fourteen of these proteins are involved in actin cytoskeleton regulation, including actin itself, 10 actin-associated proteins, and 3 regulatory proteins. Identified were two integral membrane proteins (Clmn, Nckap1) and one actin-binding protein (Mpp5) that link F-actin to the plasma membrane, five F-actin end-binding proteins (Arpc2, Arpc4, Gsn, Scin, and Capzb) involved in F-actin reorganization, and two actin adaptor proteins (Dbn1, Lasp1) that regulate actin cytoskeleton organization. There were also protease (Capn1), protein kinase (Cdc42bpb), and Rho guanine nucleotide exchange factor 2 (Arhgef2) that mediate signal-induced F-actin changes. Based on these findings, we devised a live-cell imaging method to observe vasopressin-induced F-actin dynamics in polarized mouse cortical collecting duct cells. In response to vasopressin, F-actin gradually disappeared near the center of the apical plasma membrane while consolidating laterally near the tight junction. This F-actin peripheralization was blocked by calcium ion chelation. Vasopressin-induced apical aquaporin-2 trafficking and forskolin-induced water permeability increase were blocked by F-actin disruption. In conclusion, we identified a vasopressin-regulated actin network potentially responsible for vasopressin-induced apical F-actin dynamics that could explain regulation of apical aquaporin-2 trafficking and water permeability increase.

Cellular and subcellular distribution of the type-2 vasopressin receptor in the kidney.

Arginine vasopressin (AVP) is essential for maintaining body fluid homeostasis. The antidiuretic effects of AVP are initialized by binding of AVP to the type-2 vasopressin receptor (V2R) in the kidney collecting duct (CD), resulting in the exocytic insertion of aquaporin-2 (AQP-2) water channels into the apical plasma membrane. In this study, we describe the generation and characterization of a polyclonal antibody targeted against the NH2 terminus of the rat V2R. HEK-293 cells overexpressing the rat, mouse, or human V2R showed strong intracellular immunolabeling. Additionally, immunostaining of M-1 kidney cells expressing a V2R-green fluorescent protein (GFP) fusion construct showed colocalization between GFP and antibody-specific V2R labeling. Immunoblots of rat kidney showed 43- and 47-kDa proteins in all zones that were both reduced to 34-kDa by N-glycosidase F. Protein solubilization with nonionic detergents or the use of homobifunctional cross-linkers demonstrated that the rat V2R exists as a protein complex in native kidney. Immunohistochemistry of rat and mouse kidney revealed abundant labeling of the CD. Double-labeling confocal immunofluorescence microscopy [using distal convoluted tubule/connecting tubule (CNT)-specific marker calbindin and CNT/CD-specific marker AQP-2] showed V2R labeling in both CD and CNT. There was a complete absence of labeling in vascular structures and other renal tubules, including the thick ascending limb (TAL), although RT-PCR of microdissected tubules showed expression of V2R mRNA in TAL. Confocal microscopy demonstrated that at the subcellular level, V2R labeling was predominantly intracellular in normal kidneys, although some staining was apparent in basolateral membrane domains. Confocal microscopy of isolated inner medullary collecting duct tubules showed that the V2R is expressed both intracellularly and in basolateral membrane domains.