Does Plasma Inhibit the Activity of KCl Cotransport in Red Cells From LK Sheep?

Red cells from LK sheep represent an important paradigm for control of KCl cotransport activity, as well as being important to sheep erythroid function. A previous report (Godart et al., 1997) suggested that autologous plasma markedly inhibits red cell KCC activity and identified the presence of the bicarbonate/CO2 buffer system as the probable cause. Findings were restricted, however, to red cells from patients with sickle cell disease (SCD) swollen anisotonically and carried out at a very high O2 tension (c.700 mmHg). It was therefore important to investigate the generality of the effect described and whether it was also relevant to the two main stimuli for KCC activity encountered most often by circulating red cells in vivo - low pH in active muscle beds during exercise and high urea concentrations in the renal medulla during antidiuresis. Results confirm that inhibition was significant in response to anisotonic swelling with KCC activity in MOPS-buffered saline (MBS) vs. bicarbonate-buffered saline (BBS) and in MBS vs. plasma both reduced (by about 25 and 50%, respectively). By contrast, however, inhibition was absent at low pH and in high concentrations of urea. These findings suggest therefore that red cell KCC activity represents an important membrane permeability in vivo in red cells suspended in plasma. They are relevant, in particular, to sheep red cells, and may also be important by extension to those of other species and to the abnormal red cells found in human patients with SCD.

Pathophysiological Relevance of Renal Medullary Conditions on the Behaviour of Red Cells From Patients With Sickle Cell Anaemia.

Red cells from patients with sickle cell anaemia (SCA) contain the abnormal haemoglobin HbS. Under hypoxic conditions, HbS polymerises and causes red cell sickling, a rise in intracellular Ca2+ and exposure of phosphatidylserine (PS). These changes make sickle cells sticky and liable to lodge in the microvasculature, and so reduce their lifespan. The aim of the present work was to investigate how the peculiar conditions found in the renal medulla - hypoxia, acidosis, lactate, hypertonicity and high levels of urea - affect red cell behaviour. Results show that the first four conditions all increased sickling and PS exposure. The presence of urea at levels found in a healthy medulla during antidiuresis, however, markedly reduced sickling and PS exposure and would therefore protect against red cell adherence. Loss of the ability to concentrate urine, which occurs in sickle cell nephropathy would obviate this protective effect and may therefore contribute to pathogenesis.

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.

The effect of the antisickling compound GBT1118 on the permeability of red blood cells from patients with sickle cell anemia.

Sickle cell anemia (SCA) is one of the commonest severe inherited disorders. Nevertheless, effective treatments remain inadequate and novel ones are avidly sought. A promising advance has been the design of novel compounds which react with hemoglobin S (HbS) to increase oxygen (O2 ) affinity and reduce sickling. One of these, voxelotor (GBT440), is currently in advanced clinical trials. A structural analogue, GBT1118, was investigated in the current work. As RBC dehydration is important in pathogenesis of SCA, the effect of GBT1118 on RBC cation permeability was also studied. Activities of Psickle , the Gardos channel and the KCl cotransporter (KCC) were all reduced. Gardos channel and KCC activities were also inhibited in RBCs treated with Ca2+ ionophore or the thiol reagent N-ethylmaleimide, indicative of direct effects on these two transport systems. Consistent with its action on RBC membrane transporters, GBT1118 significantly increased RBC hydration. RBC hemolysis was reduced in a nonelectrolyte lysis assay. Further to its direct effects on O2 affinity, GBT1118 was therefore found to reduce RBC shrinkage and fragility. Findings reveal important effects of GBT1118 on protecting sickle cells and suggest that this is approach may represent a useful therapy for amelioration of the clinical complications of SCA.