Combined genetic disruption of K-Cl cotransporters and Gardos channel KCNN4 rescues erythrocyte dehydration in the SAD mouse model of sickle cell disease

Abstract

Excessive red cell dehydration contributes to the pathophysiology of sickle cell disease (SCD). The densest fraction of sickle red cells (with the highest corpuscular hemoglobin concentration) undergoes the most rapid polymerization of deoxy-hemoglobin S, leading to accelerated cell sickling and increased susceptibility to endothelial activation, red cell adhesion, and vaso-occlusion. Increasing red cell volume in order to decrease red cell density can thus serve as an adjunct therapeutic goal in SCD. Regulation of circulating mouse red cell volume and density is mediated largely by the Gardos channel, KCNN4, and the K-Cl cotransporters, KCC3 and KCC1. Whereas inhibition of the Gardos channel in subjects with sickle cell disease increased red cell volume, decreased red cell density, and improved other hematological indices in subjects with SCD, specific KCC inhibitors have not been available for testing. We therefore investigated the effect of genetic inactivation of KCC3 and KCC1 in the SAD mouse model of sickle red cell dehydration, finding decreased red cell density and improved hematological indices. We describe here generation of mice genetically deficient in the three major red cell volume regulatory gene products, KCNN4, KCC3, and KCC1 in C57BL6 non-sickle and SAD sickle backgrounds. We show that combined loss-of-function of all three gene products in SAD mice leads to incrementally increased MCV, decreased CHCM and % hyperchromic cells, decreased red cell density (phthalate method), increased resistance to hypo-osmotic lysis, and increased cell K content. The data show that combined genetic deletion of the Gardos channel and K-Cl cotransporters in a mouse SCD model decreases red cell density and improves several hematological parameters, supporting the strategy of combined pharmacological inhibition of these ion transport pathways in the adjunct treatment of human SCD.

Keywords: Osmotic fragility; Potassium channel; Potassium transporter; Red blood cell; Splenomegaly.

Fig. 1.. Genetic deletion of Kcnn4 increases spleen-to-body weight ratio in all tested genetic backgrounds.

Values represent mean ± SEM for (n) mice of the indicated genotypes. One-way ANOVA, non-paired Kruskal-Wallis test. *, p<0.02 for comparison of SAD;Kcnn4^−/− vs. SAD. **, p<0.003 for comparisons of SAD;3xKO vs. SAD or vs. SAD;Kcnn4^−/−. ***, p<0.0001 for comparisons of 3xKO vs. WT^Kcnn4 or vs. Kcnn4^−/−; and for comparisons of WT^Kcnn4 vs. Kcnn4^−/− or vs. WT^SAD.

Fig. 2.. Spleen histology (hematoxylin-eosin stain) of the indicated genetic backgrounds.

Magnification 600x. Representative sections from the 3 mice of each indicated genotype examined.

Fig. 3.. Red cell phthalate density profiles as function of genotype.

A. Red cells from WT mice (black diamonds) compared with red cells from Kcc1^−/−;Kcc3^−/− mice (2xKO, open triangles) and red cells from Kcnn4^−/−;Kcc1^−/−;Kcc3^−/− mice (3xKO, open circles). B. Red cells from WT (black circles, reproduced from data of panel A) and SAD mice (open triangles) compared with Kcnn4^−/− mice (open diamonds) and SAD;Kcnn4^−/− mice (black squares). WT mice in panels A and B included WT^Kcnn4 (n=8) and WT^SAD (n=11), which were indistinguishable (not shown). C. Red cells from SAD mice (black squares, data replicated from panel B) compared with red cells from SAD;Kcc1^−/−;Kcc3^−/− mice (open circles) and red cells from SAD;Kcnn4^−/−;Kcc1^−/−;Kcc3^−/− mice (black triangles). Values are means ± SEM for (n) independently tested pooled samples from each indicated genotype.

Fig. 4.. Red cell susceptibility to osmotic lysis as function of genotype.

A. Osmotic lysis curves of red cells from WT mice (black triangles; pooled data from WT^SAD (n=3) and WT^Kcnn4 (n=4)) compared with osmotic lysis curves of red cells from Kcc1^−/−;Kcc3^−/− mice (open squares) and from Kcnn4^−/−;Kcc1^−/−;Kcc3^−/− mice (black circles). B. Osmotic lysis curves SAD mice (black squares) compared with those of red cells from SAD;Kcnn4^−/− mice (open circles), SAD;Kcc1^−/−;Kcc3^−/− mice (open triangles), and SAD;Kcnn4^−/−;Kcc1^−/−;Kcc3^−/− mice (black diamonds). Values are means ± SEM for (n) independently tested pooled samples from each genotype.

Fig. 5.. Basal and stimulated K-Cl cotransport activity in WT and SAD (B) red cells.

K-Cl cotransport activity of WT and SAD red cells was measured in isotonic medium (ISO), hypotonic medium (Hypo), isotonic medium containing 1 μM staurosporine (Stauro), and isotonic medium with added 500 mM urea (Urea). Values are means ± SEM for (n) independent experiments with blood pooled from 2–4 mice of indicated genotype. ISO WT red cells (n=5) included WT^C57 (n=2), WT^Kcnn4 (n=2) and WT^SAD (n=1). WT red cells in stimulated conditions were (for HYPO, n=10) 1 WT^C57 + 9 WT^Kcnn4; (for Stauro, n=8) 1 WT^C57 + 7 WT^Kcnn4; and (for Urea, n=9) 1 WT^C57, 1 WT^SAD, and 7 WT^Kcnn4. WT and SAD samples were compared for each condition by Mann-Whitney non-paired test.

Fig. 6.. Basal and stimulated K-Cl cotransport activity in SAD;Kcnn4^−/− red cells compared with (A) SAD red cells and with (B) Kcnn4^−/− red cells.

K-Cl cotransport activity was measured in isotonic medium (ISO), hypotonic medium (Hypo), isotonic medium containing 1 μM staurosporine (Stauro), and isotonic medium with added 500 mM urea (Urea). Values are means ± SEM for (n) independent experiments with blood pooled from 2–4 mice of indicated genotype. WT and SAD samples were compared for each condition by Mann-Whitney non-paired test. SAD mouse data in panel A is replicated from that of Figure 5.

Fig. 7.. Basal and stimulated K-Cl cotransport activity in red cells from SAD;Kcnn4^−/−;Kcc1^−/−;Kcc3^−/− mice compared with (A) red cells from SAD mice and with (B) red cells from from Kcnn4^−/−;Kcc1^−/−;Kcc3^−/− mice.

K-Cl cotransport activity was measured in isotonic medium (ISO), hypotonic medium (Hypo), isotonic medium containing 1 μM staurosporine (Stauro), and isotonic medium with added 500 mM urea (Urea). Values are means ± SEM for (n) independent experiments with blood pooled from 2–4 mice of indicated genotype. For each condition, each genotype pair was compared by Mann-Whitney non-paired test. SAD mouse data in panel A is replicated from that of Figure 5. Data from SAD;Kcnn4^−/−;Kcc1^−/−;Kcc3^−/− mouse red cells is replicated in panels A and B for ease of comparison.