Impairment in renal medulla development underlies salt wasting in Clc-k2 channel deficiency
Meng-Hsuan Lin, Jen-Chi Chen, Xuejiao Tian, Chia-Ming Lee, I-Shing Yu, Yi-Fen Lo, Shinichi Uchida, Chou-Long Huang, Bi-Chang Chen, Chih-Jen Cheng
Meng-Hsuan Lin, Jen-Chi Chen, Xuejiao Tian, Chia-Ming Lee, I-Shing Yu, Yi-Fen Lo, Shinichi Uchida, Chou-Long Huang, Bi-Chang Chen, Chih-Jen Cheng
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Research Article Nephrology

Impairment in renal medulla development underlies salt wasting in Clc-k2 channel deficiency

Abstract

The prevailing view is that the ClC-Ka chloride channel (mouse Clc-k1) functions in the thin ascending limb to control urine concentration, whereas the ClC-Kb channel (mouse Clc-k2) functions in the thick ascending limb (TAL) to control salt reabsorption. Mutations of ClC-Kb cause classic Bartter syndrome, characterized by renal salt wasting, with perinatal to adolescent onset. We studied the roles of Clc-k channels in perinatal mouse kidneys using constitutive or inducible kidney-specific gene ablation and 2D and advanced 3D imaging of optically cleared kidneys. We show that Clc-k1 and Clc-k2 were broadly expressed and colocalized in perinatal kidneys. Deletion of Clc-k1 and Clc-k2 revealed that both participated in NKCC2- and NCC-mediated NaCl reabsorption in neonatal kidneys. Embryonic deletion of Clc-k2 caused tubular injury and impaired renal medulla and TAL development. Inducible deletion of Clc-k2 beginning after medulla maturation produced mild salt wasting resulting from reduced NCC activity. Thus, both Clc-k1 and Clc-k2 contributed to salt reabsorption in TAL and distal convoluted tubule (DCT) in neonates, potentially explaining the less-severe phenotypes in classic Bartter syndrome. As opposed to the current understanding that salt wasting in adult patients with Bartter syndrome is due to Clc-k2 deficiency in adult TAL, our results suggest that it originates mainly from defects occurring in the medulla and TAL during development.

Authors

Meng-Hsuan Lin, Jen-Chi Chen, Xuejiao Tian, Chia-Ming Lee, I-Shing Yu, Yi-Fen Lo, Shinichi Uchida, Chou-Long Huang, Bi-Chang Chen, Chih-Jen Cheng

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Figure 10

Postneonatal Clc-k2 deletion causes a milder phenotype.

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Postneonatal Clc-k2 deletion causes a milder phenotype. (A) BW from the ...
(A) BW from the beginning of induction (week 3) to week 10. Results are presented as mean ± SEM, and data were analyzed by 2-way ANOVA with mixed-effects analysis. Doxycycline’s side effects likely cause the difference at weeks 5 and 6. (B) Gross appearance and H&E staining of the longitudinal section of a doxycycline-induced Clc-k2 deficient kidney (Doxycycline) and a control kidney (2% Sucrose). (C) Water deprivation and DDAVP tests were performed on 10-week-old doxycycline-induced Clc-k2–deficient mice and controls. NS between Clc-k2–deficient and control groups and between baseline and 2 hours after DDAVP (0.4 ng/g body weight) injection by 2-way ANOVA with mixed-effect analysis. (D) WB analysis of total and phosphorylated levels of Ncc and Nkcc2 in 10-week-old inducible Clc-k2–deficient kidneys and controls. Actin loading controls are shown for each blot. Protein expression was normalized to the amount of β-actin and reported relative to controls. The abundance of each band was measured by densitometry by the ImageJ program. Results shown are representative of 4 experiments with similar results. Results are presented as mean ± SEM. Mice in the same experiment were compared. *P < 0.05 and NS between Clc-k2–deficient mice (Doxycycline) and controls (2% Sucrose) using unpaired t test. (E) Hctz (12.5 mg/kg) challenge test reflects the in vivo activity of Ncc in 10-week-old inducible Clc-k2–deficient mice and controls. *P < 0.05 between Clc-k2 deficient mice and controls using unpaired t test.