Abstract: TH-PO0041
KS-WNK1 WNK Bodies Regulate mTOR Signaling in Distal Convoluted Tubules
Session Information
- Fluid, Electrolyte, and Acid-Base Disorders: Basic Research
October 22, 2026 | Location: Exhibit Hall A, 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
- McConnell, Ryan J., University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, United States
- Ballance, Heather I., University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, United States
- Kotru, Mehak, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, United States
- Cagathi, Dhriti, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, United States
- Lashway, Jared, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, United States
- Subramanya, Arohan R., University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, United States
- Boyd-Shiwarski, Cary R., University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, United States
Group or Team Name
- Boyd-Shiwarski Lab
Background
WNK bodies are biomolecular condensates that scaffold With-No-Lysine (WNK) kinase signaling within the distal convoluted tubule. Condensates enhance signaling fidelity by concentrating molecules within membraneless compartments, dynamically sequestering or releasing proteins and regulating post-translational modifications. WNK body formation requires oligomerization of Kidney-Specific WNK1 (KS-WNK1), a truncated WNK1 isoform lacking the N-terminal kinase domain. In mice, potassium restriction induces rapid WNK body formation, generating a multi-kinase hub containing L-WNK1, WNK4, SPAK, RPL22, TSC22D, and NRBP1. However, the broader composition and regulatory function of WNK bodies remain unknown. We hypothesized that KS-WNK1 interacts with diverse phosphoproteins to alter their phosphorylation, engaging both canonical WNK/SPAK signaling and previously unrecognized pathways.
Methods
We generated a stable, doxycycline-inducible Flp-In™ T-REx™293 cell line expressing KS-WNK1-mRuby, enabling titratable expression and visualization of WNK body dynamics. We performed an unbiased phosphoproteomics screen via LC-MS/MS comparing cells ±doxycycline, identifying 10,506 phosphopeptides from 3,344 proteins, alongside targeted immunoblotting of WNK pathway and mTOR pathway proteins.
Results
Phosphoproteomic analysis confirmed KS-WNK1-dependent changes in known WNK body components, including >30% fold changes in phosphorylation of WNK1, SPAK, RPL22, and TSC22D. Among 230 novel phosphopeptides identified (FDR <0.05), pathway analysis identified mTOR signaling pathway as a key novel target. Eight Tuberous Sclerosis Complex 2 (TSC2) phosphosites were altered, with S981 as the second most significantly dephosphorylated site (log2FC = -0.71). Targeted immunoblotting validated these findings: dox-induced KS-WNK1 increased total WNK1 by 36% and pSPAK S373 by 58%, confirming WNK-SPAK activation. Within the mTOR pathway, total TSC2 and S6 Kinase (S6K) were unchanged, yet pTSC2 S939 increased by 72% and pS6K T229 decreased by 58%, indicating phospho-regulation of the mTORC1 axis by KS-WNK1
Conclusion
This study identifies novel phosphoproteins regulated by WNK body formation and implicates WNK bodies in mTOR pathway modulation. Ongoing work will assess protein localization within WNK bodies, whether similar regulation occurs during dietary potassium restriction, and how WNK body–mTOR crosstalk contributes to renal potassium homeostasis.
Funding
- NIDDK Support