Abnormal thick ascending limb (THAL) NaCl reabsorption has been implicated in several forms of hypertension including salt-sensitive hypertension. We reported that THAL NaCl reabsorption is inhibited by NO and stimulated by O2- . Both reductions in NO and increases in O2- have been implicated in salt-sensitive as well as other forms of hypertension. A high-salt diet enhances luminal flow through THALs. We have shown that flow stimulates NO. Flow also enhances O2- in the absence of L-arginine (and therefore NO). In contrast, in the presence of L-arginine flow-induced O2- is reduced by >80%. Until now, it was thought that NO only reduces O2- by scavenging, producing ONOO- which is highly toxic. However, we found that the reduction in O2- caused by NO depends upon cGMP signaling. This novel finding has not been reported before. cGMP-induced reductions in O2- would be expected to be beneficial compared to scavenging because the highly toxic ONOO- is not formed as it is when NO scavenges O2- and NO is not destroyed. NO-induced reductions in O2- may result from either: 1) a decrease in O2- production by NADPH oxidase (its primary source in THALs);or 2) enhanced degradation by superoxide dismutase. A decrease in production may result from inhibiting the association of the p47phox and p67phox subunits with the catalytic subunit. This assembly is normally stimulated by protein kinase C (PKC)- dependent phosphorylation of p47phox. However, we do not know how NO regulates O2- levels, nor its effects on NaCl absorption. We hypothesize that in THALs flow-stimulated NO reduces flow-induced O2- primarily by activating cGMP-dependent protein kinase (PKG);this reduces PKC activity, which in turn prevents flow-induced activation of Nox 4-based NADPH oxidase and consequently O2--dependent NaCl absorption. Defects in the ability of NO to reduce O2- production contribute to NaCl retention and salt-sensitive hypertension. This hypothesis will be tested in four aims.
Aim 1 will test whether in THALs flow-induced O2- and therefore O2--dependent NaCl absorption is reduced by flow-stimulated NO via a cGMP-dependent mechanism. This effect is blunted in salt- sensitive hypertension.
Aim 2 will test whether flow-stimulated NO/cGMP reduces O2- production by Nox 4-based NADPH oxidase via activation of PKG II.
Aim 3 will test whether flow-stimulated NO/cGMP/PKG II reduces flow- stimulated PKC activity and hence O2- production by Nox 4-based NADPH oxidase.
Aim 4 will test whether NO- activated PKG inhibits flow-induced Nox 4-based NADPH oxidase assembly by reducing PKC activity and thereby blunting p47phox activation and translocation. NO's effect is reduced in salt-sensitive hypertension. We will use a wide range of techniques from whole animal physiology to molecular biology. Many of these techniques are highly innovative and were developed in our laboratory for these and similar studies. Successful completion of the proposed studies will further our understanding of how NO and O2- regulate renal function under physiological and pathophysiological conditions. They may also lead to new targets for the treatment of hypertension.

Public Health Relevance

Oxidative stress plays important roles in several pathological conditions, including many forms of hypertension, diabetes-induced changes in renal function, chronic renal failure and renal ischemic injury. However, the factors that regulate oxidative stress in the thick ascending limb have not been extensively studied. In contrast to oxidative stress, a compound called nitric oxide promotes natriuresis and diuresis, and its loss has been implicated in renal damage and hypertension. In this proposal, we will study whether flow-stimulated nitric oxide inhibits superoxide production, a chemical that causes most oxidative stress. If proven correct, our hypothesis may explain why: 1) decreases in nitric oxide and increases in superoxide are both implicated in salt-sensitive hypertension;and 2) there is little renal damage in the early stages of diabetes. Data from this proposal will increase our understanding of the causes of sodium retention and may result in the development of new diuretics and new therapies for hypertension-induced renal damage and chronic renal failure.

National Institute of Health (NIH)
National Heart, Lung, and Blood Institute (NHLBI)
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Hypertension and Microcirculation Study Section (HM)
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OH, Youngsuk
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Case Western Reserve University
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Saez, Fara; Hong, Nancy J; Garvin, Jeffrey L (2018) NADPH oxidase 4-derived superoxide mediates flow-stimulated NKCC2 activity in thick ascending limbs. Am J Physiol Renal Physiol 314:F934-F941
Gonzalez-Vicente, Agustin; Garvin, Jeffrey L (2017) Effects of Reactive Oxygen Species on Tubular Transport along the Nephron. Antioxidants (Basel) 6:
Monzon, Casandra M; Occhipinti, Rossana; Pignataro, Omar P et al. (2017) Nitric oxide reduces paracellular resistance in rat thick ascending limbs by increasing Na+ and Cl- permeabilities. Am J Physiol Renal Physiol 312:F1035-F1043
Saez, Fara; Hong, Nancy J; Garvin, Jeffrey L (2016) Luminal flow induces NADPH oxidase 4 translocation to the nuclei of thick ascending limbs. Physiol Rep 4:
Gonzalez-Vicente, Agustin; Saikumar, Jagannath H; Massey, Katherine J et al. (2016) Angiotensin II stimulates superoxide production by nitric oxide synthase in thick ascending limbs. Physiol Rep 4:
Monzon, Casandra M; Garvin, Jeffrey L (2015) Nitric oxide decreases the permselectivity of the paracellular pathway in thick ascending limbs. Hypertension 65:1245-50
Cabral, P D; Capurro, C; Garvin, J L (2015) TRPV4 mediates flow-induced increases in intracellular Ca in medullary thick ascending limbs. Acta Physiol (Oxf) 214:319-28
Hong, Nancy J; Garvin, Jeffrey L (2015) Endogenous flow-induced nitric oxide reduces superoxide-stimulated Na/H exchange activity via PKG in thick ascending limbs. Am J Physiol Renal Physiol 308:F444-9
Cabral, Pablo D; Garvin, Jeffrey L (2014) TRPV4 activation mediates flow-induced nitric oxide production in the rat thick ascending limb. Am J Physiol Renal Physiol 307:F666-72
Hong, Nancy J; Garvin, Jeffrey L (2014) Endogenous flow-induced superoxide stimulates Na/H exchange activity via PKC in thick ascending limbs. Am J Physiol Renal Physiol 307:F800-5

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