The Renal Sodium-Chloride Cotransporter NCC is Regulated by NDFIP1 Through a WNK-dependent Mechanism

Background: The renal NaCl cotransporter (NCC) is a key modulator of blood pressure, potassium excretion, and acid-base balance. Several mechanisms for NCC activation have been elucidated in the past decades, but little is known about NCC half-life and inhibition. It is known that NCC is a target for ubiquitination by the HECT-type ubiquitin ligase Nedd4-2 (Arroyo et al. JASN, 2011), but the mechanism remains elusive, as NCC lacks PY motifs that mediate interaction with Nedd4-2. The Nedd4-interacting protein 1 (NDFIP1) is an adaptor that could potentially explain the interaction between NCC and Nedd4-2. NDFIP1 contains three PY motifs and has previously been shown to mediate Nedd4-2–dependent degradation of proteins lacking PY motifs in renal epithelium. In the present work, we explored the effect of NDFIP1 upon NCC expression and activity. Methodology: Xenopus laevis oocytes were injected with combinations of cRNA in vitro transcribed from NCC, NDFIP1 wild-type and NDFIP1 mutant (PY-motif elimination), Nedd4-2 wild-type and its catalytically inactive mutant (Nedd4-CS), L-WNK1, KS-WNK1, WNK3, and WNK4 wild-type or chloride-insensitive mutant (WNK4 L322F). All studied groups were coinjected with NCC plus one or two of the mentioned cRNAs. Oocytes were incubated in ND96 solution for 48 hours after injection, and then NCC activity was assessed by thiazide-sensitive 22Na + tracer uptake. Protein expression was evaluated by Western blot. Membrane protein assessment was performed by surface biotinylation. Results: Each observation was compiled from at least three independent experiments. NDFIP1 or Nedd4 alone induced a significant 75% reduction of the 22Na + uptake mediated by NCC. When NDFIP1 and Nedd4-2 were coinjected, the effect was synergic, suggesting additive mechanisms. The inactive Nedd4-2-CS injected alone induced an increase in NCC activity. Coinjection of Nedd4-2-CS and NDFIP1 prevented both the positive effect of Nedd4-2-CS and the negative effect of NDFIP1 on NCC. Eliminating the three NDFIP1 PY motifs prevented the NDFIP1 effect on Nedd4-2-CS, but not the reduction of NCC activity, suggesting an NCC-inhibitory mechanism that is independent of Nedd4-2. Western blot analysis from whole lysates revealed no changes in total NCC in the presence of Nedd4-2 or NDFIP1. However, the decrease of NCC activity by Nedd4-2 was associated with reduced NCC abundance in the plasma membrane. In contrast, the decreased NCC activity caused by NDFIP1 was associated with a reduction in NCC phosphorylation, without changes in membrane expression. As expected, coinjection of NCC with L-WNK1, KS-WNK1, WNK3, WNK4 wild-type, or WNK4 L322F increased NCC activity measured by sodium uptake. However, the effect of L-WNK1, KS-WNK1, and WNK4 wild-type was abolished with the addition of NDFIP1. In contrast, this inhibitory effect was absent in the presence of WNK3 and WNK4 L322F. Further analysis revealed that total WNK protein abundance was not modified with NDFIP1 coinjection, but a decrease in WNK phosphorylation was noted in L-WNK1 and particularly in WNK4 wild-type. This reduction in phosphorylation was not observed in WNK3 or WNK4 L322F. Conclusions: NDFIP1 exhibits a negative regulatory role in NCC function. Decreased NCC activity through NDFIP1 occurs due to reduced NCC phosphorylation, apparently through a WNK-dependent process. The reduction in phosphorylation was notable in L-WNK1 and WNK4 wild-type but not in WNK3 or WNK4 L322F, suggesting a possible chloride-dependent process. This abstract was presented at the American Physiology Summit 2026 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.