The role of WNK in modulation of KCl cotransport activity in red cells from normal individuals and patients with sickle cell anaemia

Abnormal activity of red cell KCl cotransport (KCC) is involved in pathogenesis of sickle cell anaemia (SCA). KCC-mediated solute loss causes shrinkage, concentrates HbS, and promotes HbS polymerisation. Red cell KCC also responds to various stimuli including pH, volume, urea, and oxygen tension, and regulation involves protein phosphorylation. The main aim of this study was to investigate the role of the WNK/SPAK/OSR1 pathway in sickle cells. The pan WNK inhibitor WNK463 stimulated KCC with an EC50 of 10.9 ± 1.1 nM and 7.9 ± 1.2 nM in sickle and normal red cells, respectively. SPAK/OSR1 inhibitors had little effect. The action of WNK463 was not additive with other kinase inhibitors (staurosporine and N-ethylmaleimide). Its effects were largely abrogated by pre-treatment with the phosphatase inhibitor calyculin A. WNK463 also reduced the effects of physiological KCC stimuli (pH, volume, urea) and abolished any response of KCC to changes in oxygen tension. Finally, although protein kinases have been implicated in regulation of phosphatidylserine exposure, WNK463 had no effect. Findings indicate a predominant role for WNKs in control of KCC in sickle cells but an apparent absence of downstream involvement of SPAK/OSR1. A more complete understanding of the mechanisms will inform pathogenesis whilst manipulation of WNK activity represents a potential therapeutic approach.

Potassium excretion during antinatriuresis: perspective from a distal nephron model

Renal excretion of Na+ and K+ must be regulated independently within the distal nephron, but is complicated by the fact that changing excretion of one solute requires adjustments in the transport of both. It is long known that hypovolemia increases Na+ reabsorption while impairing K+ excretion, even when distal Na+ delivery is little changed. Renewed interest in this micropuncture observation came with identification of the molecular defects underlying familial hyperkalemic hypertension (FHH), which also increases distal Na+ reabsorption and impairs K+ excretion. In this work, a mathematical model of the distal nephron (Weinstein AM. Am J Physiol Renal Physiol 295: F1353–F1364, 2008), including the distal convoluted tubule (DCT), connecting segment (CNT), and collecting duct (CD), is used to examine renal K+ excretion during antinatriuresis. Within the model, Na+ avidity is represented as the modulation of DCT NaCl reabsorption, and the K+ secretion signal is an aldosterone-like effect on principal cells of the CNT and CD. The first model prediction is that changes in DCT NaCl reabsorption are not mediated by NaCl cotransporter density alone, but require additional adjustments of both peritubular Na-K-ATPase and KCl cotransport. A second observation is that the CNT response to increased DCT Na+ reabsorption should not only stabilize CD K+ delivery but also compensate for the compromise of K+ excretion downstream, as low Na+ delivery increases CD K+ reabsorption. Such anticipatory regulation is seen with the aldosterone response of hypovolemia, while the FHH phenotype manifests enhanced DCT NaCl transport but a blunted aldosterone effect. The model emphasizes the need for two distinct signals to the distal nephron, regulating Na+ excretion and K+ excretion, in contrast to a single switch apportioning NaCl reabsorption and Na+-for-K+ exchange.

ROS activate KCl cotransport in nonadherent Ehrlich ascites cells but K+ and Cl− channels in adherent Ehrlich Lettré and NIH3T3 cells

Addition of H2O2 (0.5 mM) to Ehrlich ascites tumor cells under isotonic conditions results in a substantial (22 ± 1%) reduction in cell volume within 25 min. The cell shrinkage is paralleled by net loss of K+, which was significant within 8 min, whereas no concomitant increase in the K+ or Cl− conductances could be observed. The H2O2-induced cell shrinkage was unaffected by the presence of clofilium and clotrimazole, which blocks volume-sensitive and Ca2+-activated K+ channels, respectively, and is unaffected by a raise in extracellular K+ concentration to a value that eliminates the electrochemical driving force for K+. On the other hand, the H2O2-induced cell shrinkage was impaired in the presence of the KCl cotransport inhibitor (dihydro-indenyl)oxyalkanoic acid (DIOA), following substitution of NO3− for Cl−, and when the driving force for KCl cotransport was omitted. It is suggested that H2O2 activates electroneutral KCl cotransport in Ehrlich ascites tumor cells and not K+ and Cl− channels. Addition of H2O2 to hypotonically exposed cells accelerates the regulatory volume decrease and the concomitant net loss of K+, whereas no additional increase in the K+ and Cl− conductance was observed. The effect of H2O2 on cell volume was blocked by the serine-threonine phosphatase inhibitor calyculin A, indicating an important role of serine-threonine phosphorylation in the H2O2-mediated activation of KCl cotransport in Ehrlich cells. In contrast, addition of H2O2 to adherent cells, e.g., Ehrlich Lettré ascites cells, a subtype of the Ehrlich ascites tumor cells, and NIH3T3 mouse fibroblasts increased the K+ and Cl− conductances after hypotonic cell swelling. Hence, H2O2 induces KCl cotransport or K+ and Cl− channels in nonadherent and adherent cells, respectively.

Urea stimulation of KCl cotransport induces abnormal volume reduction in sickle reticulocytes

KCl cotransport (KCC) activity contributes to pathologic dehydration in sickle (SS) red blood cells (RBCs). KCC activation by urea was measured in SS and normal (AA) RBCs as Cl-dependent Rb influx. KCC-mediated volume reduction was assessed by measuring reticulocyte cellular hemoglobin concentration (CHC) cytometrically. Urea activated KCC fluxes in fresh RBCs to levels seen in swollen cells, although SS RBCs required lower urea concentrations than did normal (AA) RBCs. Little additional KCC stimulation by urea occurred in swollen AA or SS RBCs. The pH dependence of KCC in “euvolemic” SS RBCs treated with urea was similar to that in swollen cells. Urea triggered volume reduction in SS and AA reticulocytes, establishing a higher CHC. Volume reduction was Cl dependent and was limited by the KCC inhibitor, dihydro-indenyl-oxyalkanoic acid. Final CHC depended on urea concentration, but not on initial CHC. Under all activation conditions, volume reduction was exaggerated in SS reticulocytes and produced higher CHCs than in AA reticulocytes. The sulfhydryl-reducing agent, dithiothreitol, normalized the sensitivity of KCC activation to urea in SS RBCs and mitigated the urea-stimulated volume decrease in SS reticulocytes, suggesting that the dysfunctional activity of KCC in SS RBCs was due in part to reversible sulfhydryl oxidation.

Mature Sickle and Normal Red Cells Exhibit Regulatory Volume Decrease Activated by Urea and Mediated by KCl Cotransport.

KCl Cotransport (KCC) is active in normal (AA) reticulocytes and overly active in sickle (SS) reticulocytes. Cell swelling activates KCC and induces a powerful regulatory volume decrease (RVD) in reticulocytes, which increases cellular hemoglobin concentration (CHC) to new steady state values that are higher in SS than AA cells (Blood2004;104(9):2954–60). We recently showed that urea (300–900 mM), which strongly activates KCC, also induces an intense RVD with even higher final CHC values (SS>AA) (Blood2004; 104 (11): 976a). Because KCC activity is high in reticulocyte-rich samples in both SS and AA blood, KCC activity has been assumed to be minimal in mature cells. We now report that mature RBC exhibit RVD stimulated by urea and mediated by KCC. AA and SS RBC were washed in HBS and treated with nystatin to increase cation content and decrease CHC to 22–24 gm/dl. During incubation at 37o in HBS (145 mM NaCl, 5 KCl, 1 MgCl2, 10 glucose, 20 HEPES, pH 7.4) ± 600 mM urea, timed samples were taken into iced HBS, washed, and kept on ice until analyzed later that day on an Advia 120 automated cell counter, which reports frequency distributions for CHC of both mature RBC and reticulocytes. As previously reported, within 30 min reticulocytes achieved a new steady state CHC which was higher for SS than AA cells, though the speed of RVD was similar. Surprisingly, mean CHC of mature (non-reticulocyte) RBC in both AA and SS blood also increased upon incubation with urea. RVD in mature cells was slower than in reticulocytes and was apparently incomplete after 2 hours. RVD in mature RBC was completely abrogated (CHC was stable) in the absence of Cl- (sulfamate substitution) or in the presence of 100 uM DIOA (dihydro-indenyl-oxy-alkanoic acid), both of which inhibit KCC activity. Whereas reticulocyte CHC frequency distributions after urea-stimulated KCC-mediated RVD showed a single population, CHC distributions for mature RBC revealed two distinct sub-populations: One in which CHC changed little during incubation and a second which achieved a CHC similar to that achieved by reticulocytes after RVD. The relative size of the volume regulating (high CHC) sub-population increased steadily throughout the incubation, which was responsible for the progressive increase in mean CHC values. The high CHC sub-population was not apparent when cells were incubated in Cl- free media or with DIOA, indicating that RVD was mediated by KCC. After 2 hours incubation, 67 ± 8 % of SS RBC had shifted to higher CHC, compared to 37 ± 11 % of mature AA RBC (p<<0.001 by t-test). The progressive change in CHC histograms during incubation was consistent with cells achieving the same final CHC values at various rates. In preliminary studies with biotin-labeled AA cells ageing in vivo, urea-stimulated RVD in mature cells diminished with time, but persisted through most of RBC lifespan. These data indicate that the KCl cotransporter remains in the membrane of mature AA RBC, and is capable of producing RVD under the strong stimulation of urea. In SS RBC, which have shorter lifespan, a majority of non-reticulocytes retain urea-stimulated KCC activity.

KCl cotransport mediates abnormal sulfhydryl-dependent volume regulation in sickle reticulocytes

KCl cotransport (KCC) activation by cell swelling and pH was compared in sickle (SS) and normal (AA) red blood cells (RBCs). KCC fluxes had the same relationship to mean corpuscular hemoglobin concentration (MCHC) in SS and AA RBCs when normalized to the maximal volume-stimulated (VSmax) flux (MCHC < 270 g/L [27 g/dL]). Acid-stimulated (pH 6.9) KCC flux in SS RBCs was 60% to 70% of VSmax KCC versus 20% in AA RBCs. Density gradients were used to track changes in reticulocyte MCHC during KCC-mediated regulatory volume decrease (RVD). Swelling to MCHC of 260 g/L (26 g/dL) produced Cl-dependent RVD that resulted in higher MCHC in SS than AA reticulocytes. In acid pH, RVD was also greater in SS than AA reticulocytes. Sulfhydryl reduction by dithiothreitol (DTT) lowered VSmax KCC flux in AA and SS RBCs by one third but did not alter swelling-induced RVD. DTT lowered acid-activated KCC in SS RBCs by 50% and diminished acid-induced RVD in SS reticulocytes. Thus, swelling activation of KCC is normal in SS RBCs but KCC-mediated RVD produces higher MCHC in SS than AA reticulocytes. Acid activation of KCC is exaggerated in SS RBCs and causes dehydration in SS reticulocytes. KCC response to acid stimulation was mitigated by DTT, suggesting that it arises from sulfhydryl oxidation.