Angiotensin II Stimulates Basolateral Conductance in Intercalated but Not Principal Cells of the Collecting Duct by Promoting Generation of Reactive Oxygen Species

Abstract

The renal collecting duct plays a critical role in setting urinary volume and composition by performing Na+ and K+ transport by principal cells and Cl- reabsorption via intercalated cells. Published evidence implies Angiotensin II (Ang II) is a potent activator of the apically localized epithelial Na+ channel (ENaC) and Cl-/HCO3- exchanger, pendrin, in principal and intercalated cells, respectively, particularly during hypovolemic states. However, virtually nothing is known about Ang II actions on the basolateral conductance of the collecting duct cells. To address this gap, we employed macroscopic and single channel patch clamp recordings from the basolateral membrane of freshly isolated mouse collecting duct principal and intercalated cells, pharmacological tools to delineate Ang II signaling pathway, and fluorescence-base real-time detection of Ang II-induced generation reactive oxygen species. We found that nanomolar concentrations of Ang II acutely stimulate the basolateral Cl- conductance and specifically ClC-K2 Cl- channel in intercalated cells. In contrast, similar concentration of Ang II did not affect the basolateral conductance and Kir4.1/5.1 potassium channel activity in principal cells. Inhibition of AT1 receptors with losartan abolished stimulatory actions of Ang II on ClC-K2. Deletion of AT1 receptors dramatically decreased renal ClC-K2 expression further indicating its major role in regulation of ClC-K2 function. Inhibition of either phospholipase C (PLC), phospholipase A2 (PLA2), or phosphoinositide 3 kinase (PI3-K) did not affect stimulation of ClC-K2 activity by Ang II. In contrast, we show that activation of NADPH oxidases (NOX) is the major signaling pathway downstream of Ang II-AT1R in stimulation of ClC-K2. Application of Ang II resulted in a rapid production of reactive oxygen species in both principal and intercalated cells within 5 min, which is consistent with the time course of single channel ClC-K2 activation. Overall, this study demonstrates that physiologically relevant Ang II levels stimulate ClC-K2 activity and by extension trans-cellular Cl- reabsorption in intercalated cells by triggering AT1 receptor-NOX-ROS signaling pathway. We speculate that upregulation of ClC-K2 by Ang II is critical for protection of circulatory volume during hypovolemic states, whereas over-activation of ClC-K2 might contribute to the pathophysiology of Ang II-dependent hypertension.