Elena Mironova

Kidneys play a pivotal role in long-term blood pressure regulation by controlling extracellular fluid volume through renal salt reabsorption. Discretionary control of the Epithelial Na+ Channel, ENaC, is the final step in fine-tuning renal Na+ excretion. Consequently, gain-of-function mutations in ENaC result in hypertension caused by inappropriate retention of Na+, whereas loss-of-function of this channel causes abnormal renal salt wasting. Thus, understanding how ENaC activity is regulated is important for a complete understanding of the molecular origins of hypertension. Our data show that casein kinase 2 (CK2) phosphorylates β-ENaC at a conserved motif homologous to the CK2 site in Nav1.2 and KCNQ channels, where phosphorylation controls ankyrin-3 (Ank-3) binding and channel membrane localization. In addition, CK2-dependent phosphorylation of β-ENaC is required for Ank-3 binding, which stabilizes ENaC at the apical membrane, sustains channel activity, and thereby ensures appropriate renal Na+ excretion. Using structure-guided mutagenesis of the β-ENaC C-terminus combined with advanced live-cell imaging (TIRF-FRAP, FRET) and patch-clamp electrophysiology, we found that mutation of the CK2 consensus site or disruption of the Ank-3-binding motif abolishes ENaC surface mobility and suppresses macroscopic current density. Principal cell-specific deletion of CK2 or Ank-3, or pharmacologic CK2 inhibition, eliminates ENaC activity in native collecting ducts and drives markedly accelerated urinary sodium loss in vivo. These results identify CK2-dependent Ank-3 scaffolding as an essential post-translational mechanism that maintains ENaC at the apical membrane and sustains renal Na+ reabsorption, thereby presenting a potential new targetable regulatory pathway with implications for sodium homeostasis and blood pressure control.

Plots of event-free-survival (EFS) and relative tumor volume (RTV) changes over time for hepatoblastoma models. Treatment groups: A = control, B = vobra duo: 6 mg/kg, IP, Day 0 × 1, C=SYD988: 6 mg/kg, IP, Day 0 × 1. Bold lines on RTV plots represent group median tumor volume changes.

This is the excel file with Suppl. Table S9.

Efficacy testing summary results for MRT models treated with vobra duo and SYD988.Treatment groups: A = control, B = vobra duo: 6 mg/kg, IP, Day 0 × 1, C=SYD988: 6 mg/kg, IP, Day 0 × 1.

Western blots for models WT-12 and BT-16. Antitumor activity of vobra duo and SYD988 in WT-12 (A) and BT-16 (B) xenograft models (to support Fig. 4C and D). Vobra duo (6 mg/kg, IP) and SYD988 (6 mg/kg, IP) were each administered once on day 1. Tumors were collected at 6 hours and 48 hours after treatment. Western blot analysis was performed on tumor lysates using antibodies against B7-H3 and GAPDH (loading control).

CD276 gene expression profiling. Across pediatric cancers (Top); In responding (PR, CR, MCR) versus non-responding models (PD, PD1, PD2, SD), shown by tumor panel (Bottom (A-G)). “Not tested” indicates models without available in vivo activity data.

Plots of event-free-survival (EFS) and relative tumor volume (RTV) changes over time for Wilms tumor models. Treatment groups: A = control, B = vobra duo: 6 mg/kg, IP, Day 0 × 1, C=SYD988: 6 mg/kg, IP, Day 0 × 1. Bold lines on RTV plots represent group median tumor volume changes.

Immunohistochemistry (IHC) staining of B7-H3. A, H-scores for B7-H3 protein in the cell membrane, cytoplasm, and vasculature of Wilms tumor, MRT, and hepatoblastoma models. B, Representative tumor samples showing the strongest (right column) and weakest (left column) B7-H3 protein expression in Wilms tumor (second row), MRT (third row), and hepatoblastoma (bottom row) panels. C, Example of H-scoring criteria.

Efficacy testing summary results for RMS models treated with vobra duo and SYD988.Treatment groups: A = control, B = vobra duo: 6 mg/kg, IP, Day 0 × 1, C= SYD988: 6 mg/kg, IP, Day 0 × 1.

Efficacy testing summary results for osteosarcoma models treated with vobra duo and SYD988.Treatment groups: A = control, B = vobra duo: 6 mg/kg, IP, Day 0 × 1, C=SYD988: 6 mg/kg, IP, Day 0 × 1.