High blood pressure is the leading cause of health lost worldwide, and 30% of the adult U.S. population suffers from it. Salt-sensitive hypertension is a disease in which dietary salt increases blood pressure. Increases in sodium reabsorption by the thick ascending limb of the loop of Henle (TAL) contribute to salt-sensitive hypertension, whereas loss of function mutation on the transporters in this segment leads to reduced blood pressure levels and prevention of hypertension. Na reabsorption by this segment is a 2-step process: first, NaCl enters the cells via Na/K/2 Cl cotransporter type 2 (NKCC2); and then Na exits via basolateral Na/K-ATPase while Cl exits either via Cl channels or KCl cotransporters. The role of the TAL in fluid an electrolyte regulation is underscored by the profuse diuresis induced by loop diuretics. Furthermore, we have shown that chronic infusion of suppressor doses of ANG II (< 200 ng/kg/min in rats), a salt-sensitive model of hypertension, increases net transport by the TAL.
In this model, there is a positive sodium balance during the first 3 to 7 days, and blood pressure rises up to 3 weeks before reaching a plateau. The TAL likely contributes to these effects by increasing NKCC2 activity, as confusion of furosemide prevent the increase in net transport by the TAL, and NKCC2 expression and activation markers were found enhanced at 1 week. Interestingly, in the way the blood pressure increases, a pressure-natriuretic effect counteracts the positive effects of Ang II in tubular transport. Thus in order to study the causes that leads to salt-sensitivity, it is important to study this model at early time-points, where Na balances are positive.
NO produced by NO-synthase 3 (NOS3) regulates Na transport by the TAL by inhibiting NKCC2 thereby contributing to diuresis and natriuresis. NOS3 is regulated by several mechanisms including expression and phosphorylation. Previous reports from our laboratory indicate that Ang II down regulates NOS3 expression in cultured TALs, and that the increase in TAL’s net transport in Ang II-induced hypertension depends on PKC activation. PKC targets NOS3 at the inhibitory site threonine 495 (T495). Added together these effects would reduce NO bioavailability by the TAL, and contribute to increased transport in Ang II-induced hypertension. We hypothesized that NO production in the TAL is impaired by Ang II-infusions, due to down regulation of NOS3 expression and an increased phosphorylation of T495. This would affect the capacity of the enzyme to increase NO production in response to physiological stimuli.
To test our hipothesis, we infused Ang II (200 ng/kg/min) for 5 days and measure NOS3 expression and phosphorylation at T495. Then we tested the capacity of the TALs to increase NO production in response to physiological stimuli; endothelin 1 and PIP3.
We found that chronic infusion of Ang II decreases NOS3 expression in the TAL, and increases phosphorylation of the inhibitory site T495....