Angiotensin II increases activity of the ClC-K2 Cl(−) channel in collecting duct intercalated cells by stimulating production of reactive oxygen species

The renal collecting duct plays a critical role in setting urinary volume and composition, with principal cells transporting Na(+) and K(+) and intercalated cells mediating Cl(−) reabsorption. Published evidence implies Angiotensin II (Ang II) is a potent regulator of the collecting duct apical transport systems in response to systemic volume depletion. However, virtually nothing is known about Ang II actions on the basolateral conductance of principal and intercalated cells. Here, we combined macroscopic and single channel patch clamp recordings from freshly isolated mouse collecting ducts with biochemical and fluorescence methods to demonstrate an acute stimulation of the basolateral Cl(−) conductance and specifically the ClC-K2 Cl(−) channel by nanomolar Ang II concentrations in intercalated cells. In contrast, Ang II did not exhibit measurable effects on the basolateral conductance and on K(ir)4.1/5.1 potassium channel activity in principal cells. Although both Ang II receptors AT(1) and AT(2) are expressed in collecting duct cells, we show that AT(1) receptors were essential for stimulatory actions of Ang II on ClC-K2. Moreover, AT(1)R(−/−) mice had decreased renal ClC-K2 expression. We further demonstrated that activation of NADPH oxidases is the major signaling pathway downstream of Ang II-AT(1)R that leads to stimulation of ClC-K2. Treatment of freshly isolated collecting ducts with Ang II led to production of reactive oxygen species on the same timescale as single channel ClC-K2 activation. Overall, we propose that Ang II-dependent regulation of ClC-K2 in intercalated cells is instrumental for stimulation of Cl(−) reabsorption by the collecting duct, particularly during hypovolemic states.