Dataset Information

Stretch-Induced Increases in Intracellular Ca Stimulate Thick Ascending Limb O₂^- 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 O₂^- production by thick ascending limbs (THALs). Renal O₂^- 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 O₂^- production; and stretch-stimulated, and therefore flow-induced, O₂^- production is enhanced in SS compared with SR THALs due to elevated Ca influx and increased Ca_i. Ca_i and O₂^- 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 O₂^- 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 O₂^- and eliminated differences between strains. RN1734 reduced stretch-induced O₂^- 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 O₂^- production; and (3) stretch-induced O₂^- production is enhanced in SS THALs due to greater increases in Ca_i.

SUBMITTER: Saez F

PROVIDER: S-EPMC7058965 | biostudies-literature | 2020 Feb

REPOSITORIES: biostudies-literature

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Publications

Stretch-Induced Increases in Intracellular Ca Stimulate Thick Ascending Limb O2- Production and Are Enhanced in Dahl Salt-Sensitive Rats.

Saez Fara F Hong Nancy J NJ Cabral Pablo D PD Garvin Jeffrey L JL

Hypertension (Dallas, Tex. : 1979) 20191223 2

Mechanical stretch raises intracellular Ca (Cai) in many cell types. Luminal flow-derived stretch stimulates O2- production by thick ascending limbs (THALs). Renal O2- 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 Cai in THALs; these increases in Cai are nece ...[more]

PMID: 31865796

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Project description:Hydrogen peroxide (H2O2) production in the renal outer medulla is an important determinant of renal medullary blood flow and blood pressure (BP) salt-sensitivity in Dahl salt-sensitive (SS) rats. The mechanisms and pathways responsible for these actions are poorly understood. Recently, we have discovered that the mTOR complex 2 (mTORC2) plays a critical role in BP salt-sensitivity of SS rats by regulating Na+ homeostasis. PP242, an inhibitor of mTORC1/2 pathways exhibits potent natriuretic actions and completely prevented salt-induced hypertension in SS rats. In the present study, we have found that chronic infusion of H2O2 into the single remaining kidney of Sprague Dawley (SD) rats (3 days) stimulated the functional marker (pAKTSer473/AKT) of mTORC2 activity measured by Western Blot analysis. No changes in mTORC1 activity in OM were observed as determined by pS6Ser235/236/S6. Using fluorescent microscopy and the Na+ sensitive dye Sodium Green, we have shown that H2O2 (100 µM added in the bath) increased intracellular sodium concentration ([Na+]i) in renal medullary thick ascending limbs (mTALs) isolated from SD rats. These responses were almost completely abolished by pretreatment of mTAL with 10 µM PP242, indicating that mTORC1/2 pathways were involved in the H2O2 induced increase of [Na+]i. mTAL cell volume remained unchanged (± 1%) by H2O2 as determined by 3D reconstruction confocal laser scanning microscopy techniques. Consistent with the microscopy data, Western Blot analysis of proteins obtained from freshly isolated mTAL treated with 100 µM H2O2 exhibited increased activity/phosphorylation of AKT (pAKTSer473/AKT) that was inhibited by PP242. This was associated with increased protein activity of the apical membrane cotransporter Na+-K+-2Cl- (NKCC2) and the Na/H exchanger (NHE-3). Na+-K+-ATPase activity was increased as reflected an increase in the ratio of pNa+-K+-ATPaseSer16 to total Na+-K+-ATPase. Overall, the results indicate that H2O2 mediated activation of mTORC2 plays a key role in transducing the observed increases of cytosolic [Na+]i despite associated increases of basolateral pump activity.

Dataset Information

Stretch-Induced Increases in Intracellular Ca Stimulate Thick Ascending Limb O₂^- Production and Are Enhanced in Dahl Salt-Sensitive Rats.

Publications

Stretch-Induced Increases in Intracellular Ca Stimulate Thick Ascending Limb O<sub>2</sub><sup>-</sup> Production and Are Enhanced in Dahl Salt-Sensitive Rats.

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