Aim: 

The NKCC2 cotransporter is the major salt transport pathway of the apical membrane of the mammalian kidney. In this study we analyze the role of the tetraspan-like protein MAL/VIP17 in the regulation of NKCC2.

Methods: 

For this study we used LLC-PK1 cells co-expressing NKCC2 and MAL/VIP17. Co-immunorepcipitation and co-immunolocalization experiments, cell surface biotinylation, endocytosis and phosphorylation assays were performed to achieve the goal of this study.

Results: 

We demonstrated that 1) NKCC2 and MAL/VIP17 co-localize and co-immunoprecipiate in LLC-PK1 cells as well as in rat kidney medullae 2) a 150 amino acid stretch of NKCC2 C-terminal tail is involved in the interaction with MAL/VIP17 3) MAL/VIP17 increases the cell surface retention of NKCC2 by attenuating its internalization 4) this coincides with an increase in cotransporter phosphorylation. Interestingly, over-expression of MAL/VIP17 in the kidney of transgenic mice results in cysts formation in distal nephron structures consistent with the hypothesis that MAL/VIP17 plays an important role in apical sorting or in maintaining the stability of the apical membrane. The NKCC2 expressed in these mice was highly glycosylated and phosphorylated, corroborating the results obtained in LLC-PK1 cells.

Conclusion: 

We identified the tetraspan-member family MAL/VIP17 as new player in the regulation of the functional expression of NKCC2 at the apical plasma membrane in the kidney. Indeed, the interaction between MAL/VIP17 and NKCC2 could play an important role in the physiological regulation of body salt and water homeostasis as well as in the pathogenesis of hypertension.