The molecular and physiologic effects of calcineurin inhibitors on WNK body bimolecular condensates

About This Grant

ABSTRACT The U.S. recently reached a historic milestone, surpassing one million life-saving organ transplants. For these patients, tacrolimus—a calcineurin inhibitor (CNI)—is the cornerstone of immunosuppression, prescribed to over 90% of transplant recipients due to its proven efficacy and tolerability. However, tacrolimus is associated with a distinctive triad of kidney-centered adverse effects: hypertension, hyperkalemia, and metabolic acidosis. Intriguingly, this triad resembles the rare Mendelian disorder Familial Hyperkalemia and Hypertension (FHHt), which results from over-activation of the NaCl-cotransporter (NCC) in the distal convoluted tubule. NCC activity is regulated by the WNK/SPAK (With-no-lysine and Ste20/ SPS-1 -related- proline- alanine- rich protein kinase) signaling pathway. In the distal convoluted tubule, kidney-specific WNK1 (KS-WNK1) is essential for activation of the WNK/SPAK/NCC cascade. Mice carrying human gain-of-function mutations in KS-WNK1 have over- activation of this pathway, thereby mimicking both the CNI- and FHHt-associated phenotypes. We propose that KS-WNK1 acts as a scaffold, recruiting WNK/SPAK components into biomolecular condensates known as WNK bodies, thereby amplifying NCC signaling. Biomolecular condensates are membraneless compartments that concentrate macromolecules through phase separation, creating localized environments that regulate specific biochemical reactions. The study of condensate biology has reshaped our understanding of intracellular organization and has sparked growing interest in pharmacologic strategies to target these structures. Our preliminary data indicate that tacrolimus increases the abundance of WNK bodies, suggesting a direct mechanistic link between CNIs, condensates, and NCC overactivation. The goal of this R01 is to use CNIs as both an experimental tool and clinical model to manipulate WNK body composition, dynamics, and function, while concurrently testing how CNIs alter kidney physiology through WNK bodies. Our central hypothesis is that calcineurin inhibition by tacrolimus stabilizes KS-WNK1 by increasing its phosphorylation and reducing its ubiquitylation. The resulting increase in KS-WNK1 abundance promotes WNK body assembly, amplifies WNK/SPAK/NCC signaling, and leads to the clinical triad of hypertension, hyperkalemia, and metabolic acidosis. We intend to address the hypothesis through: (1) Cellular models to define how CNIs alter KS-WNK1 post- translational modifications and WNK body dynamics; (2) Mouse models to test whether mice lacking or overexpressing WNK bodies are resistant or hypersensitive to CNI-induced side effects; and (3) Human allograft kidney biopsy samples to correlate WNK body abundance with hypertension, hyperkalemia, and acidosis. Using integrated molecular and whole-organism approaches, the expected outcomes include: (i) validation of WNK bodies as pharmacological targets of CNIs; (ii) mechanistic insight into how CNIs modulate WNK bodies to alter renal electrolyte handling; and (iii) identification of CNI-treated patients who may benefit from adjunctive therapy with thiazide diuretics to block NCC overactivation.