TRPV4 activation mediates flow-induced nitric oxide production in the rat thick ascending limb

Abstract

Nitric oxide (NO) regulates renal function. Luminal flow stimulates NO production in the thick ascending limb (TAL). Transient receptor potential vanilloid 4 (TRPV4) is a mechano-sensitive channel activated by luminal flow in different types of cells. We hypothesized that TRPV4 mediates flow-induced NO production in the rat TAL. We measured NO production in isolated, perfused rat TALs using the fluorescent dye DAF FM. Increasing luminal flow from 0 to 20 nl/min stimulated NO from 8 ± 3 to 45 ± 12 arbitrary units (AU)/min (n = 5; P < 0.05). The TRPV4 antagonists, ruthenium red (15 μmol/l) and RN 1734 (10 μmol/l), blocked flow-induced NO production. Also, luminal flow did not increase NO production in the absence of extracellular calcium. We also studied the effect of luminal flow on NO production in TALs transduced with a TRPV4shRNA. In nontransduced TALs luminal flow increased NO production by 47 ± 17 AU/min (P < 0.05; n = 5). Similar to nontransduced TALs, luminal flow increased NO production by 39 ± 11 AU/min (P < 0.03; n = 5) in TALs transduced with a control negative sequence-shRNA while in TRPV4shRNA-transduced TALs, luminal flow did not increase NO production (Δ10 ± 15 AU/min; n = 5). We then tested the effect of two different TRPV4 agonists on NO production in the absence of luminal flow. 4α-Phorbol 12,13-didecanoate (1 μmol/l) enhanced NO production by 60 ± 11 AU/min (P < 0.002; n = 7) and GSK1016790A (10 ηmol/l) increased NO production by 52 ± 15 AU/min (P < 0.03; n = 5). GSK1016790A (10 ηmol/l) did not stimulate NO production in TRPV4shRNA-transduced TALs. We conclude that activation of TRPV4 channels mediates flow-induced NO production in the rat TAL.

Keywords: TRP channels; kidney; luminal flow; mechano-transduction; nitric oxide synthase; shear stress.

Fig. 1.

Effect of increasing luminal flow on nitric oxide (NO) production by isolated thick ascending limbs (P < 0.05 compared with no flow conditions; n = 5).

Fig. 2.

Effect of increasing luminal flow on NO production by isolated thick ascending limbs in the presence of the transient receptor potential vanilloid (TRPV) antagonist ruthenium red (n = 5).

Fig. 3.

Effect of increasing luminal flow on NO production by isolated thick ascending limbs in the presence of the TRPV4 antagonist RN 1734 (n = 5).

Fig. 4.

A: representative Western blots from the left TRPV4shRNA-transduced kidney and the right nontransduced kidney. Two and five micrograms of protein were loaded and TRPV4, nitric oxide synathase (NOS) 3, and β-tubulin expressions were measured. B: TRPV4 protein expression decreased by 50.3 ± 6.3% in the left kidney (TRPV4shRNA-transduced) compared with the right kidney (nontransduced; P < 0.005; n = 4). C: NOS 3 protein expression levels did not change in the left kidney (TRPV4shRNA-transduced) compared with the right kidney (nontransduced).

Fig. 5.

Effect of increasing luminal flow on NO production by isolated thick ascending limbs from control nontransduced thick ascending limbs; TRPV4shRNA-transduced thick ascending limbs and control negative sequence shRNA-transduced thick ascending limbs (*P < 0.05 compared with no flow conditions in the same thick ascending limb; n = 5). #N.S., compared with no flow conditions in the same thick ascending limb; n = 5. N.S., nonsignificant. **P < 0.03 compared with no flow conditions in the same thick ascending limb; n = 5.

Fig. 6.

Effect of the TRPV4 agonist 4αPDD and GSK1016790A on NO production by isolated thick ascending limbs in the absence of luminal flow (*P < 0.002 compared with no flow conditions in the same nontransduced thick ascending limb; n = 7). #P < 0.03 compared with no flow conditions in the same nontransduced thick ascending limb; n = 5. **N.S., compared with no flow conditions in the same TRPV4shRNA-transduced thick ascending limb; n = 5.

Fig. 7.

Effect of increasing luminal flow on NO production by isolated thick ascending limbs in the absence of extracellular calcium (n = 5).