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Angiotensin II increases apical plasma membrane pendrin abundance and function. This study explored the role of the small GTPase Rac1 in the regulation of pendrin by angiotensin II. To do this, we generated intercalated cell (IC) Rac1 knockout mice and observed that IC Rac1 gene ablation reduced the relative abundance of pendrin in the apical region of intercalated cells in angiotensin II-treated mice but not vehicle-treated mice. Similarly, the Rac1 inhibitor EHT 1864 reduced apical pendrin abundance in angiotensin II-treated mice, through a mechanism that does not require aldosterone. This IC angiotensin II-Rac1 signaling cascade modulates pendrin subcellular distribution without significantly changing actin organization. However, NADPH oxidase inhibition with APX 115 reduced apical pendrin abundance in vivo in angiotensin II-treated mice. Moreover, superoxide dismutase mimetics reduced Cl - absorption in angiotensin II-treated cortical collecting ducts perfused in vitro. Since Rac1 is an NADPH subunit, Rac1 may modulate pendrin through NADPH oxidase-mediated reactive oxygen species production. Because pendrin gene ablation blunts the pressor response to angiotensin II, we asked if pendrin blunts the angiotensin II-induced increase in kidney superoxide. Although kidney superoxide was similar in vehicle-treated wild-type and pendrin knockout mice, it was lower in angiotensin II-treated pendrin-null kidneys than in wild-type kidneys. We conclude that angiotensin II acts through Rac1, independently of aldosterone, to increase apical pendrin abundance. Rac1 may stimulate pendrin, at least partly, through NADPH oxidase. This increase in pendrin abundance contributes to the increment in blood pressure and kidney superoxide content seen in angiotensin II-treated mice. NEW & NOTEWORTHY This study defines a new signaling mechanism by which angiotensin II modulates oxidative stress and blood pressure.

Pendrin is a Cl-/HCO3- exchanger expressed in the apical regions of renal intercalated cells. Following pendrin gene ablation, blood pressure falls, in part, from reduced renal NaCl absorption. We asked if pendrin is expressed in vascular tissue and if the lower blood pressure observed in pendrin null mice is accompanied by reduced vascular reactivity. Thus, the contractile responses to KCl and phenylephrine (PE) were examined in isometrically mounted thoracic aortas from wild-type and pendrin null mice. Although pendrin expression was not detected in the aorta, pendrin gene ablation changed contractile protein abundance and increased the maximal contractile response to PE when normalized to cross sectional area (CSA). However, the contractile sensitivity to this agent was unchanged. The increase in contractile force/cross sectional area observed in pendrin null mice was due to reduced cross sectional area of the aorta and not from increased contractile force per vessel. The pendrin-dependent increase in maximal contractile response was endothelium- and nitric oxide-independent and did not occur from changes in Ca2+ sensitivity or chronic changes in catecholamine production. However, application of 100 nM angiotensin II increased force/CSA more in aortas from pendrin null than from wild type mice. Moreover, angiotensin type 1 receptor inhibitor (candesartan) treatment in vivo eliminated the pendrin-dependent changes contractile protein abundance and changes in the contractile force/cross sectional area in response to PE. In conclusion, pendrin gene ablation increases aorta contractile force per cross sectional area in response to angiotensin II and PE due to stimulation of angiotensin type 1 receptor-dependent signaling. The angiotensin type 1 receptor-dependent increase in vascular reactivity may mitigate the fall in blood pressure observed with pendrin gene ablation.

We reported previously that angiotensin II (AngII) increases net Cl(-) absorption in mouse cortical collecting duct (CCD) by transcellular transport across type B intercalated cells (IC) via an H(+)-ATPase-and pendrin-dependent mechanism. Because intracellular trafficking regulates both pendrin and H(+)-ATPase, we hypothesized that AngII induces the subcellular redistribution of one or both of these exchangers. To answer this question, CCD from furosemide-treated mice were perfused in vitro, and the subcellular distributions of pendrin and the H(+)-ATPase were quantified using immunogold cytochemistry and morphometric analysis. Addition of AngII in vitro did not change the distribution of pendrin or H(+)-ATPase within type B IC but within type A IC increased the ratio of apical plasma membrane to cytoplasmic H(+)-ATPase three-fold. Moreover, CCDs secreted bicarbonate under basal conditions but absorbed bicarbonate in response to AngII. In summary, angiotensin II stimulates H(+) secretion into the lumen, which drives Cl(-) absorption mediated by apical Cl(-)/HCO(3)(-) exchange as well as generates more favorable electrochemical gradient for ENaC-mediated Na(+) absorption.

Pendrin (Slc26a4) localizes to type B and non-A, non-B intercalated cells in the distal convoluted tubule, the connecting tubule, and the cortical collecting duct (CCD), where it mediates apical Cl(-)/HCO(3)(-) exchange. The purpose of this study was to determine whether angiotensin II increases transepithelial net chloride transport, J(Cl), in mouse CCD through a pendrin-dependent mechanism. J(Cl) and transepithelial voltage, V(T), were measured in CCDs perfused in vitro from wild-type and Slc26a4 null mice ingesting a NaCl-replete diet or a NaCl-replete diet and furosemide. In CCDs from wild-type mice ingesting a NaCl-replete diet, V(T) and J(Cl) were not different from zero either in the presence or absence of angiotensin II (10(-8) M) in the bath. Thus further experiments employed mice given the high-NaCl diet and furosemide to upregulate renal pendrin expression. CCDs from furosemide-treated wild-type mice had a lumen-negative V(T) and absorbed Cl(-). With angiotensin II in the bath, Cl(-) absorption doubled although V(T) did not become more lumen negative. In contrast, in CCDs from furosemide-treated Slc26a4 null mice, Cl(-) secretion and a V(T) of approximately 0 were observed, neither of which changed with angiotensin II application. Inhibiting ENaC with benzamil abolished V(T) although J(Cl) fell only approximately 50%. Thus substantial Cl(-) absorption is observed in the absence of an electromotive force. Attenuating apical anion exchange with the peritubular application of the H(+)-ATPase inhibitor bafilomycin abolished benzamil-insensitive Cl(-) absorption. In conclusion, angiotensin II increases transcellular Cl(-) absorption in the CCD through a pendrin- and H(+)-ATPase-dependent process.

ANG II increases Cl- absorption in the mouse CCD by increasing transcellular transport across type B intercalated cells, through an H+-ATPase and Cl-/HCO3- exchanger (pendrin)-dependent mechanism. Thus, we asked if ANG 11 induces subcellular redistribution of pendrin or the H+-ATPase, using quantitative immunogold cytochemistry of mouse CCDs perfused in vitro. ANG II (10 nM) application to the bath did not change the subcellular distribution of pendrin or the H+-ATPase in B cells, but increased apical plasma membrane H+-ATPase expression 3-fold in A intercalated cells. To determine if ANG II increases H+-ATPase-mediated H+ secretion, Jt·CO2 was measured under identical conditions. Under basal conditions, HCO3- secretion was observed (L-co: = -4.2 + 2,2 pmol/mm/min), while HCO3- absorption (Jt-CO2 = 2.5 ± 1.1 pmol/mm/min, P < 0.05) was observed with ANG II applied to the bath, consistent with apical H+-ATPase activation. Finally, application of the H+-ATPase inhibitor, bafilomycin, generated a more lumen-negative transepithelial voltage, VT, in the presence, but not in the absence of ANG II. Conclusions: ANG II increases apical plasma membrane H+-ATPase expression and function. Increased H+ secretion may increase NaCl absorption by: 1) stimulating apical Cl/HCO3- exchange by reducing luminal HCO3- concentration and increasing CO2 formation and 2) shunting Vτ generated by ENaC-mediated Na+ absorption. [ABSTRACT FROM AUTHOR]