Shear stress increases nitric oxide production in thick ascending limbs

Abstract

We showed that luminal flow stimulates nitric oxide (NO) production in thick ascending limbs. Ion delivery, stretch, pressure, and shear stress all increase when flow is enhanced. We hypothesized that shear stress stimulates NO in thick ascending limbs, whereas stretch, pressure, and ion delivery do not. We measured NO in isolated, perfused rat thick ascending limbs using the NO-sensitive dye DAF FM-DA. NO production rose from 21 ± 7 to 58 ± 12 AU/min (P < 0.02; n = 7) when we increased luminal flow from 0 to 20 nl/min, but dropped to 16 ± 8 AU/min (P < 0.02; n = 7) 10 min after flow was stopped. Flow did not increase NO in tubules from mice lacking NO synthase 3 (NOS 3). Flow stimulated NO production by the same extent in tubules perfused with ion-free solution and physiological saline (20 ± 7 vs. 24 ± 6 AU/min; n = 7). Increasing stretch while reducing shear stress and pressure lowered NO generation from 42 ± 9 to 17 ± 6 AU/min (P < 0.03; n = 6). In the absence of shear stress, increasing pressure and stretch had no effect on NO production (2 ± 8 vs. 8 ± 8 AU/min; n = 6). Similar results were obtained in the presence of tempol (100 μmol/l), a O(2)(-) scavenger. Primary cultures of thick ascending limb cells subjected to shear stresses of 0.02 and 0.55 dyne/cm(2) produced NO at rates of 55 ± 10 and 315 ± 93 AU/s, respectively (P < 0.002; n = 7). Pretreatment with the NOS inhibitor l-NAME (5 mmol/l) blocked the shear stress-induced increase in NO production. We concluded that shear stress rather than pressure, stretch, or ion delivery mediates flow-induced stimulation of NO by NOS 3 in thick ascending limbs.

Fig. 1.

Effect of increasing and decreasing luminal flow on nitric oxide (NO) production by isolated thick ascending limbs (TALs; *P < 0.02 compared with 0 nl/min, period 1; **P < 0.02 compared with 20 nl/min, period 1; #P < 0.04 compared with 0 nl/min, period 2; n = 7). There was no difference between flow-stimulated NO production during periods 1 and 2.

Fig. 2.

Effect of ion removal on flow-stimulated NO production by isolated, perfused TALs (n = 7). Tubules were perfused with either physiologic saline or an ion-free solution as described in the text. Luminal flow was 20 nl/min in the 2 periods.

Fig. 3.

Transmitted light images of an isolated and perfused TAL (×100 immersion oil objective). A: isolated and perfused TAL before collagenase treatment. B: isolated and perfused TAL after collagenase treatment.

Fig. 4.

Effect of increasing stretch while reducing pressure and shear stress on flow-stimulated NO production by isolated, perfused TALs. Luminal flow was maintained at 20 nl/min. Treatment of TALs with collagenase (collag) increased stretch (seen as a 24 ± 2% increase in diameter), lowering both shear stress and pressure (*P < 0.03; n = 6).

Fig. 5.

Effect of increasing pressure and stretch but not shear stress on NO production by isolated TALs (n = 6). Distal ends of tubules were pinched closed and pressurized. Thus, stretch and pressure increased but not shear stress. P, pressure; Str, stretch.

Fig. 6.

Transmitted light image of primary culture of rat medullary TAL cells (×40 immersion oil objective).

Fig. 7.

Effect of increasing shear stress on NO production by primary cultures of TAL cells (*P < 0.002; n = 7).