Assessment of the Renin–Angiotensin System in Cellular Organelle: New Arenas for Study in the Mitochondria

  • Bryan A. Wilson
  • Mark C. ChappellEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1614)


The renin–angiotensin system (RAS) is an important hormonal system composed of various protein and peptide components that contribute to blood pressure regulation. Although originally characterized as a circulating system, there is increasing evidence for the intracellular expression of RAS elements on the nucleus and mitochondria that may function in concert with or independent of the circulating system. The present chapter describes several experimental approaches to quantify the expression of RAS components in isolated mitochondria from the kidney. These approaches are intended to provide a framework to understand the mitochondrial RAS within a cell-free environment.

Key words

Mitochondria Renin–angiotensin system Subcellular fractionation Peptide metabolism Ang II Ang-(1–7) Renin Mas protein Neprilysin Thimet oligopeptidase 



The authors gratefully acknowledge Nancy Pirro, Eric LeSaine, and Pamela Dean for technical and surgical support. During the completion of this work, B.A. Wilson was supported by an American Heart Association (AHA) predoctoral fellowship grant (15PRE25120007). Additional support for these studies was provided by National Institutes of Health Grants HD-047584, HD-017644, HD-084227, HL-51952, T32 grant (HL091797); the Groskert Heart Fund, the Wake Forest Venture Fund, and the Farley-Hudson Foundation (Jacksonville, NC).


  1. 1.
    Campbell DJ (2014) Clinical relevance of local renin angiotensin systems. Front Endocrinol (Lausanne) 5:113. doi: 10.3389/fendo.2014.00113 Google Scholar
  2. 2.
    Chappell MC (2016) Biochemical evaluation of the renin-angiotensin system: the good, bad, and absolute? Am J Physiol Heart Circ Physiol 310(2):H137–H152. doi: 10.1152/ajpheart.00618.2015 CrossRefPubMedGoogle Scholar
  3. 3.
    Santos RA (2014) Angiotensin-(1–7). Hypertension 63(6):1138–1147. doi: 10.1161/HYPERTENSIONAHA.113.01274 CrossRefPubMedGoogle Scholar
  4. 4.
    Abadir PM, Foster DB, Crow M, Cooke CA, Rucker JJ, Jain A, Smith BJ, Burks TN, Cohn RD, Fedarko NS, Carey RM, O'Rourke B, Walston JD (2011) Identification and characterization of a functional mitochondrial angiotensin system. Proc Natl Acad Sci U S A 108(36):14849–14854. doi: 10.1073/pnas.1101507108 CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Gwathmey TM, Shaltout HA, Pendergrass KD, Pirro NT, Figueroa JP, Rose JC, Diz DI, Chappell MC (2009) Nuclear angiotensin II type 2 (AT2) receptors are functionally linked to nitric oxide production. Am J Physiol Renal Physiol 296(6):F1484–F1493. doi: 10.1152/ajprenal.90766.2008 CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Pendergrass KD, Gwathmey TM, Michalek RD, Grayson JM, Chappell MC (2009) The angiotensin II-AT1 receptor stimulates reactive oxygen species within the cell nucleus. Biochem Biophys Res Commun 384(2):149–154. doi: 10.1016/j.bbrc.2009.04.126 CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Wilson BA, Nautiyal M, Gwathmey TM, Rose JC, Chappell MC (2015) Evidence for a mitochondrial angiotensin-(1–7) system in the kidney. Am J Physiol Renal Physiol 00479:02015. doi: 10.1152/ajprenal.00479.2015 Google Scholar
  8. 8.
    Abadir PM, Walston JD, Carey RM (2012) Subcellular characteristics of functional intracellular renin-angiotensin systems. Peptides 38(2):437–445. doi: 10.1016/j.peptides.2012.09.016 CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    De Mello WC (1998) Intracellular angiotensin II regulates the inward calcium current in cardiac myocytes. Hypertension 32(6):976–982CrossRefPubMedGoogle Scholar
  10. 10.
    Gwathmey TM, Pendergrass KD, Reid SD, Rose JC, Diz DI, Chappell MC (2010) Angiotensin-(1–7)-angiotensin-converting enzyme 2 attenuates reactive oxygen species formation to angiotensin II within the cell nucleus. Hypertension 55(1):166–171. doi: 10.1161/HYPERTENSIONAHA.109.141622 CrossRefPubMedGoogle Scholar
  11. 11.
    Gwathmey TM, Westwood BM, Pirro NT, Tang L, Rose JC, Diz DI, Chappell MC (2010) Nuclear angiotensin-(1–7) receptor is functionally coupled to the formation of nitric oxide. Am J Physiol Renal Physiol 299(5):F983–F990. doi: 10.1152/ajprenal.00371.2010 CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Kumar R, Thomas CM, Yong QC, Chen W, Baker KM (2012) The intracrine renin-angiotensin system. Clin Sci (Lond) 123(5):273–284. doi: 10.1042/CS20120089 CrossRefGoogle Scholar
  13. 13.
    Kurdi M, De Mello WC, Booz GW (2005) Working outside the system: an update on the unconventional behavior of the renin-angiotensin system components. Int J Biochem Cell Biol 37(7):1357–1367. doi: 10.1016/j.biocel.2005.01.012 CrossRefPubMedGoogle Scholar
  14. 14.
    Re RN, Cook JL (2011) Noncanonical intracrine action. J Am Soc Hypertens 5(6):435–448. doi: 10.1016/j.jash.2011.07.001 CrossRefPubMedGoogle Scholar
  15. 15.
    de Cavanagh EM, Inserra F, Ferder M, Ferder L (2007) From mitochondria to disease: role of the renin-angiotensin system. Am J Nephrol 27(6):545–553. doi: 10.1159/000107757 CrossRefPubMedGoogle Scholar
  16. 16.
    Haller H, Lindschau C, Erdmann B, Quass P, Luft FC (1996) Effects of intracellular angiotensin II in vascular smooth muscle cells. Circ Res 79(4):765–772CrossRefPubMedGoogle Scholar
  17. 17.
    Zhuo JL, Li XC, Garvin JL, Navar LG, Carretero OA (2006) Intracellular ANG II induces cytosolic Ca2+ mobilization by stimulating intracellular AT1 receptors in proximal tubule cells. Am J Physiol Renal Physiol 290(6):F1382–F1390. doi: 10.1152/ajprenal.00269.2005 CrossRefPubMedGoogle Scholar
  18. 18.
    Li XC, Zhuo JL (2008) Intracellular ANG II directly induces in vitro transcription of TGF-beta1, MCP-1, and NHE-3 mRNAs in isolated rat renal cortical nuclei via activation of nuclear AT1a receptors. Am J Physiol Cell Physiol 294(4):C1034–C1045. doi: 10.1152/ajpcell.00432.2007 CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    de Cavanagh EM, Piotrkowski B, Basso N, Stella I, Inserra F, Ferder L, Fraga CG (2003) Enalapril and losartan attenuate mitochondrial dysfunction in aged rats. FASEB J 17(9):1096–1098. doi: 10.1096/fj.02-0063fje PubMedGoogle Scholar
  20. 20.
    Nautiyal M, Katakam PV, Busija DW, Gallagher PE, Tallant EA, Chappell MC, Diz DI (2012) Differences in oxidative stress status and expression of MKP-1 in dorsal medulla of transgenic rats with altered brain renin-angiotensin system. Am J Physiol Regul Integr Comp Physiol 303(8):R799–R806. doi: 10.1152/ajpregu.00566.2011 CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    Rajapakse N, Shimizu K, Payne M, Busija D (2001) Isolation and characterization of intact mitochondria from neonatal rat brain. Brain Res Brain Res Protoc 8(3):176–183CrossRefPubMedGoogle Scholar
  22. 22.
    Taugner R, Buhrle CP, Nobiling R, Kirschke H (1985) Coexistence of renin and cathepsin B in epithelioid cell secretory granules. Histochemistry 83(2):103–108CrossRefPubMedGoogle Scholar
  23. 23.
    Clausmeyer S, Sturzebecher R, Peters J (1999) An alternative transcript of the rat renin gene can result in a truncated prorenin that is transported into adrenal mitochondria. Circ Res 84(3):337–344CrossRefPubMedGoogle Scholar
  24. 24.
    Wanka H, Kessler N, Ellmer J, Endlich N, Peters BS, Clausmeyer S, Peters J (2009) Cytosolic renin is targeted to mitochondria and induces apoptosis in H9c2 rat cardiomyoblasts. J Cell Mol Med 13(9A):2926–2937. doi: 10.1111/j.1582-4934.2008.00448.x CrossRefPubMedGoogle Scholar
  25. 25.
    Ishigami T, Kino T, Chen L, Minegishi S, Araki N, Umemura M, Abe K, Sasaki R, Yamana H, Umemura S (2014) Identification of bona fide alternative renin transcripts expressed along cortical tubules and potential roles in promoting insulin resistance in vivo without significant plasma renin activity elevation. Hypertension 64(1):125–133. doi: 10.1161/HYPERTENSIONAHA.114.03394 CrossRefPubMedGoogle Scholar
  26. 26.
    Takahashi S, Hori K, Ogasawara H, Hiwatashi K, Sugiyama T (2006) Effects of nucleotides on the interaction of renin with GlcNAc 2-epimerase (renin binding protein, RnBP). J Biochem 140(5):725–730. doi: 10.1093/jb/mvj201 CrossRefPubMedGoogle Scholar
  27. 27.
    Kim SM, Kim YG, Jeong KH, Lee SH, Lee TW, Ihm CG, Moon JY (2012) Angiotensin II-induced mitochondrial Nox4 is a major endogenous source of oxidative stress in kidney tubular cells. PLoS One 7(7):e39739. doi: 10.1371/journal.pone.0039739 CrossRefPubMedPubMedCentralGoogle Scholar
  28. 28.
    Shaltout HA, Westwood BM, Averill DB, Ferrario CM, Figueroa JP, Diz DI, Rose JC, Chappell MC (2007) Angiotensin metabolism in renal proximal tubules, urine, and serum of sheep: evidence for ACE2-dependent processing of angiotensin II. Am J Physiol Renal Physiol 292(1):F82–F91. doi: 10.1152/ajprenal.00139.2006 CrossRefPubMedGoogle Scholar
  29. 29.
    Astin R, Bentham R, Djafarzadeh S, Horscroft JA, Kuc RE, Leung PS, Skipworth JR, Vicencio JM, Davenport AP, Murray AJ, Takala J, Jakob SM, Montgomery H, Szabadkai G (2013) No evidence for a local renin-angiotensin system in liver mitochondria. Sci Rep 3:2467. doi: 10.1038/srep02467 CrossRefPubMedPubMedCentralGoogle Scholar
  30. 30.
    Campbell DJ, Nussberger J, Stowasser M, Danser AH, Morganti A, Frandsen E, Menard J (2009) Activity assays and immunoassays for plasma renin and prorenin: information provided and precautions necessary for accurate measurement. Clin Chem 55(5):867–877. doi: 10.1373/clinchem.2008.118000 CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  1. 1.McAllister Heart InstituteUniversity of North Caroliina Chapel HillWinston-SalemUSA
  2. 2.Hypertension and Vascular Research, Department of SurgeryCardiovascular Scineces CenterWinston-SalemUSA

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