In Vivo Knockdown of TRPV4 Decreases Flow‐Induced Calcium Influx in the Thick Ascending Limb

Abstract

The thick ascending limb (TAL) plays a critical role in the regulation of salt homeostasis by reabsorbing 30 % of the filtered NaCl. Mechanical stimulation exerted by elevating luminal flow in this segment causes increases in intracellular calcium (Ca(i)). The mechanosensitive Transient Receptor Potential Vanilloid 4 (TRPV4) channel mediates calcium influx in response to luminal flow in other cell types. We hypothesize that flow‐induced calcium influx is mediated by TRPV4 in the TAL. To test our hypothesis we measured Ca(i) using the ratiometric calcium sensitive dye Fura‐ 2 in isolated and perfused TALs. The role of TRPV4 in response to elevations in luminal flow was addressed by targeting the channel using a shRNA against TRPV4. In the absence of luminal flow, activating TRPV4 using the selective agonist GSK1016790A (10 nM) increased Ca(i) levels from 72 ± 16 to 255 ± 50 nM (p<0.04; n=8). We next tested the role of TRPV4 in flow‐induced increases in Ca(i). In non‐transduced control TALs, luminal flow elevated Ca(i) levels by 104 ± 20 nM. However, in TALs transduced with a shRNA‐TRPV4, increasing luminal flow elevated Ca(i) levels only by 56 ± 8 nM (p<0.05, n=9). We conclude that in vivo knockdown of TRPV4 decreases flow‐induced calcium influx in the TAL.National Heart, Lung and Blood Institute of the NIH to J.L.G (HL 070985; HL 090550 and HL 028982).American Heart Association to P.D.C (11POST7490010).