The renin-angiotensin system (RAS) is undisputedly one of the most prominent endocrine (tissue-to-tissue), paracrine (cell-to-cell) and intracrine (intracellular/nuclear) vasoactive systems in the physiological regulation of neural, cardiovascular, blood pressure, and kidney function. The importance of the RAS in the development and pathogenesis of cardiovascular, hypertensive and kidney diseases has now been firmly established in clinical trials and practice using renin inhibitors, angiotensin-converting enzyme (ACE) inhibitors, type 1 (AT1) angiotensin II (ANG II) receptor blockers (ARBs), or aldosterone receptor antagonists as major therapeutic drugs. The major mechanisms of actions for these RAS inhibitors or receptor blockers are mediated primarily by blocking the detrimental effects of the classic angiotensinogen/renin/ACE/ANG II/AT1/aldosterone axis. However, the RAS has expanded from this classic axis to include several other complex biochemical and physiological axes, which are derived from the metabolism of this classic axis. Currently, at least five axes of the RAS have been described, with each having its key substrate, enzyme, effector peptide, receptor, and/or downstream signaling pathways. These include the classic angiotensinogen/renin/ACE/ANG II/AT1 receptor, the ANG II/APA/ANG III/AT2/NO/cGMP, the ANG I/ANG II/ACE2/ANG (1–7)/Mas receptor, the prorenin/renin/prorenin receptor (PRR or Atp6ap2)/MAP kinases ERK1/2/V-ATPase, and the ANG III/APN/ANG IV/IRAP/AT4 receptor axes. Since the roles and therapeutic implications of the classic angiotensinogen/renin/ACE/ANG II/AT1 receptor axis have been extensively reviewed, this article will focus primarily on reviewing the roles and therapeutic implications of the vasoprotective axes of the RAS in cardiovascular, hypertensive and kidney diseases.

We and others have previously shown that high levels of ANG II are accumulated in the rat kidney via a type 1 (AT(1)) receptor-mediated mechanism, but it is not known which AT(1) receptor is involved in this process in rodents. We tested the hypothesis that AT(1a) receptor-deficient mice (Agtr1a-/-) are unable to accumulate ANG II intracellularly in the kidney because of the absence of AT(1a) receptor-mediated endocytosis. Adult male wild-type (Agtr1a+/+), heterozygous (Agtr1a+/-), and Agtr1a-/- were treated with vehicle, ANG II (40 ng/min ip via osmotic minipump), or ANG II plus the AT(1) antagonist losartan (10 mg.kg(-1).day(-1) po) for 2 wk. In wild-type mice, ANG II induced hypertension (168 +/- 4 vs. 113 +/- 3 mmHg, P < 0.001), increased kidney-to-body weight ratio (P < 0.01), caused pressure natriuresis (P < 0.05), and elevated plasma and whole kidney ANG II levels (P < 0.001). Concurrent administration of ANG II with losartan attenuated these responses to ANG II. In contrast, Agtr1a-/- mice had lower basal systolic pressures (P < 0.001), smaller kidneys with much fewer AT(1b) receptors (P < 0.001), higher basal 24-h urinary sodium excretion (P < 0.01), as well as basal plasma and whole kidney ANG II levels (P < 0.01). However, intracellular ANG II levels in the kidney were lower in Agtr1a-/- mice. In Agtr1a-/- mice, ANG II slightly increased systolic pressure (P < 0.05) but had no effect on the kidney weight, urinary sodium excretion, and whole kidney ANG II levels. Losartan restored systolic pressure to basal levels and decreased whole kidney ANG II levels by approximately 20% (P < 0.05). These results demonstrate a predominant role of AT(1a) receptors in blood pressure regulation and in the renal responses to long-term ANG II administration, that AT(1b) receptors may play a limited role in blood pressure control and mediating intrarenal ANG II accumulation in the absence of AT(1a) receptors.

Although renin, the rate-limiting enzyme of the renin-angiotensin system (RAS), was first discovered by Robert Tigerstedt and Bergman more than a century ago, the research on the RAS still remains stronger than ever. The RAS, once considered to be an endocrine system, is now widely recognized as dual (circulating and local/tissue) or multiple hormonal systems (endocrine, paracrine and intracrine). In addition to the classical renin/angiotensin I-converting enzyme (ACE)/angiotensin II (Ang II)/Ang II receptor (AT1/AT2) axis, the prorenin/(Pro)renin receptor (PRR)/MAP kinase axis, the ACE2/Ang (1-7)/Mas receptor axis, and the Ang IV/AT4/insulin-regulated aminopeptidase (IRAP) axis have recently been discovered. Furthermore, the roles of the evolving RAS have been extended far beyond blood pressure control, aldosterone synthesis, and body fluid and electrolyte homeostasis. Indeed, novel actions and underlying signaling mechanisms for each member of the RAS in physiology and diseases are continuously uncovered. However, many challenges still remain in the RAS research field despite of more than one century's research effort. It is expected that the research on the expanded RAS will continue to play a prominent role in cardiovascular, renal and hypertension research. The purpose of this article is to review the progress recently being made in the RAS research, with special emphasis on the local RAS in the kidney and the newly discovered prorenin/PRR/MAP kinase axis, the ACE2/Ang (1-7)/Mas receptor axis, the Ang IV/AT4/IRAP axis, and intracrine/intracellular Ang II. The improved knowledge of the expanded RAS will help us better understand how the classical renin/ACE/Ang II/AT1 receptor axis, extracellular and/or intracellular origin, interacts with other novel RAS axes to regulate blood pressure and cardiovascular and kidney function in both physiological and diseased states.

Long-term angiotensin II (ANG II) administration is associated with increased ANG II accumulation in the kidney, but intrarenal compartment(s) involved in this response remains to be determined. We tested the hypothesis that 1) extracellular ANG II is taken up by proximal tubule cells (PTCs) through AT(1) receptor-mediated endocytosis, 2) this process is regulated by cytoskeleton microtubule- and tyrosine phosphatase-dependent mechanisms, and 3) AT(1) receptor-mediated endocytosis of ANG II has a functional relevance by modulating intracellular cAMP signaling. In cultured PTCs, [(125)I]Tyr-labeled ANG II and fluorescein labeled-ANG II were internalized in a time-dependent manner and colocalized with the endosome marker Alexa Fluor 594-transferrin. Endocytosis of extracellular ANG II was inhibited by the AT(1) receptor blocker losartan (16.5 +/- 4.6%, P < 0.01 vs. ANG II, 78.3 +/- 6.2%) and by the tyrosine phosphatase inhibitor phenylarsine oxide (PAO; 30.0 +/- 3.5%, P < 0.05 vs. ANG II). Intracellular ANG II levels were increased by approximately 58% (basal, 229.8 +/- 11.4 vs. ANG II, 361.3 +/- 11.8 pg ANG II/mg protein, P < 0.01), and the responses were blocked by losartan (P < 0.01), the cytoskeleton microtubule inhibitor colchicine (P < 0.05), and PAO (P < 0.01), whereas depletion of clathrin-coated pits with hyperosmotic sucrose had no effect (356.1 +/- 25.5 pg ANG II/mg protein, not significant). ANG II accumulation was associated with significant inhibition of both basal (control, 15.5 +/- 2.8 vs. ANG II, 9.1 +/- 2.4 pmol/mg protein, P < 0.05) and forskolin-stimulated cAMP signaling (forskolin, 68.7 +/- 8.6 vs. forskolin + ANG II, 42.8 +/- 13.8 pmol/mg protein, P < 0.01). These effects were blocked by losartan and PAO. We conclude that extracellular ANG II is internalized in PTCs through AT(1) receptor-mediated endocytosis and that internalized ANG II may play a functional role in proximal tubule cells by inhibiting intracellular cAMP signaling.

Intracellular ANG II induces biological effects in nonrenal cells, but it is not known whether it plays a physiological role in renal proximal tubule cells (PTCs). PTCs express angiotensinogen, renin, and angiotensin-converting enzyme mRNAs, suggesting the presence of high levels of intracellular ANG II. We determined if microinjection of ANG II directly in single PTCs increases intracellular calcium concentration ([Ca2+]i) and, if so, elucidated the cellular mechanisms involved. Changes in [Ca2+]i responses were studied by fluorescence imaging using the Ca2+ indicator fluo 3. ANG II (1 nM) was microinjected directly in the cells, whereas cell-surface angiotensin type 1 (AT1) receptors were blocked by losartan (10 microM). When ANG II (1 nM) was added to the perfusate, there was a marked increase in [Ca2+]i that was blocked by extracellular losartan. With losartan in the perfusate, intracellular microinjection of ANG II elicited a robust increase in cytoplasmic [Ca2+]i that peaked at 30 s (basal: 2.2 +/- 0.3 vs. ANG II: 14.9 +/- 0.4 relative fluorescence units; P < 0.01). Chelation of extracellular Ca2+ with EGTA (2 mM) did not alter microinjected ANG II-induced [Ca2+]i responses (Ca2+ free + ANG II: 12.3 +/- 2.6 relative fluorescence units, not significant vs. ANG II); however, pretreatment with thapsigargin to deplete intracellular Ca2+ stores or with U-73122 to inhibit phospholipase C (1 microM each) markedly attenuated microinjected ANG II-induced [Ca2+]i responses. Combined microinjection of ANG II and losartan abolished [Ca2+]i responses, whereas a combination of ANG II and PD-123319 had no effect. These data demonstrate for the first time that direct microinjection of ANG II in single PTCs increases [Ca2+]i by stimulating intracellular AT1 receptors and releases Ca2+ from intracellular stores, suggesting that intracellular ANG II may play a physiological role in PTC function.