Regulation of thick ascending limb transport by angiotensin II

Hypertension is the leading cause of “loss of health” world-wide. Activation of angiotensin II type 1 receptors (AT₁) promotes salt retention by stimulating nephron transport and constricting renal vessels ^{38, 39}, while stimulating type 2 receptors (AT₂) promotes salt excretion. Imbalances favoring the former promote increases in blood pressure (blood pressure). Thick ascending limbs reabsorb 25-30% of filtered NaCl through both trans- and paracellular pathways. We and others reported that AT₁ activation stimulated protein kinase C and O₂^-, both of which usually enhance thick ascending limb NaCl absorption. We also showed that stimulating AT₂ enhanced NO production. NO inhibits NaCl absorption by activating phosphodiesterase II (PDE II), which degrades cAMP, and by reducing O₂^-. However, the role of AT₁ in stimulating thick ascending limb NaCl absorption is not well understood, and whether AT₂ activation inhibits transport is unknown. Elevated thick ascending limb NaCl absorption and defects in thick ascending limb NO production and signaling contribute to salt-sensitive hypertension and renal injury, but we do not know whether thick ascending limb AT₂-induced NO signaling is defective as well.  We hypothesize that in animals on a normal diet the stimulatory effects of AT₁ activation on thick ascending limb NaCl absorption are blunted by AT₂-induced NO which reduces cAMP and O₂^-. Defects in thick ascending limb AT₂ signaling promote increases in blood pressure and salt-sensitive hypertension.  Understanding how angiotensin II regulates thick ascending limb NaCl absorption will help form an integrated model that explains how angiotensin II regulates urinary volume (UV) and urinary Na excretion (U_NaV) and thus blood pressure and renal damage. It may lead to new treatments for salt-sensitive hypertension and renal injury.

To test this hypothesis we will measure the effect of angiotensin II on thick ascending limb NaCl absorption in vivo and in vitro. First in vivo we will measure UV and U_NaV after an acute salt and volume load in: 1) wild-type mice; 2) thick ascending limb-specific AT_1a -/- mice; 3) global AT₂ -/- mice; and 4) AT₂ -/- that have had AT₂ restored to only the thick ascending limbs. Next we will measure the effect of angiotensin II on net NaCl absorption, Na/K/2 Cl cotransporter type 2 activity, Na/K ATPase activity and paracellular permeability and selectivity in isolated, perfused thick ascending limbs in the presence and absence of AT₁ and AT₂ antagonists. Next we will measure the effect of angiotensin II in the presence and absence of an AT₁ or AT₂ antagonist and an AT₂ agonist on NO, cGMP and cAMP levels using fluorescence microscopy and a fluorescence resonance energy transfer (FRET) activity reporter. Then we will measure the effect of an AT₂ agonist and angiotensin II on O₂^- production in thick ascending limb suspensions with and without inhibiting NO production. We will also measure the effect of angiotensin II on net NaCl absorption when O₂^- levels are reduced. Finally we will measure the effect of angiotensin II on net NaCl absorption in thick ascending limbs from Dahl salt-sensitive  and salt-resistant rats in the presence of: 1) vehicle; 2) an AT₁ antagonist; 3) an AT₂ antagonist; 4) L-NAME; or 5) L-NAME and an AT₂ antagonist.