Angiotensin II stimulates trafficking of NHE3, NaPi2, and associated proteins into the proximal tubule microvilli

Abstract

(ANG II) stimulates proximal tubule (PT) sodium and water reabsorption. We showed that treating rats acutely with the angiotensin-converting enzyme inhibitor captopril decreases PT salt and water reabsorption and provokes rapid redistribution of the Na ϩ /H ϩ exchanger isoform 3 (NHE3), Na ϩ /Pi cotransporter 2 (NaPi2), and associated proteins out of the microvilli. The aim of the present study was to determine whether acute ANG II infusion increases the abundance of PT NHE3, NaPi2, and associated proteins in the microvilli available for reabsorbing NaCl. Male Sprague-Dawley rats were infused with a dose of captopril (12 g/min for 20 min) that increased PT flow rate ϳ20% with no change in blood pressure (BP) or glomerular filtration rate (GFR). When ANG II (20 ng⅐kg Ϫ1 ⅐min Ϫ1 for 20 min) was added to the captopril infusate, PT volume flow rate returned to baseline without changing BP or GFR. After captopril, NHE3 was localized to the base of the microvilli and NaPi2 to subapical cytoplasmic vesicles; after 20 min ANG II, both NHE3 and NaPi2 redistributed into the microvilli, assayed by confocal microscopy and density gradient fractionation. Additional PT proteins that redistributed into low-density microvilli-enriched membranes in response to ANG II included myosin VI, DPPIV, NHERF-1, ezrin, megalin, vacuolar H ϩ -ATPase, aminopeptidase N, and clathrin. In summary, in response to 20 min ANG II in the absence of a change in BP or GFR, multiple proteins traffic into the PT brush-border microvilli where they likely contribute to the rapid increase in PT salt and water reabsorption. hypertension; captopril ANGIOTENSIN II (ANG II), a potent vasoconstrictor and sodiumretaining hormone, is crucial for the regulation of sodium transport in the kidneys and therefore for blood pressure (BP) homeostasis. Strong evidence highlights the contribution of high ANG II levels to the development of cardiovascular and renal diseases (23, 37). Consequently, drugs affecting the renin-angiotensin system (RAS), and in particular angiotensinconverting enzyme inhibitors (ACEI) and angiotensin receptor blockers, are commonly used for the treatment of high BP.The angiotensin type I receptor (AT1R) is responsible for the Na ϩ -retaining effects of ANG II. The renal proximal tubule (PT) expresses AT1R on both the apical and basolateral membranes and ANG II is delivered via the general circulation or filtered at the glomerulus. In addition, the PT cells synthesize all the components necessary to produce and secrete ANG II into its lumen: angiotensinogen, renin, and ACE (14, 29). ANG II has been shown to increase PT sodium and water reabsorption, and ACE inhibitors and AT1R blockers decrease PT sodium and water reabsorption (10, 13). The sodium hydrogen exchanger isoform 3 (NHE3) is the main transporter mediating sodium reabsorption in the PT (20) and cultured kidney cell studies indicate that ANG II can rapidly increase NHE3 abundance and activity in the plasma membrane (9).We recently investigated the molecular mechanisms responsible for th...

Effects of ACE inhibition and ANG II stimulation on renal Na-Cl cotransporter distribution, phosphorylation, and membrane complex properties

“…Previous studies (41,45) demonstrated that these doses of captopril and ANG II did not change mean arterial pressure or glomerular filtration rate and that urine output increased during captopril treatment in one study (45) but not significantly in another (41), as in the current analysis (Fig. 5).…”

“…As previously described in detail (41), kidneys were perfusionfixed via the dorsal aorta with PLP fixative (2% paraformaldehyde, 75 mM lysine, and 10 mM Na-periodate, pH 7.4) at a rate that did not alter blood pressure (measured via carotid cannula), and were then post-fixed in PLP for another 2-3 h. The fixed tissue was cryoprotected by incubation overnight in 30% sucrose in PBS, embedded in Tissue-Tek OCT compound (Sakura Finetek, Torrance, CA), and frozen in liquid nitrogen. Cryosections (5 m) were cut and transferred to Fisher Superfrost Plus-charged glass slides and air dried.…”

The absence of intrarenal ACE protects against hypertension

“…Importantly, the absence of these changes in the ACE 10/10 mice implies that removal of kidney ACE prevents the effect of Ang II infusion that shifts the pressure-natriuresis relationship toward hypertension. The initial drop in sodium and urine excretion in wild-type mice may be explained by the higher plasma Ang II during the first 24 hours in this group and its acute effects on renal hemodynamics, NHE3 activity, or both (28). It is unclear why plasma Ang II levels were initially higher in wild-type mice.…”

“…Cryosections were cut, and sections of both uninfused and Ang II-infused mice were collected on the same charged object glass slide, side by side, to assure comparable treatments. Samples were subjected to antigen retrieval and incubated with primary and secondary antibodies (Supplemental Table 1), as described in detail elsewhere (28). Samples were viewed with a Leica DMI 6000 inverted microscope and imaged using a Leica TCS SP5 confocal fluorescence imaging system powered by a Chameleon Ultra-II MP laser (Coherent Inc.) and visible RGB lasers (Leica Microsystems).…”

Regulation of proximal tubule vacuolar H+-ATPase by PKA and AMP-activated protein kinase

“…Another important regulator of proton secretion in the proximal tubule is angiotensin II (ANG II), as this hormone stimulates NHE3 (40). ANG II can induce decreases in cAMP levels in the proximal tubule (10,52), yet it can increase V-ATPase activity via PKC (12). While long-term regulation of the V-ATPase by ANG II in proximal tubule cells appears to be dependent on mechanisms involving tyrosine kinases and PI-3-kinase (11), others have found that ANG II stimulates V-ATPase-dependent proton extrusion in the proximal tubule by insertion of vesicles into the brush-border membrane (64).…”