Role of Myosins II and VI in Mediating Hormonal Regulation of NHE3 Activity in the Rat Renal Proximal Tubule

Abstract

Rapid regulation of NHE3 activity may occur through redistribution of the transporter between the body and the base of the proximal tubule microvilli (PT MV). Myosins are motor proteins that move along actin filaments. The present study tested the hypothesis that myosins II (myo II), which moves cargo away from cell center, and VI (myo VI), which moves cargo toward cell center, may mediate NHE3 redistribution within the MV in response to stimuli. Renal PT NHE3 activity was evaluated in Wistar rats by net bicarbonate reabsorption (JHCO3−) in the presence or absence of 10−10 M angiotensin II (Ang II), 10−4 M forskolin (FSK), a cAMP‐elevating agent, 10−5 M blebbistatin (a specific myo II inhibitor) or 4×10−6 M 2,4,6‐triiodophenol (TIP) (a myo VI inhibitor). As expected, Ang II increased [4.36 ± 0.18 nmol/cm2.s (N = 5)] whereas FSK reduced [1.26 ± 0.10 nmol/cm2.s (N = 5)] JHCO3− compared to CTRL [2.74 ± 0.13 nmol/cm2.s (N = 4)]. Simultaneous tubular perfusion of Ang II with blebbistatin prevent Ang II‐induced NHE3 stimulation [2.92 ± 0.14 nmol/cm2.s (N = 5), P < 0.001]. Simultaneous tubular perfusion of FSK with TIP reduced the inhibition with FSK alone [1.78 ± 0.15 (N = 4) vs. 1.26 ± 0.10 nmol/cm2.s (N = 5), P < 0.05] on JHCO3−. The localization of NHE3 and myosins IIB and VI was assessed by confocal immunofluorescence in Wistar rats kidney after a 30 min pressor dose of Ang II (50 ng/kg/min). NHE3 and myo VI shift from base to body of MV after Ang II; myo IIB remains mostly enriched at the MV base. To assess NHE3 activity in OKP cells, experimental pH recovery points were fitted to simple exponential decay curves (y = A·e(−kt) + y0) in order to predict the rate constant (k) of each recovery curve. FSK reduced NHE3 activity [0.025 ± 0.003 s−1 (N = 4)] compared to CTRL [0.048 ± 0.007 s−1 (N = 5)]. siRNA for myo VI efficiently reduced its expression by 64 %; the inhibitory effect of FSK on NHE3 activity was completely abolished by siRNA myo VI [0.043 ± 0.005 s−1 (N = 4)]. These results provide further evidence for myosin II and VI regulation of NHE3 redistribution in the rat kidney proximal tubule.Support or Funding InformationFAPESPThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.