Aquaporin-2 and Na+/H+ exchanger isoform 1 modulate the efficiency of renal cell migration

RIVAROLA V; FORD P; DI GIUSTO G; BELTRAMONE N; CAPURRO C; PIZZONI A; WHITE A

Rosario

Congreso; Reunión Anual de la Sociedad Argentina de Fisiología; 2019

Sociedad Argentina de Fisiología

Aquaporin-2 (AQP2), in addition to its canonical role as a water channel, promotes renal cell migration by the modulation of integrin β1 trafficking and the turnover of focal adhesions. This novel role described for AQP2 opens the possibility to further investigate if AQP2 also works in concert with other components of the cell migration machinery. Na+/H+ exchanger isoform 1 (NHE1) is a well-known protein involved in the regulation of cell migration, which is proposed to act in the leading-edge membrane to direct migration. NHE1 activity is highly modulated by Ca2+ and we recently showed a physical interaction between AQP2 and the Ca2+ channel TRPV4. Then, the aim of our work was to investigate the possible crosstalk between AQP2, its mechanosensitive partner TRPV4, and NHE1 to regulate cell migration. We used two renal cell models: one not expressing AQPs and another one expressing AQP2. We performed wound healing and cell tracking assays to evaluate cell migration; immunofluorescence assays to evaluate lamellipodia volume, focal adhesions, and assembly of F-actin; and fluorescence videomicroscopy to measure lamellipodia pHi and NHE activity. Our results confirm that AQP2 promotes renal cell migration and during wound closure, AQP2-expressing cells follow a less tortuous route compared with AQP2-null cells. Lamellipodia of AQP2-expressing cells exhibit significantly smaller volumes and size of focal adhesions and more alkaline pHi due to increased NHE1 activity than AQP2-null cells. The blockage of AQP2 or TRPV4 significantly reduced lamellipodia NHE1 activity. Also, the blockage of NHE1 significantly reduced the rate of cell migration, the number of lamellipodia and the assembly of F-actin only in AQP2-expressing cells. Altogether these results let us propose that during lamellipodia protrusion the presence of AQP2 activates it´s partner TRPV4, leading to Ca2+ entry and to the consequent activation of NHE1. It is likely that the interplay between AQP2, TRPV4, and NHE1 defines the pH dependent-actin polymerization, providing mechanical stability to delineate lamellipodia structure and consequently the speed and directionality of cells, promoting the migration.