Disruption of erythroid K-Cl cotransporters alters erythrocyte volume and partially rescues erythrocyte dehydration in SAD mice
Marco B. Rust, … , Thomas J. Jentsch, Christian A. Hübner
Marco B. Rust, … , Thomas J. Jentsch, Christian A. Hübner
Published June 1, 2007
Citation Information: J Clin Invest. 2007;117(6):1708-1717. https://doi.org/10.1172/JCI30630.
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Research Article Hematology

Disruption of erythroid K-Cl cotransporters alters erythrocyte volume and partially rescues erythrocyte dehydration in SAD mice

Abstract

K-Cl cotransport activity in rbc is a major determinant of rbc volume and density. Pathologic activation of erythroid K-Cl cotransport activity in sickle cell disease contributes to rbc dehydration and cell sickling. To address the roles of individual K-Cl cotransporter isoforms in rbc volume homeostasis, we disrupted the Kcc1 and Kcc3 genes in mice. As rbc K-Cl cotransport activity was undiminished in Kcc1–/– mice, decreased in Kcc3–/– mice, and almost completely abolished in mice lacking both isoforms, we conclude that K-Cl cotransport activity of mouse rbc is mediated largely by KCC3. Whereas rbc of either Kcc1–/– or Kcc3–/– mice were of normal density, rbc of Kcc1–/–Kcc3–/– mice exhibited defective volume regulation, including increased mean corpuscular volume, decreased density, and increased susceptibility to osmotic lysis. K-Cl cotransport activity was increased in rbc of SAD mice, which are transgenic for a hypersickling human hemoglobin S variant. Kcc1–/–Kcc3–/– SAD rbc lacked nearly all K-Cl cotransport activity and exhibited normalized values of mean corpuscular volume, corpuscular hemoglobin concentration mean, and K+ content. Although disruption of K-Cl cotransport rescued the dehydration phenotype of most SAD rbc, the proportion of the densest red blood cell population remained unaffected.

Authors

Marco B. Rust, Seth L. Alper, York Rudhard, Boris E. Shmukler, Rubén Vicente, Carlo Brugnara, Marie Trudel, Thomas J. Jentsch, Christian A. Hübner

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Figure 1

Generation of Kcc1–/– mice.

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Generation of Kcc1–/– mice. (A) Partial genomic organiza...
(A) Partial genomic organization of Slc12a4 (top) and the targeting construct (middle). Exons are shown as vertical bars, loxP sites as arrowheads. Transient Cre expression resulted in excision of exons 4 and 5 (bottom), producing a frameshift and premature stop. DTA, diphtheria toxin A cassette. (B) An additional EcoRV site was exploited for Southern blot analysis and resulted in the approximately 6.5-kb KO compared with the approximately 10-kb Kcc1+/+ fragment with the probe shown in A. (C) Northern blot analysis of Kcc1+/+, Kcc1+/–, and Kcc1–/– liver tissue with a full-length KCC1 probe revealed some residual aberrant transcript. (D) A membrane protein immunoblot with a KCC1 antibody confirmed the absence of KCC1 in tissues of Kcc1–/– mice. (E) rbc membrane protein immunoblot demonstrated KCC3b expression in WT rbc and absence of KCC3a, KCC2, and KCC4 in erythrocytes of WT, Kcc1–/–Kcc3–/–, and SADKcc1–/–Kcc3–/– mice. Actin served as a loading control. Kidney served as a positive control for KCC1 and KCC3b, brain for KCC2 and KCC3a, and lung for KCC4. (F and G) Expression levels of KCC1 and KCC3 proteins in rbc ghosts of various genotypes. (F) KCC3b protein level was upregulated in ghosts lacking KCC1, but KCC1 levels were unchanged in KCC3-KO ghosts. (G) Levels of both KCC1 and KCC3b proteins were increased in ghosts from SAD mice. Protein levels were determined by Western blot analysis (see Supplemental Figure 1) and normalized to actin. Bars represent arithmetic means from 6 mice normalized to WT. Error bars represent SEM. *P < 0.05, **P < 0.005 compared with WT.