ENaC activity in collecting ducts modulates NCC in cirrhotic mice.

Cirrhosis is a frequent and severe disease, complicated by renal sodium retention leading to ascites and oedema. A better understanding of the complex mechanisms responsible for renal sodium handling could improve clinical management of sodium retention. Our aim was to determine the importance of the amiloride-sensitive epithelial sodium channel (ENaC) in collecting ducts in compensate and decompensate cirrhosis. Bile duct ligation was performed in control mice (CTL) and collecting duct-specific αENaC knockout (KO) mice, and ascites development, aldosterone plasma concentration, urinary sodium/potassium ratio and sodium transporter expression were compared. Disruption of ENaC in collecting ducts (CDs) did not alter ascites development, urinary sodium/potassium ratio, plasma aldosterone concentrations or Na,K-ATPase abundance in CCDs. Total αENaC abundance in whole kidney increased in cirrhotic mice of both genotypes and cleaved forms of α and γ ENaC increased only in ascitic mice of both genotypes. The sodium chloride cotransporter (NCC) abundance was lower in non-ascitic KO, compared to non-ascitic CTL, and increased when ascites appeared. In ascitic mice, the lack of αENaC in CDs induced an upregulation of total ENaC and NCC and correlated with the cleavage of ENaC subunits. This revealed compensatory mechanisms which could also take place when treating the patients with diuretics. These compensatory mechanisms should be considered for future development of therapeutic strategies

Sodium retention in rats with liver cirrhosis is associated with increased renal abundance of NaCl cotransporter (NCC)

Background. Liver cirrhosis is associated with enhanced renal tubular sodium retention, the mechanism of which is still debated. We hypothesized that liver cirrhosis is associated with increased expression of renal epithelial sodium transporter(s). Methods. Liver cirrhosis was induced by bile duct ligation (BDL) in rats. Steady state mRNA of ENaC subunits α, β, γ serum and glucocorticoid inducible kinase (Sgk1) were measured by TaqMan PCR in kidney homogenates at week 1, 2, 3 and 4 after BDL. Renal protein content of ENaC subunits, ubiquitin-protein-ligase Nedd4-2 and NaCl cotransporter (NCC) were assessed by western blot. Subcellular localization of ENaC subunits and NCC were analysed by immunohistochemistry. Results. Steady state mRNA of ENaC α, β and γ were unchanged during the 4 weeks investigated, while ENaC protein decreased most prominently at week 2 (control vs BDL; α, −46%; β, −81%; and γ, −63%; n = 6). Subcellular localization of ENaC subunits was not altered at week 2. Sgk1 mRNA did not change, whereas Nedd4-2 protein was reduced by >50% 2-4 weeks after BDL. NCC protein significantly increased at week 1 (control vs BDL: +66%, n = 6, P<0.05) and decreased at week 3 (control vs BDL: −85%, n = 6, P<0.0005). Conclusions. Enhanced abundance of NCC was observed in the initial stage after BDL, followed by a marked decrease. ENaC transcription, translation or cell surface abundance was not increased after BD

Severe hyperkalemia is rescued by low-potassium diet in renal βENaC-deficient mice.

In adulthood, an induced nephron-specific deficiency of αENaC (Scnn1a) resulted in pseudohypoaldosteronism type 1 (PHA-1) with sodium loss, hyperkalemia, and metabolic acidosis that is rescued through high-sodium/low-potassium (HNa &lt;sup&gt;+&lt;/sup&gt; /LK &lt;sup&gt;+&lt;/sup&gt; ) diet. In the present study, we addressed whether renal βENaC expression is required for sodium and potassium balance or can be compensated by remaining (α and γ) ENaC subunits using adult nephron-specific knockout (Scnn1b &lt;sup&gt;Pax8/LC1&lt;/sup&gt; ) mice. Upon induction, these mice present a severe PHA-1 phenotype with weight loss, hyperkalemia, and dehydration, but unlike the Scnn1a &lt;sup&gt;Pax8/LC1&lt;/sup&gt; mice without persistent salt wasting. This is followed by a marked downregulation of STE20/SPS1-related proline-alanine-rich protein kinase (SPAK) and Na &lt;sup&gt;+&lt;/sup&gt; /Cl &lt;sup&gt;-&lt;/sup&gt; co-transporter (NCC) protein expression and activity. Most of the experimental Scnn1b &lt;sup&gt;Pax8/LC1&lt;/sup&gt; mice survived with a HNa &lt;sup&gt;+&lt;/sup&gt; /LK &lt;sup&gt;+&lt;/sup&gt; diet that partly normalized NCC phosphorylation, but not total NCC expression. Since salt loss was minor, we applied a standard-sodium/LK &lt;sup&gt;+&lt;/sup&gt; diet that efficiently rescued these mice resulting in normokalemia and normalization of NCC phosphorylation, but not total NCC expression. A further switch to LNa &lt;sup&gt;+&lt;/sup&gt; /standard-K &lt;sup&gt;+&lt;/sup&gt; diet induced again a severe PHA-1-like phenotype, but with only transient salt wasting indicating that low-K &lt;sup&gt;+&lt;/sup&gt; intake is critical to decrease hyperkalemia in a NCC-dependent manner. In conclusion, while the βENaC subunit plays only a minor role in sodium balance, severe hyperkalemia results in downregulation of NCC expression and activity. Our data demonstrate the importance to primarily correct the hyperkalemia with a low-potassium diet that normalizes NCC activity

Extracellular K(+) rapidly controls NaCl cotransporter phosphorylation in the native distal convoluted tubule by Cl(-) -dependent and independent mechanisms.

High dietary potassium (K(+) ) intake dephosphorylates and inactivates the NaCl cotransporter (NCC) in the renal distal convoluted tubule (DCT). Using several ex vivo models, we show that physiological changes in extracellular K(+) , similar to those occurring after a K(+) rich diet, are sufficient to promote a very rapid dephosphorylation of NCC in native DCT cells. Although the increase of NCC phosphorylation upon decreased extracellular K(+) appears to depend on cellular Cl(-) fluxes, the rapid NCC dephosphorylation in response to increased extracellular K(+) is not Cl(-) -dependent. The Cl(-) -dependent pathway involves the SPAK/OSR1 kinases, whereas the Cl(-) independent pathway may include additional signalling cascades. A high dietary potassium (K(+) ) intake causes a rapid dephosphorylation, and hence inactivation, of the thiazide-sensitive NaCl cotransporter (NCC) in the renal distal convoluted tubule (DCT). Based on experiments in heterologous expression systems, it was proposed that changes in extracellular K(+) concentration ([K(+) ]ex ) modulate NCC phosphorylation via a Cl(-) -dependent modulation of the with no lysine (K) kinases (WNK)-STE20/SPS-1-44 related proline-alanine-rich protein kinase (SPAK)/oxidative stress-related kinase (OSR1) kinase pathway. We used the isolated perfused mouse kidney technique and ex vivo preparations of mouse kidney slices to test the physiological relevance of this model on native DCT. We demonstrate that NCC phosphorylation inversely correlates with [K(+) ]ex , with the most prominent effects occurring around physiological plasma [K(+) ]. Cellular Cl(-) conductances and the kinases SPAK/OSR1 are involved in the phosphorylation of NCC under low [K(+) ]ex . However, NCC dephosphorylation triggered by high [K(+) ]ex is neither blocked by removing extracellular Cl(-) , nor by the Cl(-) channel blocker 4,4'-diisothiocyano-2,2'-stilbenedisulphonic acid. The response to [K(+) ]ex on a low extracellular chloride concentration is also independent of significant changes in SPAK/OSR1 phosphorylation. Thus, in the native DCT, [K(+) ]ex directly and rapidly controls NCC phosphorylation by Cl(-) -dependent and independent pathways that involve the kinases SPAK/OSR1 and a yet unidentified additional signalling mechanism

High glucose up-regulates ENaC and SGK1 expression in HCD-cells

Background/Aim: Diabetic nephropathy is associated with progressive renal damage, leading to impaired function and end-stage renal failure. Secondary hypertension stems from a deranged ability of cells within the kidney to resolve and appropriately regulate sodium resorption in response to hyperglycaemia. However, the mechanisms by which glucose alters sodium re-uptake have not been fully characterised. Methods: Here we present RT-PCR, western blot and immunocytochemistry data confirming mRNA and protein expression of the serum and glucocorticoid inducible kinase (SGK1) and the a conducting subunit of the epithelial sodium channel (ENaC) in a model in vitro system of the human cortical collecting duct (HCD). We examined changes in expression of these elements in response to glucose challenge, designed to mimic hyperglycaemia associated with type 2 diabetes mellitus. Changes in Na+ concentration were assessed using single-cell microfluorimetry. Results: Incubation with glucose, the Ca2+-ionophore ionomycin and the cytokine TGF-beta 1 were all found to evoke significant and time-dependent increases in both SGK1 and alpha ENaC protein expression. These molecular changes were correlated to an increase in Na+-uptake at the single-cell level. Conclusion: Together these data offer a potential explanation for glucose-evoked Na+-resorption and a potential contributory role of SGK1 and ENaCs in development of secondary hypertension, commonly linked to diabetic nephropathy

Hand and Eye Dominance in Sport: Are Cricket Batters Taught to Bat Back-to-Front?

Background: When first learning to bimanually use a tool to hit a target (e.g., when chopping wood or hitting a golf ball), most people assume a stance that is dictated by their dominant hand. By convention, this means that a ‘right-handed’ or ‘left-handed’ stance that places the dominant hand closer to the striking end of the tool is adopted in many sports. Objective: The aim of this study was to investigate whether the conventional stance used for bimanual hitting provides the best chance of developing expertise in that task. Methods: Our study included 43 professional (international/first-class) and 93 inexperienced (<5 years’ experience) cricket batsmen. We determined their batting stance (plus hand and eye dominance) to compare the proportion of batters who adopted a reversed stance when batting (that is, the opposite stance to that expected based on their handedness). Results: We found that cricket batsmen who adopted a reversed stance had a stunning advantage, with professional batsmen 7.1 times more likely to adopt a reversed stance than inexperienced batsmen, independent of whether they batted right or left handed or the position of their dominant eye. Conclusion: Findings imply that batsmen who adopt a conventional stance may inadvertently be batting ‘back-to-front’ and have a significant disadvantage in the game. Moreover, the results may generalize more widely, bringing into question the way in which other bimanual sporting actions are taught and performed

Proliferation of Acid-Secretory Cells in the Kidney during Adaptive Remodelling of the Collecting Duct

The renal collecting duct adapts to changes in acid-base metabolism by remodelling and altering the relative number of acid or alkali secreting cells, a phenomenon termed plasticity. Acid secretory A intercalated cells (A-IC) express apical H+-ATPases and basolateral bicarbonate exchanger AE1 whereas bicarbonate secretory B intercalated cells (B-IC) express basolateral (and apical) H+-ATPases and the apical bicarbonate exchanger pendrin. Intercalated cells were thought to be terminally differentiated and unable to proliferate. However, a recent report in mouse kidney suggested that intercalated cells may proliferate and that this process is in part dependent on GDF-15. Here we extend these observations to rat kidney and provide a detailed analysis of regional differences and demonstrate that differentiated A-IC proliferate massively during adaptation to systemic acidosis. We used markers of proliferation (PCNA, Ki67, BrdU incorporation) and cell-specific markers for A-IC (AE1) and B-IC (pendrin). Induction of remodelling in rats with metabolic acidosis (with NH4Cl for 12 hrs, 4 and 7 days) or treatment with acetazolamide for 10 days resulted in a larger fraction of AE1 positive cells in the cortical collecting duct. A large number of AE1 expressing A-IC was labelled with proliferative markers in the cortical and outer medullary collecting duct whereas no labeling was found in B-IC. In addition, chronic acidosis also increased the rate of proliferation of principal collecting duct cells. The fact that both NH4Cl as well as acetazolamide stimulated proliferation suggests that systemic but not urinary pH triggers this response. Thus, during chronic acidosis proliferation of AE1 containing acid-secretory cells occurs and may contribute to the remodelling of the collecting duct or replace A-IC due to a shortened life span under these conditions

Dietary sodium induces a redistribution of the tubular metabolic workload.

Body Na &lt;sup&gt;+&lt;/sup&gt; content is tightly controlled by regulated urinary Na &lt;sup&gt;+&lt;/sup&gt; excretion. The intrarenal mechanisms mediating adaptation to variations in dietary Na &lt;sup&gt;+&lt;/sup&gt; intake are incompletely characterized. We confirmed and expanded observations in mice that variations in dietary Na &lt;sup&gt;+&lt;/sup&gt; intake do not alter the glomerular filtration rate but alter the total and cell-surface expression of major Na &lt;sup&gt;+&lt;/sup&gt; transporters all along the kidney tubule. Low dietary Na &lt;sup&gt;+&lt;/sup&gt; intake increased Na &lt;sup&gt;+&lt;/sup&gt; reabsorption in the proximal tubule and decreased it in more distal kidney tubule segments. High dietary Na &lt;sup&gt;+&lt;/sup&gt; intake decreased Na &lt;sup&gt;+&lt;/sup&gt; reabsorption in the proximal tubule and increased it in distal segments with lower energetic efficiency. The abundance of apical transporters and Na &lt;sup&gt;+&lt;/sup&gt; delivery are the main determinants of Na &lt;sup&gt;+&lt;/sup&gt; reabsorption along the kidney tubule. Tubular O &lt;sub&gt;2&lt;/sub&gt; consumption and the efficiency of sodium reabsorption are dependent on sodium diet. Na &lt;sup&gt;+&lt;/sup&gt; excretion by the kidney varies according to dietary Na &lt;sup&gt;+&lt;/sup&gt; intake. We undertook a systematic study of the effects of dietary salt intake on glomerular filtration rate (GFR) and tubular Na &lt;sup&gt;+&lt;/sup&gt; reabsorption. We examined the renal adaptive response in mice subjected to 7 days of a low sodium diet (LSD) containing 0.01% Na &lt;sup&gt;+&lt;/sup&gt; , a normal sodium diet (NSD) containing 0.18% Na &lt;sup&gt;+&lt;/sup&gt; and a moderately high sodium diet (HSD) containing 1.25% Na &lt;sup&gt;+&lt;/sup&gt; . As expected, LSD did not alter measured GFR and increased the abundance of total and cell-surface NHE3, NKCC2, NCC, α-ENaC and cleaved γ-ENaC compared to NSD. Mathematical modelling predicted that tubular Na &lt;sup&gt;+&lt;/sup&gt; reabsorption increased in the proximal tubule but decreased in the distal nephron because of diminished Na &lt;sup&gt;+&lt;/sup&gt; delivery. This prediction was confirmed by the natriuretic response to diuretics targeting the thick ascending limb, the distal convoluted tubule or the collecting system. On the other hand, HSD did not alter measured GFR but decreased the abundance of the aforementioned transporters compared to NSD. Mathematical modelling predicted that tubular Na &lt;sup&gt;+&lt;/sup&gt; reabsorption decreased in the proximal tubule but increased in distal segments with lower transport efficiency with respect to O &lt;sub&gt;2&lt;/sub&gt; consumption. This prediction was confirmed by the natriuretic response to diuretics. The activity of the metabolic sensor adenosine monophosphate-activated protein kinase (AMPK) was related to the changes in tubular Na &lt;sup&gt;+&lt;/sup&gt; reabsorption. Our data show that fractional Na &lt;sup&gt;+&lt;/sup&gt; reabsorption is distributed differently according to dietary Na &lt;sup&gt;+&lt;/sup&gt; intake and induces changes in tubular O &lt;sub&gt;2&lt;/sub&gt; consumption and sodium transport efficiency

Renal tubular SGK1 deficiency causes impaired K+ excretion via loss of regulation of NEDD4-2/WNK1 and ENaC.

The stimulation of postprandial K(+) clearance involves aldosterone-independent and -dependent mechanisms. In this context, serum- and glucocorticoid-induced kinase (SGK)1, a ubiquitously expressed kinase, is one of the primary aldosterone-induced proteins in the aldosterone-sensitive distal nephron. Germline inactivation of SGK1 suggests that this kinase is fundamental for K(+) excretion under conditions of K(+) load, but the specific role of renal SGK1 remains elusive. To avoid compensatory mechanisms that may occur during nephrogenesis, we used inducible, nephron-specific Sgk1(Pax8/LC1) mice to assess the role of renal tubular SGK1 in K(+) regulation. Under a standard diet, these animals exhibited normal K(+) handling. When challenged by a high-K(+) diet, they developed severe hyperkalemia accompanied by a defect in K(+) excretion. Molecular analysis revealed reduced neural precursor cell expressed developmentally downregulated protein (NEDD)4-2 phosphorylation and total expression. γ-Epithelial Na(+) channel (ENaC) expression and α/γENaC proteolytic processing were also decreased in mutant mice. Moreover, with no lysine kinase (WNK)1, which displayed in control mice punctuate staining in the distal convoluted tubule and diffuse distribution in the connecting tubule/cortical colleting duct, was diffused in the distal convoluted tubule and less expressed in the connecting tubule/collecting duct of Sgk(Pax8/LC1) mice. Moreover, Ste20-related proline/alanine-rich kinase phosphorylation, and Na(+)-Cl(-) cotransporter phosphorylation/apical localization were reduced in mutant mice. Consistent with the altered WNK1 expression, increased renal outer medullary K(+) channel apical localization was observed. In conclusion, our data suggest that renal tubular SGK1 is important in the regulation of K(+) excretion via the control of NEDD4-2, WNK1, and ENaC