Luminal flow induces eNOS activation and translocation in the rat thick ascending limb

Department of Internal Medicine, Henry Ford Hospital, Detroit, Michigan, United States
American journal of physiology. Renal physiology (Impact Factor: 3.3). 08/2004; 287(2):F274-80. DOI: 10.1152/ajprenal.00382.2003
Source: PubMed

ABSTRACT Nitric oxide (NO) produced by endothelial NO synthase (eNOS) acts as an autacoid to inhibit NaCl absorption in the thick ascending limb of the loop of Henle (THAL). In the vasculature, shear stress activates eNOS. We hypothesized that increasing luminal flow activates eNOS and enhances NO production in the THAL. We measured NO production by isolated, perfused THALs using a NO-sensitive microelectrode. Increasing luminal flow from 0 to 20 nl/min increased NO production by 43.1 +/- 4.1 pA/mm of tubule (n = 10, P < 0.05), and this response was blunted (92%) by the NOS inhibitor L-(omega)nitro-methylarginine (P < 0.05). We studied the effect of flow on eNOS subcellular localization. In the absence of flow, eNOS was diffusely localized throughout the cell (basolateral = 33 +/- 4%; middle = 27 +/- 3%; apical = 40 +/- 4% of total eNOS). Increasing luminal flow induced eNOS translocation to the apical membrane, as evidenced by a 60% increase in eNOS immunoreactivity in the apical membrane (from 40 +/- 4 to 65 +/- 2%; n = 6; P < 0.05). Disrupting the actin cytoskeleton with cytochalasin D (10 microM) reduced flow-induced NO production by 62% (from 37.1 +/- 3.4 to 14.0 +/- 2.4 pA/mm tubule, n = 7, P < 0.04) and blocked flow-induced eNOS translocation. Flow also increased the amount of phosphorylated eNOS (Ser1179) at the apical membrane (from 25 +/- 2 to 56 +/- 2%; P < 0.05). We conclude that increasing luminal flow induces eNOS activation and translocation to the apical membrane in THALs. These are the first data showing that flow regulates eNOS in epithelial cells. This may be an important mechanism for regulation of NO levels in the renal medulla.

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    ABSTRACT: Thick ascending limbs reabsorb 30% of the filtered NaCl load. Nitric oxide (NO) produced by NO synthase 3 (NOS3) inhibits NaCl transport by this segment. In contrast, chronic angiotensin II (Ang II) infusion increases net thick ascending limb transport. NOS3 activity is regulated by changes in expression and phosphorylation at threonine 495 (T495) and serine 1177 (S1177), inhibitory and stimulatory sites respectively. We hypothesized that NO production by thick ascending limbs is impaired by chronic Ang II-infusion, due to reduced NOS3 expression, increased phosphorylation of T495 and decreased phosphorylation of S1177. Rats were infused with 200 ng/kg/min Ang II or vehicle for 1 and 5 days. Ang II infusion for 5 days decreased NOS3 expression by 40 ± 12% (p < 0.007; n = 6) and increased T495 phosphorylation by 147 ± 26 % (p < 0.008; n = 6). One-day Ang-II infusion had no significant effect. NO production in response to endothelin-1 was blunted in thick ascending limbs from Ang II-infused animals (Ang II -0.01 ± 0.06 AFU/min vs. 0.17 ± 0.02 AFU/min in controls; p<0.01). This was not due to endothelin-1 receptor expression. Phosphatidylinositol 3,4,5-triphosphate (PIP3)-induced NO production was also reduced in Ang II-infused rats (Ang II -0.07 ± 0.06 AFU/min vs. 0.13 ± 0.04 AFU/min in controls; p<0.03), and this correlated with an impaired ability of PIP3 to increase S1177 phosphorylation. We conclude that in Ang II-induced hypertension NO production by thick ascending limbs is impaired due to decreased NOS3 expression and altered phosphorylation.-
    American journal of physiology. Renal physiology 11/2014; 308(2):ajprenal.00279.2014. DOI:10.1152/ajprenal.00279.2014 · 3.30 Impact Factor
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    ABSTRACT: Luminal flow stimulates endogenous NO and O2(-) production by renal thick ascending limbs (TALs). The delicate balance between these two factors regulates Na transport in TALs; NO enhances natriuresis whereas O2(-) augments Na absorption. Endogenous, flow-stimulated O2(-) enhances Na/H exchange (NHE). Flow-stimulated NO reduces flow-induced O2(-), a process mediated by cGMP-dependent protein kinase (PKG). However, whether flow-stimulated, endogenously-produced NO diminishes O2(-) -stimulated NHE activity and the signaling pathway involved are unknown. We hypothesized that flow-induced NO reduces the stimulation of NHE activity caused by flow-induced O2(-) via PKG in TALs. Intracellular pH recovery after an acid-load was measured as an indicator of NHE activity in isolated, perfused rat TALs. L-arginine, the NO synthase substrate, decreased NHE activity by 34 ± 5% (n = 5; P < 0.04). The O2(-) scavenger tempol decreased NHE activity by 46 ± 8% (n = 6; P < 0.004) in the absence of NO. In the presence of L-arginine, the inhibitory effect of tempol on NHE activity was reduced to -19 ± 6% (n = 6; P < 0.03). The soluble guanylate cyclase inhibitor LY-83583 blocked the effect of L-arginine thus restoring tempol's effect on NHE activity to -42 ± 4% (n = 6; P < 0.0005). The PKG inhibitor KT-5823 also inhibited L-arginine's effect on tempol-reduced NHE activity (-43 ± 5%; n = 5; P < 0.03). We conclude that flow-induced NO reduces the stimulatory effect of endogenous, flow-induced O2(-) on NHE activity in TALs via an increase in cGMP and PKG activation. Copyright © 2014, American Journal of Physiology - Renal Physiology.
    American journal of physiology. Renal physiology 12/2014; DOI:10.1152/ajprenal.00583.2014 · 3.30 Impact Factor
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    ABSTRACT: Null mutations in the p67(phox) subunit of nicotinamide adenine dinucleotide phosphate-oxidase confer protection from salt sensitivity on Dahl salt-sensitive rats. Here, we track the sequential changes in medullary blood flow (MBF), glomerular filtration rate (GFR), urinary protein, and mean arterial pressure in SSp67(phox) null rats and wild-type littermates during 21 days of 4.0% NaCl high-salt (HS) diet. Optical fibers were implanted in the renal medulla and MBF was measured in conscious rats by laser Doppler flowmetry. Separate groups of rats were prepared with femoral venous catheters and GFR was measured by the transcutaneous assessment of fluorescein isothiocyanate-sinistrin disappearance curves. Mean arterial blood pressure was measured by telemetry. In wild-type rats, HS caused a rapid reduction in MBF, which was significantly lower than control values by HS day-6. Reduced MBF was associated with a progressive increase in mean arterial pressure, averaging 170±5 mm Hg by HS salt day-21. A significant reduction in GFR was evident on day-14 HS, after the onset of hypertension and reduced MBF. In contrast, HS had no significant effect on MBF in SSp67(phox) null rats and the pressor response to sodium was blunted, averaging 150±3 mm Hg on day-21 HS. GFR was maintained throughout the study and proteinuria was reduced. In summary, when p67(phox) is not functional in the salt-sensitive rats, HS does not cause reduced MBF and salt-sensitive hypertension is attenuated, and consequently renal injury is reduced and GFR is maintained. © 2014 American Heart Association, Inc.
    Hypertension 12/2014; 65(3). DOI:10.1161/HYPERTENSIONAHA.114.04468 · 7.63 Impact Factor

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