Deletion of the angiotensin II type 1 receptor–associated protein enhances renal sodium reabsorption and exacerbates angiotensin II–mediated hypertension
Authors: Masato Ohsawa, Kouichi Tamura, Hiromichi Wakui, Akinobu Maeda, Toru Dejima, Tomohiko Kanaoka, Kengo Azushima, Kazushi Uneda, Yuko Tsurumi-Ikeya, Ryu Kobayashi, Miyuki Matsuda, Shinichi Uchida, Yoshiyuki Toya, Hiroyuki Kobori, Akira Nishiyama, Akio Yamashita, Yoshihiro Ishikawa, Satoshi Umemura
Journal: Kidney International (2014)

Angiotensin II type 1 receptor (AT1R)–associated protein (ATRAP) promotes AT1R internalization along with suppression of pathological activation of tissue AT1R signaling. However, the functional significance of ATRAP in renal sodium handling and blood pressure regulation under pathological stimuli is not fully resolved. Here we show the blood pressure of mice with a gene-targeted disruption of ATRAP was comparable to that of wild-type mice at baseline. However, in ATRAP-knockout mice, angiotensin II–induced hypertension was exacerbated and the extent of positive sodium balance was increased by angiotensin II. Renal expression of the sodium-proton antiporter 3, a major sodium transporter in the proximal tubules, urinary pH, renal angiotensinogen production, and angiotensin II content was unaffected. Stimulation of the renal expression and activity of the epithelial sodium channel (ENaC), a major sodium transporter in the distal tubules, was significantly enhanced by chronic angiotensin II infusion. The circulating and urinary aldosterone levels were comparable. The blood pressure response and renal ENaC expression by aldosterone were not affected. Thus, ATRAP deficiency exacerbated angiotensin II–mediated hypertension by pathological activation of renal tubular AT1R by angiotensin II. This directly stimulates ENaC in the distal tubules and enhances sodium retention in an aldosterone-independent manner. Angiotensin II type 1 receptor (AT1R)–associated protein (ATRAP) promotes AT1R internalization along with suppression of pathological activation of tissue AT1R signaling. However, the functional significance of ATRAP in renal sodium handling and blood pressure regulation under pathological stimuli is not fully resolved. Here we show the blood pressure of mice with a gene-targeted disruption of ATRAP was comparable to that of wild-type mice at baseline. However, in ATRAP-knockout mice, angiotensin II–induced hypertension was exacerbated and the extent of positive sodium balance was increased by angiotensin II. Renal expression of the sodium-proton antiporter 3, a major sodium transporter in the proximal tubules, urinary pH, renal angiotensinogen production, and angiotensin II content was unaffected. Stimulation of the renal expression and activity of the epithelial sodium channel (ENaC), a major sodium transporter in the distal tubules, was significantly enhanced by chronic angiotensin II infusion. The circulating and urinary aldosterone levels were comparable. The blood pressure response and renal ENaC expression by aldosterone were not affected. Thus, ATRAP deficiency exacerbated angiotensin II–mediated hypertension by pathological activation of renal tubular AT1R by angiotensin II. This directly stimulates ENaC in the distal tubules and enhances sodium retention in an aldosterone-independent manner. Hypertension is the most common chronic disease worldwide. It has a complex etiology in which genetic and environmental factors are intricately intertwined. Although the mechanisms underlying hypertension are difficult to fully elucidate, the renin–angiotensin system (RAS) has a pivotal role in it. Among tissue RAS forms, activation of angiotensin II type 1 receptor (AT1R) signaling in the renal tubules is suggested to have a key role in altered renal sodium handling that occurs in hypertension. According to Guyton's hypothesis, it is maintained that hypertension is caused by a disproportionate suppression of sodium excretion from kidney due to arterial pressure.1.Guyton A.C. Blood pressure control—special role of the kidneys and body fluids.Science. 1991; 252: 1813-1816Crossref PubMed Scopus (744) Google Scholar Therefore, pathological activation of renal tubule AT1R signaling provokes defective renal sodium handling, with a consequent dysregulation of body fluid volume that, in turn, leads to development of hypertension. The AT1R-associated protein (ATRAP/Agtrap) has been identified as a specific AT1R-binding protein.2.Daviet L. Lehtonen J.Y. Tamura K. et al.Cloning and characterization of ATRAP, a novel protein that interacts with the angiotensin II type 1 receptor.J Biol Chem. 1999; 274: 17058-17062Crossref PubMed Scopus (143) Google Scholar,3.Lopez-Ilasaca M. Liu X. Tamura K. et al.The angiotensin II type I receptor-associated protein, ATRAP, is a transmembrane protein and a modulator of angiotensin II signaling.Mol Biol Cell. 2003; 14: 5038-5050Crossref PubMed Scopus (91) Google Scholar Previous studies have shown that ATRAP selectively suppresses angiotensin II (Ang II)–mediated pathological activation of AT1R in cardiovascular cells, and that cardiac ATRAP enhancement ameliorates cardiac hypertrophy in Ang II–infused mice without any effect on baseline cardiovascular functions, including blood pressure (BP).4.Tanaka Y. Tamura K. Koide Y. et al.The novel angiotensin II type 1 receptor (AT1R)-associated protein ATRAP downregulates AT1R and ameliorates cardiomyocyte hypertrophy.FEBS Lett. 2005; 579: 1579-1586Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar, 5.Azuma K. Tamura K. 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Masuda S. et al.Enhanced angiotensin receptor-associated protein in renal tubule suppresses angiotensin-dependent hypertension.Hypertension. 2013; 61: 1203-1210Crossref PubMed Scopus (40) Google Scholar Based on these observations, we hypothesized that a downregulation of tissue ATRAP enhances pathological activation of tissue AT1R in response to certain stimuli without any evident effect on baseline physiological AT1R signaling. ATRAP is abundantly distributed along the renal tubules.9.Masuda S. Tamura K. Wakui H. et al.Expression of angiotensin II type 1 receptor-interacting molecule in normal human kidney and IgA nephropathy.Am J Physiol Renal Physiol. 2010; 299: F720-F731Crossref PubMed Scopus (25) Google Scholar,10.Tsurumi Y. Tamura K. Tanaka Y. et al.Interacting molecule of AT1 receptor, ATRAP, is colocalized with AT1 receptor in the mouse renal tubules.Kidney Int. 2006; 69: 488-494Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar Therefore, we investigated the effects of chronic Ang II infusion on BP and renal sodium handling in the context of gene-specific disruption of ATRAP (ATRAP-knockout (KO) mice). The ATRAP mRNA was widely distributed in the tissues of littermate wild-type control mice (WT mice) with the highest expression in kidney (Figure 1a). In contrast, no ATRAP expression was detected in any tissues, including the kidney, in ATRAP-KO mice (Figure 1a and b). On immunohistochemical analysis with consecutive sections using markers specific to the tubular segments, such as megalin (proximal convoluted tubules), calbindin-D (distal convoluted tubules and connecting tubules), and aquaporin 2 (collecting ducts), ATRAP immunostaining was broadly detected in nephron segments from proximal convoluted tubules to collecting ducts of WT mice (Figure 1c). In contrast, there was no ATRAP immunostaining in nephron segments of ATRAP-KO mice (Figure 1d). Although genetic inactivation of other RAS components, such as angiotensinogen (AGT), renin, and AT1R, has been reported to result in renal morphological alteration even under baseline conditions,11.Tanimoto K. Sugiyama F. Goto Y. et al.Angiotensinogen-deficient mice with hypotension.J Biol Chem. 1994; 269: 31334-31337Abstract Full Text PDF PubMed Google Scholar, 12.Niimura F. Labosky P.A. Kakuchi J. et al.Gene targeting in mice reveals a requirement for angiotensin in the development and maintenance of kidney morphology and growth factor regulation.J Clin Invest. 1995; 96: 2947-2954Crossref PubMed Scopus (319) Google Scholar, 13.Kihara M. Umemura S. Sumida Y. et al.Genetic deficiency of angiotensinogen produces an impaired urine concentrating ability in mice.Kidney Int. 1998; 53: 548-555Abstract Full Text PDF PubMed Scopus (70) Google Scholar, 14.Oliverio M.I. Kim H.S. Ito M. et al.Reduced growth, abnormal kidney structure, and type 2 (AT2) angiotensin receptor-mediated blood pressure regulation in mice lacking both AT1A and AT1B receptors for angiotensin II.Proc Natl Acad Sci USA. 1998; 95: 15496-15501Crossref PubMed Scopus (279) Google Scholar, 15.Yanai K. Saito T. Kakinuma Y. et al.Renin-dependent cardiovascular functions and renin-independent blood-brain barrier functions revealed by renin-deficient mice.J Biol Chem. 2000; 275: 5-8Crossref PubMed Scopus (133) Google Scholar ATRAP-KO mice at baseline exhibited no apparent alterations in renal morphology (Figure 1e) or creatinine clearance (Figure 1f, 260.8±21.7 in WT vs. 277.6±13.2 in ATRAP-KO, μl/min, P=0.918; unpaired t-test). Furthermore, there was no significant difference in AT1R mRNA expression between WT and ATRAP-KO mice (Figure 1g). Genetic deficiency of the RAS components (that is, AGT, renin, and AT1R) has also been reported to result in significant decreases in baseline BP.11.Tanimoto K. Sugiyama F. Goto Y. et al.Angiotensinogen-deficient mice with hypotension.J Biol Chem. 1994; 269: 31334-31337Abstract Full Text PDF PubMed Google Scholar, 12.Niimura F. Labosky P.A. Kakuchi J. et al.Gene targeting in mice reveals a requirement for angiotensin in the development and maintenance of kidney morphology and growth factor regulation.J Clin Invest. 1995; 96: 2947-2954Crossref PubMed Scopus (319) Google Scholar, 13.Kihara M. Umemura S. Sumida Y. et al.Genetic deficiency of angiotensinogen produces an impaired urine concentrating ability in mice.Kidney Int. 1998; 53: 548-555Abstract Full Text PDF PubMed Scopus (70) Google Scholar, 14.Oliverio M.I. Kim H.S. Ito M. et al.Reduced growth, abnormal kidney structure, and type 2 (AT2) angiotensin receptor-mediated blood pressure regulation in mice lacking both AT1A and AT1B receptors for angiotensin II.Proc Natl Acad Sci USA. 1998; 95: 15496-15501Crossref PubMed Scopus (279) Google Scholar, 15.Yanai K. Saito T. Kakinuma Y. et al.Renin-dependent cardiovascular functions and renin-independent blood-brain barrier functions revealed by renin-deficient mice.J Biol Chem. 2000; 275: 5-8Crossref PubMed Scopus (133) Google Scholar However, baseline 24-h mean systolic BP, diastolic BP, and heart rate, measured by radiotelemetric method, were similar in WT and ATRAP-KO mice (Figure 2a, systolic BP 120±2 vs. 123±4, mmHg, P=0.545; Figure 2b, diastolic BP 92±5 vs. 95±4, mmHg, P=0.793; Figure 2c, HR 541±18 vs. 547±10 beat/min, P=0.598; unpaired t-test). Urinary pH, which reflects the activity of sodium-proton antiporter 3 (NHE3), was also similar in WT and ATRAP-KO mice (Figure 2d). As ATRAP-KO mice did not display any alternation in baseline BP, we next examined the effects of ATRAP deficiency on BP response to chronic Ang II infusion (500 or 2000ng/kg/min). Whereas systolic BP was increased during the period of chronic Ang II infusion in WT mice, Ang II–induced elevation of systolic BP was significantly exacerbated in ATRAP-KO mice compared with WT mice, irrespective of dose (Figure 3a; Ang II, 500ng/kg/min, F=6.117, P=0.048; Figure 3b, Ang II, 2000ng/kg/min, F=86.758, P<0.001, two-way repeated measures analysis of variance (ANOVA)). We employed a higher dose of Ang II (2000ng/kg/min) for further experiments to characterize ATRAP-KO mice in comparison with WT mice. The ATRAP-KO mice also exhibited a significantly increased ratio of heart weight/body weight compared with WT mice by Ang II (Figure 3c, P=0.040; analysis of variance (ANOVA). Similarly, urinary albumin excretion was elevated in ATRAP-KO mice compared with WT mice (Figure 3d; P<0.001. ANOVA). The mortality rate was comparable in WT and ATRAP-KO mice during Ang II infusion (2000ng/kg/min) for 14 days (5.7 vs. 7.3%, P=0.576, χ2-test). As the highest ATRAP expression was observed in renal tubules among analyzed tissues, we hypothesized that ATRAP deficiency might enhance Ang II–mediated hypertension by affecting renal sodium handling. To examine this, we performed metabolic cage analysis (Figure 4a–d). Day-by-day sodium intake was similar in WT and ATRAP-KO mice (Figure 4a, F=0.559, P=0.469, two-way repeated measures ANOVA), and urinary sodium excretion was comparable in WT and ATRAP-KO mice (Figure 4b, F=1.690, P=0.218, two-way repeated measures ANOVA). However, day-by-day sodium balance was significantly increased in ATRAP-KO mice compared with WT mice over the entire Ang II infusion period (Figure 4c, F=4.892, P=0.047, two-way repeated measures ANOVA). Furthermore, the extent of cumulative sodium balance during 14 days of Ang II infusion was significantly increased in ATRAP-KO mice as compared with WT mice (Figure 4d, P=0.047, unpaired t-test), consistent with blunted natriuresis as a mechanism for the exacerbation of Ang II-induced hypertension. To investigate the mechanisms involved in the suppression of urinary sodium excretion in response to Ang II in ATRAP-KO mice, we compared renal expression of major sodium transporters (NHE3, sodium–potassium–two chloride cotransporter (NKCC2), Na+–Cl- cotransporter (NCC), and ENaC subunits). Age-matched WT and ATRAP-KO mice were divided into four groups: (1) vehicle-infused WT mice, (2) Ang II–infused WT mice, (3) vehicle-infused ATRAP-KO mice, and (4) Ang II–infused ATRAP-KO mice. There were no differences in the expression levels of NHE3 or NKCC mRNA and protein between WT and ATRAP-KO mice with or without Ang II infusion for 14 days (Figures 5a and b and 6a and b). On the other hand, although Ang II–mediated increase in NCC mRNA was significant only in ATRAP-KO mice (Figure 5c), renal expression of phosphorylated NCC, which is the activated NCC protein, was similarly increased by Ang II infusion in both groups (Figure 6c).Figure 6Angiotensin II type 1 receptor (AT1R)–associated protein (ATRAP) deficiency enhances upregulation of renal α-subunit of the epithelial sodium channel (αENaC) protein expression by chronic angiotensin II (Ang II) infusion. Effects of Ang II (2000ng/kg/min) infusion for 14 days on protein expression of the major sodium transporters (a, sodium-proton antiporter 3 (NHE3); b, phosphorylated sodium–potassium–two chloride cotransporter (NKCC2) on Thr96; c, phosphorylated Na+–Cl- cotransporter (NCC) on Ser71; d, αENaC; e, βENaC; and f, γENaC) in the kidneys of wild-type (WT) and ATRAP-knockout (KO) mice. Values are expressed as mean±s.e. (n=6–8 in each group). *P<0.05 versus vehicle, **P<0.01 versus vehicle, †P<0.05 versus WT mice.View Large Image Figure ViewerDownload (PPT) However, Ang II–mediated upregulation of αENaC, but not of βENaC or γENaC mRNA, was significantly enhanced in ATRAP-KO mice compared with WT mice (Figure 5d–f). Furthermore, although αENaC protein expression did not differ between WT and ATRAP-KO mice with vehicle, there was a significant Ang II–mediated increase in renal αENaC protein expression in ATRAP-KO mice (P=0.007 relative to vehicle-infused ATRAP-KO mice, P=0.042 relative to Ang II–infused WT mice, ANOVA; Figure 6d). Protein levels of βENaC and γENaC were similar in WT and ATRAP-KO mice (Figure 6e and f). The promoting effect of Ang II infusion on expression of renal RAS components including AGT in proximal tubules has been reported.16.Kobori H. Harrison-Bernard L.M. Navar L.G. Expression of angiotensinogen mRNA and protein in angiotensin II-dependent hypertension.J Am Soc Nephrol. 2001; 12: 431-439Crossref PubMed Google Scholar,17.Gonzalez-Villalobos R.A. Seth D.M. Satou R. et al.Intrarenal angiotensin II and angiotensinogen augmentation in chronic angiotensin II-infused mice.Am J Physiol Renal Physiol. 2008; 295: F772-F779Crossref PubMed Scopus (85) Google Scholar Thus, to investigate the mechanisms involved in Ang II–induced renal αENaC expression in ATRAP-KO mice, renal AGT mRNA expression, urinary AGT excretion, and renal Ang II levels were analyzed. However, renal AGT mRNA expression and urinary AGT excretion were similar in WT and ATRAP-KO mice with vehicle, and exhibited no apparent changes by Ang II (Figure 7a and b). In addition, renal Ang II level was comparable in WT and ATRAP-KO mice with vehicle, and was significantly and similarly increased by Ang II in both WT and ATRAP-KO mice (Figure 7c). These results indicate that ATRAP deficiency exerts no apparent effect on proximal tubule AGT production or the renal Ang II levels. We next examined the plasma aldosterone concentration and urinary aldosterone excretion to investigate a possible role of aldosterone in the enhanced αENaC expression in response to Ang II. Under vehicle infusion, plasma aldosterone concentration (Figure 8a, 208.7±38.1 vs. 197.5±29.3pg/ml, P=1.000, ANOVA) and urinary aldosterone excretion (Figure 8b, 5.2±0.6 vs. 4.6±0.8ng/day, P=0.974, ANOVA) were comparable in WT and ATRAP-KO mice. Furthermore, Ang II infusion significantly and similarly increased plasma aldosterone concentration (Figure 8a, 1154.0±170.5 vs. 1464.5± 267.9pg/ml, P=0.401, ANOVA) and urinary aldosterone excretion (Figure 8b, 19.2±0.5 vs. 15.9±2.1ng/day, P=0126, ANOVA) in both WT and ATRAP-KO mice. We also administered exogenous aldosterone subcutaneously to WT and ATRAP-KO mice. Systolic BP measured by the tail-cuff method was similar in WT and ATRAP-KO mice by vehicle administration, and was similarly increased by chronic aldosterone infusion in both groups (Figure 9a). Furthermore, renal αENaC protein levels did not exhibit any significant difference between WT and ATRAP-KO mice with or without aldosterone (Figure 9b). These results would support the notion that ATRAP deficiency enhances renal αENaC expression in an aldosterone-independent manner. To examine whether ENaC activity was affected by the enhancement of renal αENaC expression in ATRAP-KO mice, diuretic test using a potent and specific ENaC inhibitor amiloride was performed, and the effects of ATRAP deficiency on the functional transport activity of ENaC were examined. Urinary sodium excretion and urinary volume after intraperitoneal injection of amiloride were significantly increased in the Ang II–infused ATRAP-KO as mice compared with the Ang II–infused WT mice (Figure 10a and b). This finding indicated that the ENaC is functionally activated in response to chronic Ang II simulation so as to increase renal sodium retention in ATRAP-KO mice. Although the renal αENaC protein expression in ATRAP-KO mice was significantly increased as compared with WT mice by chronic Ang II infusion for 14 days (Figure 6d), the αENaC protein expression levels just after the start of Ang II infusion (on day 1) did not differ significantly between WT and ATRAP-KO mice (Figure 11). On the other hand, telemetric BP showed a trend toward enhanced Ang II–induced BP elevation in ATRAP-KO mice, concomitant with a tendency of increase in day-by-day sodium balance in ATRAP-KO mice as compared with WT mice already during the early period of Ang II infusion (Figures 3 and 4). Based on the above observations, we examined the vasoconstrictor response of artery rings to Ang II to investigate whether there would be an enhancement of vascular response to Ang II as one of the contributing factors to the exaggerated BP elevation in the initial phase of Ang II infusion in ATRAP-KO mice. Histological analysis of aortic sections revealed a normal vascular structure in ATRAP-KO mice, in spite of no vascular ATRAP immunostaining, without any alteration in aortic medial thickness in ATRAP-KO mice (Figure 12a and b). However, Ang II provoked an exaggerated vasoconstrictor response of vascular rings of ATRAP-KO mice compared with WT mice upon vessel wire myograph analysis (Figure 12c, F=8.583, P=0.015, two-way repeated measures ANOVA). This suggests a possible involvement of increased vasoconstriction in the initial stage of the exacerbation of Ang II–mediated hypertension in ATRAP-KO mice. In this study it was demonstrated that systemic ATRAP deficiency does not exert any evident influence on baseline BP upon radiotelemetry or on renal structure and function. This is in sharp contrast to the reported phenotypic changes in the genetic deletion of other RAS components (that is, AGT, renin, and AT1R). These RAS-inactivated mice displayed significant decreases in baseline BP as well as pathological alterations in renal structure and function compared with the control mice.11.Tanimoto K. Sugiyama F. Goto Y. et al.Angiotensinogen-deficient mice with hypotension.J Biol Chem. 1994; 269: 31334-31337Abstract Full Text PDF PubMed Google Scholar,12.Niimura F. Labosky P.A. Kakuchi J. et al.Gene targeting in mice reveals a requirement for angiotensin in the development and maintenance of kidney morphology and growth factor regulation.J Clin Invest. 1995; 96: 2947-2954Crossref PubMed Scopus (319) Google Scholar,13.Kihara M. Umemura S. Sumida Y. et al.Genetic deficiency of angiotensinogen produces an impaired urine concentrating ability in mice.Kidney Int. 1998; 53: 548-555Abstract Full Text PDF PubMed Scopus (70) Google Scholar,14.Oliverio M.I. Kim H.S. Ito M. et al.Reduced growth, abnormal kidney structure, and type 2 (AT2) angiotensin receptor-mediated blood pressure regulation in mice lacking both AT1A and AT1B receptors for angiotensin II.Proc Natl Acad Sci USA. 1998; 95: 15496-15501Crossref PubMed Scopus (279) Google Scholar,15.Yanai K. Saito T. Kakinuma Y. et al.Renin-dependent cardiovascular functions and renin-independent blood-brain barrier functions revealed by renin-deficient mice.J Biol Chem. 2000; 275: 5-8Crossref PubMed Scopus (133) Google Scholar With respect to a possible effect of genetic alteration in ATRAP on BP regulation, a previous study examined phenotypic changes in systemic ATRAP gene-trap mice.18.Oppermann M. Gess B. Schweda F. et al.Atrap deficiency increases arterial blood pressure and plasma volume.J Am Soc Nephrol. 2010; 21: 468-477Crossref PubMed Scopus (47) Google Scholar These mice have a gene-trap cassette with splicing acceptor in ATRAP intron 4 and exhibit fusion of ATRAP exons 1 to 4 with β-galactosidase/neomycin resistance gene, with an elevated baseline BP concomitant with an increased plasma volume and lowered urinary pH.18.Oppermann M. Gess B. Schweda F. et al.Atrap deficiency increases arterial blood pressure and plasma volume.J Am Soc Nephrol. 2010; 21: 468-477Crossref PubMed Scopus (47) Google Scholar This previous study suggested an augmented baseline NHE3-dependent sodium reabsorption in proximal tubules of these mice, and the authors proposed a regulatory role for proximal tubule ATRAP in modulation of renal sodium handling and baseline BP.18.Oppermann M. Gess B. Schweda F. et al.Atrap deficiency increases arterial blood pressure and plasma volume.J Am Soc Nephrol. 2010; 21: 468-477Crossref PubMed Scopus (47) Google Scholar However, the phenotypic difference in these ATRAP gene-trap mice might be because of aberrant ATRAP-gal/neo protein production. In addition, the strain of ATRAP gene-trap and control mice used for comparison were not reported,18.Oppermann M. Gess B. Schweda F. et al.Atrap deficiency increases arterial blood pressure and plasma volume.J Am Soc Nephrol. 2010; 21: 468-477Crossref PubMed Scopus (47) Google Scholar thereby making it difficult to properly interpret their BP data. Furthermore, the previously reported systemic ATRAP transgenic mice (TGM) produced separately in both our laboratory and a distinct laboratory exhibited no baseline BP changes.19.Oshita A. Iwai M. Chen R. et al.Attenuation of inflammatory vascular remodeling by angiotensin II type 1 receptor-associated protein.Hypertension. 2006; 48: 671-676Crossref PubMed Scopus (45) Google Scholar,20.Wakui H. Tamura K. Matsuda M. et al.Intrarenal suppression of angiotensin II type 1 receptor binding molecule in angiotensin II-infused mice.Am J Physiol Renal Physiol. 2010; 299: F991-F1003Crossref PubMed Scopus (31) Google Scholar These findings support the results of the present study, showing an absence of BP change at baseline in ATRAP-KO mice. In contrast to the lack of any evident change in baseline BP in ATRAP-KO mice, Ang II–mediated hypertension was significantly exacerbated in ATRAP-KO mice concomitant with an increased sodium balance compared with WT mice. With respect to the mechanisms involved in Ang II–induced hypertension, previous studies using a series of kidney cross-transplant experiments showed that activation of the intrarenal Ang II–AT1R axis is critically important for both Ang II–dependent hypertension and end-organ damage.21.Coffman T.M. Crowley S.D. Kidney in hypertension: guyton redux.Hypertension. 2008; 51: 811-816Crossref PubMed Scopus (103) Google Scholar, 22.Crowley S.D. Gurley S.B. Herrera M.J. et al.Angiotensin II causes hypertension and cardiac hypertrophy through its receptors in the kidney.Proc Natl Acad Sci USA. 2006; 103: 17985-17990Crossref PubMed Scopus (523) Google Scholar, 23.Crowley S.D. Zhang J. Herrera M. et al.Role of AT receptor-mediated salt retention in angiotensin II-dependent hypertension.Am J Physiol Renal Physiol. 2011; 301: F1124-F1130Crossref PubMed Scopus (40) Google Scholar Sodium reabsorption through renal proximal and distal tubules is reportedly regulated by the activity of the intrarenal Ang II–AT1R axis.24.Kobori H. Nangaku M. Navar L.G. et al.The intrarenal renin-angiotensin system: from physiology to the pathobiology of hypertension and kidney disease.Pharmacol Rev. 2007; 59: 251-287Crossref PubMed Scopus (933) Google Scholar The proximal tubule AT1R directly affects BP regulation because a major portion of the glomerular filtrate is reabsorbed in proximal tubules, and proximal tubule sodium transport is considered a major determinant of the pressure natriuresis response.25.McDonough A.A. Leong P.K. Yang L.E. Mechanisms of pressure natriuresis: how blood pressure regulates renal sodium transport.Ann NY Acad Sci. 2003; 986: 669-677Crossref PubMed Scopus (64) Google Scholar The proximal tubule–specific AT1R-deficient mice exhibited a partial suppression of Ang II–induced BP elevation as well as a decrease in baseline BP compared with control mice, concomitant with Ang II–induced reduction in NHE3 expression.26.Gurley S.B. Riquier-Brison A.D. Schnermann J. et al.AT1A angiotensin receptors in the renal proximal tubule regulate blood pressure.Cell Metab. 2011; 13: 469-475Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar On the other hand, another type of proximal tubule–specific AT1R-deficient mouse exhibited a significant reduction in baseline BP, but without any evident inhibition of Ang II–induced hypertension in comparison with control mice.27.Li H. Weatherford E.T. Davis D.R. et al.Renal proximal tubule angiotensin AT1A receptors regulate blood pressure.Am J Physiol Regul Integr Comp Physiol. 2011; 301: R1067-R1077Crossref PubMed Scopus (65) Google Scholar These results indicate that activation of AT1R signaling in renal proximal tubules is unable to fully account for the positive effect of Ang II on BP increase. Regarding the role of distal tubules in renal sodium handling by the Ang II–AT1R axis, previous studies showed that distal nephron segments also have a crucial role in the regulation of sodium reabsorption via intrarenal RAS.28.Navar L.G. Kobori H. Prieto M.C. et al.Intratubular renin-angiotensin system in hypertension.Hypertension. 2011; 57: 355-362Crossref PubMed Scopus (170) Google Scholar,29.Prieto-Carrasquero M.C. Botros F.T. Kobori H. et al.Collecting duct renin: a major player in angiotensin II-dependent hypertension.J Am Soc Hypertens. 2009; 3: 96-104Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar The modulation of sodium reabsorption in response to Ang II is mediated by NCC and ENaC in the distal nephron.30.Castaneda-Bueno M. Cervantes-Perez L.G. Vazquez N. et al.Activation of the renal Na+:Cl- cotransporter by angiotensin II is a WNK4-dependent process.Proc Natl Acad Sci USA. 2012; 109: 7929-7934Crossref PubMed Scopus (194) Google Scholar, 31.Christensen B.M. Perrier R. Wang Q. et al.Sodium and potassium balance depends on alphaENaC expression in connecting tubule.J Am Soc Nephrol. 2010; 21: 1942-1951Crossref PubMed Scopus (85) Google Scholar, 32.Peti-Peterdi J. Warnock D.G. Bell P.D. 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Enhanced distal nephron sodium reabsorption in chronic angiotensin II-infused mice.Hypertension. 2009; 54: 120-126Crossref PubMed Scopus (67) Google Scholar These findings support the notion that the renal distal tubules also have a role in sodium reabsorption that takes place in response to Ang II. Recently, we generated re