Angiotensin II causes hypertension and cardiac
hypertrophy through its receptors in the kidney
Steven D. Crowley*, Susan B. Gurley*, Maria J. Herrera*, Phillip Ruiz†, Robert Griffiths*, Anil P. Kumar*,
Hyung-Suk Kim‡, Oliver Smithies‡, Thu H. Le*, and Thomas M. Coffman*§
*Department of Medicine, Duke University Medical Center and Durham Veterans Affairs Medical Center, Durham, NC 27710;†Department of Pathology,
University of Miami, Miami, FL 33136; and‡Department of Pathology, University of North Carolina, Chapel Hill, NC 27599
Edited by Richard P. Lifton, Yale University School of Medicine, New Haven, CT, and approved September 27, 2006 (received for review July 3, 2006)
Essential hypertension is a common disease, yet its pathogenesis is
not well understood. Altered control of sodium excretion in the
kidney may be a key causative feature, but this has been difficult
to test experimentally, and recent studies have challenged this
hypothesis. Based on the critical role of the renin-angiotensin
system (RAS) and the type I (AT1) angiotensin receptor in essential
hypertension, we developed an experimental model to separate
AT1 receptor pools in the kidney from those in all other tissues.
Although actions of the RAS in a variety of target organs have the
potential to promote high blood pressure and end-organ damage,
we show here that angiotensin II causes hypertension primarily
through effects on AT1receptors in the kidney. We find that renal
AT1 receptors are absolutely required for the development of
angiotensin II-dependent hypertension and cardiac hypertrophy.
When AT1receptors are eliminated from the kidney, the residual
repertoire of systemic, extrarenal AT1receptors is not sufficient to
induce hypertension or cardiac hypertrophy. Our findings demon-
strate the critical role of the kidney in the pathogenesis of hyper-
tension and its cardiovascular complications. Further, they suggest
that the major mechanism of action of RAS inhibitors in hyperten-
sion is attenuation of angiotensin II effects in the kidney.
transgenic mice ? kidney transplantation ? blood pressure
disease) are a major public health problem (1). Despite decades of
scrutiny, the precise pathogenesis of essential hypertension has
defective handling of sodium by the kidney and consequent dys-
regulation of body fluid volumes is a requisite, final common
studies of Lifton and associates showing that virtually all of the
Mendelian disorders with major impact on blood pressure ho-
meostasis are caused by genetic variants affecting salt and water
reabsorption by the distal nephron (3). On the other hand, several
recent studies have suggested that primary vascular defects may
cause hypertension by impacting peripheral resistance without
direct involvement of renal excretory functions (4–7).
Among the various regulatory systems that impact blood pres-
sure, the RAS has a key role. Inappropriate activation of the RAS,
as in renal artery stenosis, leads to profound hypertension and
cardiovascular morbidity (8). Moreover, in patients with essential
inhibitors and angiotensin receptor blockers (ARBs) effectively
(9–11), suggesting that dysregulation of the RAS contributes to
their elevated blood pressure.
At the cellular level, responsiveness to angiotensin II (Ang
II) is conferred by the expression of the two classes of
angiotensin receptors (AT1and AT2). The effects of Ang II to
increase blood pressure are mediated by AT1receptors (12),
and these receptors are expressed in a variety of organ systems
thought to play key roles in blood pressure homeostasis,
igh blood pressure (BP) is a highly prevalent disorder, and its
complications (including heart disease, stroke, and kidney
including the heart, kidney, blood vessels, adrenal glands, and
cardiovascular control centers in the brain (13). For example,
in the vascular system, stimulation of AT1 receptors causes
potent vasoconstriction (14, 15). In the adrenal cortex, their
activation stimulates the release of aldosterone (16) that in
turn promotes sodium reabsorption in the mineralocorticoid-
responsive segments of the distal nephron (17). In the brain,
intraventricular injection of Ang II causes a dramatic pressor
response that is mediated by AT1A receptors (18). In the
kidney, activation of AT1 receptors is associated with renal
vasoconstriction and antinatriuresis (19, 20). Nevertheless,
whether angiotensin actions in these individual tissue sites
contribute in vivo to the pathogenesis of hypertension and its
complications is not clear.
To address this question, we used a kidney cross-transplantation
strategy to separate the actions of AT1receptor pools in the kidney
from those in systemic tissues. Our findings suggest that AT1
receptors expressed in the kidney are the primary determinants
of hypertension and end-organ damage in Ang II-dependent
Kidney Cross-Transplantation Model. We used a kidney cross-
transplantation strategy to separate the actions of AT1receptor
pools in the kidney from those in systemic tissues, as we have
described previously (21). Kidney transplantation was carried out
between genetically matched F1(C57BL?6 ? 129) wild-type mice
and F1(C57BL?6 ? 129) mice homozygous for a targeted disrup-
tion of the Agtr1a gene locus encoding the AT1Areceptor (14). The
AT1Areceptor is the major AT1receptor isoform in the mouse and
generated four groups of animals in which renal function was
provided entirely by the single transplanted kidney. The Wild-type
group consisted of wild-type mice transplanted with kidneys from
wild-type donors and thus have normal expression of AT1Arecep-
tors in the kidney transplant and in all systemic tissues. For the
Systemic KO group, AT1Areceptor-deficient recipients were trans-
planted with kidneys from wild-type donors; these animals lack
AT1Areceptors in all tissues except the kidney. Kidney KO animals
are wild-type recipients of AT1Areceptor-deficient kidneys, thus
lacking expression of AT1Areceptors only in the kidney, but with
normal expression of receptors in all systemic, nonrenal tissues,
including the adrenal gland. Finally, the Total KO group consists of
AT1Areceptor-deficient recipients of AT1Areceptor-deficient kid-
neys and therefore completely lacking AT1Areceptors in all tissues.
Author contributions: S.D.C., O.S., T.H.L., and T.M.C. designed research; S.D.C., S.B.G.,
M.J.H., P.R., R.G., A.P.K., and H.-S.K. performed research; S.D.C., S.B.G., P.R., T.H.L., and
T.M.C. analyzed data; and S.D.C., O.S., T.H.L., and T.M.C. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS direct submission.
§To whom correspondence should be addressed at: Duke University Medical Center,
Box 3014, Durham, NC 27710. E-mail: firstname.lastname@example.org.
© 2006 by The National Academy of Sciences of the USA
November 21, 2006 ?
vol. 103 ?
no. 47 ?
ing the mouse blood pressure telemetry device (TA11PA-C20), are
described in ref. 62. Six to 8 days after the kidney transplantation
procedure, the pressure catheter was implanted in the left carotid
artery as described in ref. 63.
Telemetric Blood Pressure Analysis. Data were collected, stored, and
analyzed by using Dataquest A.R.T. software (Transoma Medical).
Blood pressures were measured on unanesthetized, unrestrained
animals beginning 7 days after the catheter implantation when the
mice had reestablished normal circadian rhythms (63). Telemetry
Experimental Protocol. Baselinebloodpressuremeasurementswere
determined on 3 consecutive days while the animals ingested a
normal diet containing 0.4% sodium chloride. After these baseline
recordings, an osmotic minipump (Alzet Model 2004; DURECT)
was implanted s.c. as described in ref. 23, and blood pressure
measurements continued for 21 days.
Metabolic Balance Studies. One week after transplantation, the
mice were fed 10 gm?day gelled 0.25% NaCl diet that contained all
nutrients and water (Nutra-gel; Bio-Serv, Frenchtown, NJ). After
1 week of baseline collections, the animals were implanted with
osmotic minipumps infusing Ang II as described above and were
returned to the metabolic cage for 5 more days. Urinary sodium
content was determined by using an IL943 Automatic Flame
photometer per the manufacturer’s instructions (Instrumentation
Laboratory, Lexington, MA).
Histopathologic Analysis. After 28 days of Ang II infusion, hearts
were harvested, weighed, fixed in formalin, sectioned, and stained
with Masson trichrome. All of the tissues were examined by a
pathologist (P.R.) without knowledge of genotypes. Pathology was
graded based on the presence and severity of component abnor-
malities, including cellular infiltrate, myocardial cell injury, vessel
wall thickening, and fibrosis. Grading for each component was
performed by using a semiquantitative scale where 0 was normal
cardiac injury score for each heart was a summation of the
component injury scores.
Quantification of Cardiac mRNA Expression. Hearts were harvested,
and total RNA was isolated by using an RNeasy mini kit per the
manufacturer’s instructions (Qiagen, Valencia, CA). The gene
expression levels of ANP, BNP, ?-MHC, and ?-MHC in cardiac
tissue were determined by real-time quantitative RT-PCR as
reported in ref. 64.
Statistical Analysis. The values for each parameter within a group
are expressed as the mean ? SEM. For comparisons between
groups with normally distributed data, statistical significance was
assessed by using ANOVA followed by an unpaired t test; within
groups, a paired t test was used. For nonparametric comparisons,
the Mann–Whitney U test was used between groups, and the
Wilcoxon signed rank test was used within groups.
We acknowledge outstanding administrative support from Ms. Norma
Barrow and technical support from Mr. Chris Best. This work was
supported by National Institutes of Health Grants HL49277 and
HL56122 and by funding from the Medical Research Service of the
Department of Veterans Affairs.
1. Chobanian AV, Bakris GL, Black HR, Cushman WC, Green LA, Izzo JL, Jr, Jones DW,
Materson BJ, Oparil S, Wright JT, Jr, et al. (2003) Hypertension 42:1206–1252.
2. Guyton AC (1991) Science 252:1813–1816.
3. Lifton RP, Gharavi AG, Geller DS (2001) Cell 104:545–556.
4. Brenner R, Perez GJ, Bonev AD, Eckman DM, Kosek JC, Wiler SW, Patterson AJ, Nelson
MT, Aldrich RW (2000) Nature 407:870–876.
5. Zhu Y, Bian Z, Lu P, Karas RH, Bao L, Cox D, Hodgin J, Shaul PW, Thoren P, Smithies
O, et al. (2002) Science 295:505–508.
6. Huang PL, Huang Z, Mashimo H, Bloch KD, Moskowitz MA, Bevan JA, Fishman MC
(1995) Nature 377:239–242.
7. Tang MK, Wang GR, Lu P, Karas RH, Aronovitz M, Heximer SP, Kaltenbronn KM, Blumer
KJ, Siderovski DP, Zhu Y, et al. (2003) Nat Med 9:1506–1512.
8. Lonn EM, Yusuf S, Jha P, Montague TJ, Teo KK, Benedict CR, Pitt B (1994) Circulation
B, de Faire U, Morlin C, et al. (1999) Lancet 353:611–616.
10. Dahlof B, Devereux RB, Kjeldsen SE, Julius S, Beevers G, de Faire U, Fyhrquist F, Ibsen
H, Kristiansson K, Lederballe-Pedersen O, et al. (2002) Lancet 359:995–1003.
12. Crowley SD, Tharaux PL, Audoly LP, Coffman TM (2004) Acta Physiol Scand 181:561–570.
13. Shanmugam S, Sandberg K (1996) Cell Biol Int 20:169–176.
14. Ito M, Oliverio MI, Mannon PJ, Best CF, Maeda N, Smithies O, Coffman TM (1995) Proc
Natl Acad Sci USA 92:3521–3525.
15. Oliverio MI, Best CF, Kim HS, Arendshorst WJ, Smithies O, Coffman TM (1997) Am J
16. Aguilera G (1992) Mol Cell Endocrinol 90:53–60.
17. Masilamani S, Kim GH, Mitchell C, Wade JB, Knepper MA (1999) J Clin Invest
18. Davisson RL, Oliverio MI, Coffman TM, Sigmund CD (2000) J Clin Invest 106:103–106.
19. Ichikawa I, Brenner BM (1980) J Clin Invest 65:1192–1201.
20. Navar LG, Carmines PK, Huang WC, Mitchell KD (1987) Kidney Int Suppl 20:S81–S88.
21. Crowley SD, Gurley SB, Oliverio MI, Pazmino AK, Griffiths R, Flannery PJ, Spurney RF,
Kim HS, Smithies O, Le TH, et al. (2005) J Clin Invest 115:1092–1099.
22. Zhan Y, Brown C, Maynard E, Anshelevich A, Ni W, Ho IC, Oettgen P (2005) J Clin Invest
24. Vecchione C, Patrucco E, Marino G, Barberis L, Poulet R, Aretini A, Maffei A, Gentile
MT, Storto M, Azzolino O, et al. (2005) J Exp Med 201:1217–1228.
25. Oliverio MI, Best CF, Smithies O, Coffman TM (2000) Hypertension 35:550–554.
26. Koren MJ, Devereux RB, Casale PN, Savage DD, Laragh JH (1991) Ann Intern Med
27. Levy D, Garrison RJ, Savage DD, Kannel WB, Castelli WP (1990) N Engl J Med
28. Schmieder RE, Martus P, Klingbeil A (1996) J Am Med Assoc 275:1507–1513.
29. Mathew J, Sleight P, Lonn E, Johnstone D, Pogue J, Yi Q, Bosch J, Sussex B, Probstfield
J, Yusuf S (2001) Circulation 104:1615–1621.
30. Devereux RB, Dahlof B, Gerdts E, Boman K, Nieminen MS, Papademetriou V, Rokkedal
J, Harris KE, Edelman JM, Wachtell K (2004) Circulation 110:1456–1462.
31. Lee RT, Bloch KD, Pfeffer JM, Pfeffer MA, Neer EJ, Seidman CE (1988) J Clin Invest
32. Nakagawa O, Ogawa Y, Itoh H, Suga S, Komatsu Y, Kishimoto I, Nishino K, Yoshimasa
T, Nakao K (1995) J Clin Invest 96:1280–1287.
33. Jones WK, Grupp IL, Doetschman T, Grupp G, Osinska H, Hewett TE, Boivin G, Gulick
J, Ng WA, Robbins J (1996) J Clin Invest 98:1906–1917.
34. D’Angelo DD, Sakata Y, Lorenz JN, Boivin GP, Walsh RA, Liggett SB, Dorn GW, II (1997)
Proc Natl Acad Sci USA 94:8121–8126.
35. Sadoshima J, Izumo S (1993) Circ Res 73:413–423.
36. Bendall JK, Cave AC, Heymes C, Gall N, Shah AM (2002) Circulation 105:293–296.
37. Brunner HR, Laragh JH, Baer L, Newton MA, Goodwin FT, Krakoff LR, Bard RH, Buhler
FR (1972) N Engl J Med 286:441–449.
38. Gavras H, Lever AF, Brown JJ, Macadam RF, Robertson JI (1971) Lancet 2:19–22.
39. Brunner HR (2001) Am J Cardiol 87:3C–9C.
40. Mazzolai L, Pedrazzini T, Nicoud F, Gabbiani G, Brunner HR, Nussberger J (2000)
41. Gasc JM, Shanmugam S, Sibony M, Corvol P (1994) Hypertension 24:531–537.
43. Schuster VL, Kokko JP, Jacobson HR (1984) J Clin Invest 73:507–515.
44. Geibel J, Giebisch G, Boron WF (1990) Proc Natl Acad Sci USA 87:7917–7920.
45. Kwon TH, Nielsen J, Kim YH, Knepper MA, Frokiaer J, Nielsen S (2003) Am J Physiol
46. Wang T, Giebisch G (1996) Am J Physiol 271:F143–F149.
47. Barreto-Chaves ML, Mello-Aires M (1996) Am J Physiol 271:F977–F984.
48. Tojo A, Tisher CC, Madsen KM (1994) Am J Physiol 267:F1045–F1051.
49. Peti-Peterdi J, Warnock DG, Bell PD (2002) J Am Soc Nephrol 13:1131–1135.
50. Arendshorst WJ, Brannstrom K, Ruan X (1999) J Am Soc Nephrol 10(Suppl 11):S149–S161.
52. Hall JE (1986) Am J Physiol 250:R960–R972.
53. Faubert PF, Chou SY, Porush JG (1987) Kidney Int 32:472–478.
54. Sadoshima J, Xu Y, Slayter HS, Izumo S (1993) Cell 75:977–984.
55. Schunkert H, Sadoshima J, Cornelius T, Kagaya Y, Weinberg EO, Izumo S, Riegger G,
Lorell BH (1995) Circ Res 76:489–497.
56. Svensson P, de Faire U, Sleight P, Yusuf S, Ostergren J (2001) Hypertension 38:E28–E32.
57. Nataraj C, Oliverio MI, Mannon RB, Mannon PJ, Audoly LP, Amuchastegui CS, Ruiz P,
Smithies O, Coffman TM (1999) J Clin Invest 104:1693–1701.
58. Brilla CG, Pick R, Tan LB, Janicki JS, Weber KT (1990) Circ Res 67:1355–1364.
59. Sun Y, Ramires FJ, Weber KT (1997) Cardiovasc Res 35:138–147.
60. Zhai P, Yamamoto M, Galeotti J, Liu J, Masurekar M, Thaisz J, Irie K, Holle E, Yu X,
Kupershmidt S, et al. (2005) J Clin Invest 115:3045–3056.
61. Luft FC (2001) Hypertension 37:594–598.
62. Mills PA, Huetteman DA, Brockway BP, Zwiers LM, Gelsema AJ, Schwartz RS, Kramer
K (2000) J Appl Physiol 88:1537–1544.
63. Butz GM, Davisson RL (2001) Physiol Genomics 5:89–97.
64. Kim H-S, Lee G, John SWM, Maeda N, Smithies O (2002) Proc Natl Acad Sci USA
www.pnas.org?cgi?doi?10.1073?pnas.0605545103Crowley et al.