Kidney Week

Abstract: SA-PO0503

Gain-of-Function WNK4 Rescues Cell Cycle and Proliferation Defects in ClC-Kb-Deficient Thick Ascending Limb

Session Information

• Fluid, Electrolyte, and Acid-Base Disorders: Basic Research
  November 08, 2025 | Location: Exhibit Hall, Convention Center
  Abstract Time: 10:00 AM - 12:00 PM

Category: Fluid, Electrolytes, and Acid-Base Disorders

• 1101 Fluid, Electrolyte, and Acid-Base Disorders: Basic

Authors

• Hsieh, Chiao-Hui, The University of Iowa Roy J and Lucille A Carver College of Medicine, Iowa City, Iowa, United States

Chiao-Hui Hsieh, PhD

• Chen, Yu Jen, Taichung Veterans General Hospital, Taichung, Taichung City, Taiwan

Yu Jen Chen, MD

• Morrison, Emily, The University of Iowa Roy J and Lucille A Carver College of Medicine, Iowa City, Iowa, United States

Emily Morrison

• Huang, Chou-Long, The University of Iowa Roy J and Lucille A Carver College of Medicine, Iowa City, Iowa, United States

Chou-Long Huang, MD, PhD

• Cheng, Chih-Jen, The University of Iowa Roy J and Lucille A Carver College of Medicine, Iowa City, Iowa, United States

Chih-Jen Cheng, MD, PhD

Background

ClC-Kb deficiency impairs the transport and development of the thick ascending limbs (TALs) and distal convoluted tubules (DCTs). Conversely, activation of with-no-lysine 4 (WNK4) kinase stimulates salt reabsorption and cell hypertrophy in DCTs. This study investigates how ClC-Kb deficiency affects gene expression and cellular state in TALs, and whether gain-of-function WNK4^D561A/+ can reverse these changes.

Methods

TALs isolated from Clc-k2 (mouse ClC-Kb ortholog)-knockout (Clc-k2^-/-), Wnk4^D561A/+ rescued (Wnk4^D561A/+ /Clc-k2-ko), and control mice were subjected to transcriptome analysis. Primary cultured TAL or HEK cells overexpressing ClC-Kb/barttin were treated with NPPB, a chloride channel blocker, or vehicle for cell cycle and proliferation assays. Clc-k2^-/- mice were interbred with WNK4^D561A/+ knockin or Kcc4-knockout (Kcc4^-/-) mice to decide whether WNK4^D561A/+ and Kcc4 improve the phenotype of Clc-k2^-/- mice.

Results

Deletion of Clc-k2 led to transcriptomic changes in cell cycle regulation, cell proliferation, and early injury responses in TALs. Primary cultured Clc-k2^-/- TALs proliferated 40% less efficiently than wild-type cells. NPPB induced G2-M cell cycle arrest and impaired cell proliferation in ClC-Kb-overexpressed HEK cells. The phenotype of WNK4^D561A/+/Clc-k2^-/- mice was analyzed and compared to Clc-k2^-/- mice receiving salt supplementation. While both approaches prevented neonatal death in Clc-k2^-/- mice, only WNK4^D561A/+/Clc-k2^-/- mice showed improvements in growth, activity of sodium cotransporters, urine concentration, and renal fibrosis. Moreover, WNK4^D561A/+ corrected the transcriptomic alterations induced by Clc-k2 deficiency, indicating that enhancing transport activity is achievable and therapeutic in Clc-k2-deficient renal tubules. Double deletion of Clc-k2 and Kcc4 exacerbated neonatal mortality, growth delay, and renal salt wasting, suggesting that Kcc4 partially compensates for Clc-k2 in the TAL/DCT.

Conclusion

Clc-k2 regulates the cell cycle and proliferation of TALs, likely by affecting transport activity and cellular growth. The gain-of-function Wnk4 significantly improves the phenotype, renal histology, and cell proliferation of Clc-k2^-/- mice through restoring salt reabsorption in TALs/DCTs. Kcc4 provides alternative basolateral chloride access for Clc-k2-deficient TALs/DCTs.

Funding

• NIDDK Support

Digital Object Identifier (DOI)

• doi: 10.1681/ASN.20257zrfwqjh