Stretch-Induced Increases in Intracellular Ca Stimulate Thick Ascending Limb O 2 − Production and Are Enhanced in Dahl Salt-Sensitive Rats

Abstract

Mechanical stretch raises intracellular Ca (Ca i ) in many cell types. Luminal flow-derived stretch stimulates O 2 − production by thick ascending limbs (THALs). Renal O 2 − is greater in Dahl salt-sensitive (SS) than salt-resistant (SR) rats. We hypothesized that mechanical stretch stimulates Ca influx via TRPV4 (transient receptor potential vanilloid type 4) which in turn raises Ca i in THALs; these increases in Ca i are necessary for stretch to augment O 2 − production; and stretch-stimulated, and therefore flow-induced, O 2 − production is enhanced in SS compared with SR THALs due to elevated Ca influx and increased Ca i . Ca i and O 2 − were measured in SS and SR THALs from rats on normal salt using Fura2-acetoxymethyl ester and dihydroethidium, respectively. Stretch raised Ca i in SS by 270.4±48.9 nmol/L and by 123.6±27.0 nmol/L in SR THALs ( P <0.02). Removing extracellular Ca eliminated the increases and differences in Ca i between strains. Knocking down TRPV4 in SS THALs reduced stretch-induced Ca i to SR levels (SS: 92.0±15.9 nmol/L; SR: 123.6±27.0 nmol/L). RN1734, a TRPV4 inhibitor, blunted stretch-elevated Ca i by ≈75% and ≈66% in SS ( P <0.03) and SR ( P <0.04), respectively. Stretch augmented O 2 − production by 58.6±10.2 arbitrary fluorescent units/min in SS and by 24.4±2.6 arbitrary fluorescent units/min in SR THALs ( P <0.05). Removal of extracellular Ca blunted stretch-induced increases in O 2 − and eliminated differences between strains. RN1734 reduced stretch-induced O 2 − by ≈70% in SS ( P <0.005) and ≈60% in SR ( P <0.01). Conclusions are as follows: (1) stretch activates TRPV4, which raises Ca i in THALs; (2) the increase in Ca i stimulates O 2 − production; and (3) stretch-induced O 2 − production is enhanced in SS THALs due to greater increases in Ca i .