Kidney Week

Abstract: SA-PO1043

Differential Regulation of L-WNK1 and KS-WNK1 by the Ubiquitin Ligases Nedd4-2 and Kelch-CUL3 Complex

Session Information

• Na+, K+, Cl-
  November 04, 2017 | Location: Hall H, Morial Convention Center
  Abstract Time: 10:00 AM - 10:00 AM

Category: Fluid, Electrolytes, and Acid-Base

• 703 Na+, K+, Cl- Basic

Authors

• Ostrosky-Frid, Mauricio, Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán, Tlalpan, Mexico City, Mexico

Mauricio Ostrosky-Frid,

• Argaiz, Eduardo R., Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán, Tlalpan, Mexico City, Mexico

Eduardo R. Argaiz,

• Gallardo, Fabiola, Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán, Tlalpan, Mexico City, Mexico

Fabiola Gallardo,

• Chavez-Canales, Maria, Instituto de Investigaciones Biomédicas, UNAM, Mexico City, Mexico

Maria Chavez-Canales,

• Vázquez, Norma Hilda, Instituto de Investigaciones Biomédicas, UNAM, Mexico City, Mexico

Norma Hilda Vázquez,

• Ellison, David H., Oregon Health & Science University, Portland, Oregon, United States

David H. Ellison,

• Gamba, Gerardo, Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán, Tlalpan, Mexico City, Mexico

Gerardo Gamba,

Background

It has been suggested that the full-length form of WNK1 kinase (L-WNK1) is a target for ubiquitylation by the Nedd4-2 and the Kelch-Cul3 ubiquitin ligases. The major transcript of WNK1 gene in the renal tissue is KS-WNK1, the truncated kidney-specific isoform that is almost exclusively expressed in the distal convoluted tubule, where its transcript is 80 times more abundant than L-WNK1. The higher transcript expression could be due to a different sensibility to the ubiquitin-induced degradation. The effect of ubiquitin ligases in the KS-WNK1 variant, however, is not known.

Methods

The effect of Nedd4-2 and Kelch3-CUL3 complex upon L-WNK1 and KS-WNK1 was assessed by western blot and immunoprecipitation (IP) two days after microinjection of Xenopus oocytes with Myc-tagged KS-WNK1-Δ11 and/or L-WNK1-Δ11 cRNA, in the absence or presence of Flag-tagged Nedd4-2 or Flag-tagged-Kelch3 cRNA. The NCC activity was measured by functional expression in the oocytes using tracer ²²Na⁺ uptake. We used the variants lacking the exon 11 (Δ11) because this is the most abundant form in the kidney. Immunoprecipitation between WNK1 variants and Nedd4-2 or Kelch3 was corroborated using a c-myc precipitation kit.

Results

We observed that KS-WNK1-Δ11, was completely degraded by Kelch-CUL3 complex (N=10). In contrast, only about 20% of the L-WNK1-Δ11 was degraded in the presence of Kelch3. The IP confirmed that the Kelch-CUL3 complex is co-precipitated with KS-WNK1, but not with L-WNK1-Δ11 (N=3). Functionally, the positive effect of KS-WNK1-Δ11, but not that of L-WNK1-Δ11, on NCC was prevented by the coinjection with Kelch3 (n=3). Interestingly, the effect of Nedd4-2 was the opposite. Nedd4-2 induced degradation of L-WNK-Δ11 and reduced its effect on NCC, while it had no effect on KS-WNK1-Δ11. Using the same expression system, we observed that WNK4 was also highly sensitive to Kelch3.

Conclusion

Although KS-WNK1 and L-WNK1 exhibit the same interaction site for ubiquitin ligases, KS-WNK1-Δ11 is sensitive to Kelch3, but not to Nedd4-2, while L-WNK1-Δ11 is sensitive to Nedd4-2, but not to Kelch3. Given the differential expression of KS-WNK1 and L-WNK1 along the distal nephron, modulation of these kinases abundance by Nedd4-2 and Kelch3 could have an implication in the fine tune modulation of ion transport.

Funding

• NIDDK Support