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The complexity of the intrarenal renin-angiotensin system (RAS) continues to reveal itself as evidence accumulates demonstrating its robust independent regulation in the interstitial and intratubular compartments within the kidney1–3. Early reports demonstrating the presence of angiotensin II (Ang II) receptors on the brush border of proximal tubules suggested physiological roles4. However, because of the abundance of degradating enzymes on the brush border, the concentrations of angiotensin peptides were considered to be relatively low. Nevertheless, the abundance of luminal Ang II receptors throughout proximal and distal nephron segments sustained interest in its luminal actions5,6. Tubular perfusion studies indicating that luminal Ang II alters tubular sodium and volume reabsorption rate 1,5,7,8 supported an important physiological role for luminal Ang II receptors9.
A paradigm shift occurred when it was discovered that the proximal intratubular concentrations of Ang I and II were much greater than their corresponding plasma concentrations7,10,11. In addition, when proximal tubular fluid was incubated with excess renin, the resultant formation of Ang I indicated very high angiotensinogen (AGT) substrate availability in this segment7,12. Furthermore, tubular fluid collected from downstream segments of perfused tubules also had Ang II concentrations similar to those in non-perfused tubules thus supporting a local origin11. These findings, along with the demonstration that proximal tubule cells express AGT mRNA and protein13,14, established the foundation for the existence of a robust physiologically important tubular RAS.
The principal AT receptor in adult kidneys is the AT1 receptor5, although AT2 receptors are upregulated in certain conditions 15,16 and may also play a role in renin synthesis17. Nevertheless overall renal AT1 receptor abundance far exceeds AT2 receptor levels 5,18 and AT1 receptors are widely distributed on luminal membranes throughout the nephron segments including proximal tubule, thick ascending limb of Loop of Henle, macula densa, distal tubule and collecting ducts (CD)5,19. The regulation of intrarenal AT1 receptors is complex as vascular AT1 receptors are downregulated while tubular AT1 receptors are either sustained or upregulated by elevated Ang II levels1,6,20,21.
The presence of AT1 receptors on luminal membranes of various nephron segments generated interest in the Ang II concentrations available to activate the receptors7,22–24. Proximal tubule fluid concentrations of Ang I and Ang II are in the range of 5–10 pmol/ml 2,7 which are similar to renal interstitial fluid concentrations25. The tubular Ang II concentrations remain elevated in hypertension models including Ang II infused hypertension26, Goldblatt hypertension 27 and TGR(mRen2) rats 28 suggesting their sustained actions on proximal reabsorption rate. The critical importance of kidney AT1 receptors in the regulation of normal blood pressure and development of hypertension has been demonstrated by studies showing that AT1a receptors in the kidneys are essential for normal blood pressure regulation and for mediating the hypertensive response to Ang II infusions29,30. Furthermore, AT1a knockout mice fail to develop hypertension in response to unilateral renal arterial constriction31,32.
The tubular fluid Ang II concentrations in other nephron segments have not been measured due to difficulty in collecting sufficient fluid for analysis. Measurements made from urine samples collected under conditions where the major distal nephron transport systems were pharmacologically blocked, suggest CD concentrations in the range of 0.5 pmol/ml for control mice with about a two fold increase in Ang II infused hypertensive mice33. Increased urinary excretion rates of Ang II also occur in chronic Ang II infused rats 34,35 and these were decreased during treatment with AT1 receptor blockers even though the circulating Ang II concentrations were increased35. These recent studies indicate that distal nephron Ang II is formed locally in the tubules at concentrations that are sufficiently high to influence distal nephron transport function which has been shown to respond to Ang I and Ang II7,22,23. Distal nephron Ang II was recently shown to enhance the sensitivity of the “connecting tubule glomerular feedback mechanism” that communicates signals between the connecting tubule (CNT) and the afferent arteriole36. In contrast to the macula densa tubular glomerular feedback mechanism where Ang II augments its vasoconstriction capability5,37, the effect of Ang II on the CNT feedback mechanism is afferent vasodilatation36.
AT1 receptors are also responsible for internalizing Ang II and the presence of substantial Ang II in endosones in both control and Ang II infused hypertensive rats supports their internalization into a protected compartment that prevents degradation of some of the internalized Ang II20. AT1 receptor blockade prevents the internalization of the Ang II. Intracellular Ang II may activate various signaling pathways and also contribute to fibrogenic proliferative responses and microthrombosis38–40. Internalized Ang II may also migrate to the nucleus to exert transcriptional effects38,41. Ang II binding sites have been shown on nuclear membranes 41,42 and co-localization with nuclear markers suggests migration of the receptor complex to the nucleus38,43.
The seminal findings that AGT mRNA and protein are present in proximal tubule cells generated a great deal of interest regarding its intrarenal function13,44–46. Chronic Ang II infusions augment intrarenal AGT mRNA and protein in proximal tubule cells in rats and mice13,14,47,48. This effect is mediated via activation of AT1 receptors as it is prevented by treatment with ARBs48,49. Ang II also stimulates AGT production in proximal tubule cell cultures50. Thus, chronic infusions of Ang II in rats and mice lead to an augmentation of AGT expression leading to greater generation and intrarenal production of Ang II (Figure 1). Importantly, this process appears to be self limiting as higher Ang II infusions do not stimulate AGT mRNA 48 and complex signaling mechanisms are activated to prevent uncontrolled positive feedback51,52.
Because the level of AGT is close to the Michaelis-Menten constant for renin, AGT levels can also control RAS activity; thus, upregulation of AGT levels may lead to elevated angiotensin peptide levels53. Studies on rat and mouse models of hypertension have documented the effect of augmented AGT in the activation of the RAS54–58. Genetic manipulations that lead to overexpression of the AGT gene cause hypertension55,59. In human genetic studies, a linkage has been established between the AGT gene and hypertension60–63. Enhanced intrarenal AGT mRNA and/or protein levels occur in experimental models of hypertension and diabetes including Ang II-dependent hypertensive rats 13,14,47,49 and mice48,56,64, Dahl salt-sensitive hypertensive rats65, and spontaneously hypertensive rats66, as well as in kidney diseases including diabetic nephropathy67–69, IgA nephropathy70–72, and radiation nephropathy73. Thus, increased intrarenal AGT contributes to the development and progression of hypertension and may be useful as a predictor of developing kidney disease1,74. While clearly related to activation of AT1 receptors49, the mechanism by which Ang II stimulates AGT mRNA and protein is complex and appears to require interactions with inflammatory factors including interleukin 6, and increased oxidative stress75–77.
Urinary excretion rates of AGT provide an index of intratubular RAS status and are correlated with kidney Ang II levels in Ang II-dependent hypertensive rats78,79. Furthermore, mice overexpressing AGT only in proximal tubules have increased urinary Ang II excretion77. Because of its potential importance in identifying Ang II dependent hypertension in human subjects, direct quantitative methods to measure urinary AGT using human/mouse/rat AGT ELISA were recently developed80,81. Using this system, urinary excretion rates of AGT have been used as an index of intrarenal RAS status in patients with chronic kidney disease74,82,83, diabetes mellitus84,85, and hypertension86–88. In a cross-sectional study, we reported that urinary AGT levels are significantly greater in hypertensive patients not treated with RAS blockers compared with normotensive subjects (Figure 2). Moreover, patients treated with RAS blockers showed reduced urinary AGT levels87. In a population study, we showed that urinary AGT levels are correlated with high blood pressure in humans88. Urinary AGT levels were significantly correlated with systolic and diastolic blood pressures and high correlations between urinary AGT and blood pressure were shown in male subjects, especially in male African-American subjects88. These recent translational studies strengthen the hypothesis that intratubular AGT exerts a crucial role in the development and progression of hypertension and kidney disease. The augmentation of proximal tubule AGT leads to spillover into the distal nephron segments providing substrate for additional generation of Ang I and subsequent formation of Ang II (Figure 3).
Renin is also produced by the principal cells of CNT and cortical and medullary CD of mouse, rat, and human kidneys89–91. Renin co-localizes with aquaporin 291. In response to chronic Ang II infusions, renin mRNA and protein levels increase in CNT and CD91. This effect contrasts with the effect of Ang II to suppress JG renin92, but is also an AT1 receptor-mediated process93. As shown in Figure 4, the stimulation of CD renin during Ang II-dependent hypertension occurs independently of blood pressure since both non-clipped and clipped kidneys of Goldblatt hypertensive rats exhibit augmentation of renin synthesis and renin activity in the renal medulla, which is devoid of JG cells94. Thus, CD renin is increased by Ang II in association with increased AGT spillover from the proximal tubules95. In hypertensive models, the increased renin is primarily active renin 94 while in diabetic models, the increased CD renin is primarily (pro)renin90. There is also an enhancement of ACE and inhibition of ACE2 gene expression associated with decreases in intrarenal Ang 1–7 levels 96,97 suggesting that suppression of ACE2 activity contributes to augmentation of intrarenal Ang II.
The (pro)renin receptor, (P)RR, a 350-amino acid protein with a single transmembrane domain which binds renin or (pro)renin, increases the catalytic activity of renin and fully activates (pro)renin98. (P)RR activation also elicits intracellular signals via extracellular signal-regulated kinase (ERK)1 and ERK2 mitogen-activated protein (MAP) kinase. (P)RR has been localized in glomerular mesangial cells, subendothelium of renal arteries, podocytes, macula densa cells, distal tubules and collecting ducts98,99. (P)RR is predominantly expressed at the apex of the intercalated cells100. An example of this localization is depicted in Figure 5. Recent findings have also shown that the full length form of (P)RR can be processed intracellularly by cleavage leading to a soluble form (s(P)RR) that can be secreted into the plasma and consequently bind renin101. While the function of (P)RR or s(P)RR in hypertensive conditions has not been established102, (P)RR data from various models suggest its contribution to hypertension, diabetes and associated cardiovascular and renal diseases90,103,104. These observations are of relevance in light of CD renin upregulation in Ang II-dependent hypertensive rats91,93,94, and renin and/or (pro)renin secretion by CD cells89,90,94.
ACE is responsible for most conversion of Ang I to Ang II and is expressed on endothelial cells of the vasculature, on brush border of proximal tubule cells, glomeruli and distal nephron segments including inner medullary CD6,23,105–107. ACE knockout mice display very low arterial pressures coupled with an impaired capacity to generate Ang II, that is reflected as low levels of circulating and intrarenal Ang II, high levels of circulating Ang I108, and failure to show increases in blood pressure in response to Ang I infusions109.
As shown in Figure 6, mice treated chronically with an ACE inhibitor show markedly attenuated responses in arterial pressure and lower intrarenal Ang II levels with low dose infusions of Ang II htat elicit a slow pressor response64. Thus, endogenous ACE-derived Ang II formation contributes to the development of high local Ang II levels and hypertension induced by chronic Ang II infusions. To further determine the ability of kidney-specific ACE to augment intrarenal Ang II content and blood pressure, mice expressing ACE exclusively in the kidneys were infused chronically with Ang I110. Kidney specific ACE-derived Ang II formation increased Ang II content and led to the progressive development of hypertension, indicating that intrarenal ACE is a major contributor to the development of hypertension and increased intrarenal Ang II levels. Indeed, ACE expression is sustained or even augmented during Ang II-dependent hypertension 6,106 and other models of kidney injury111.
The results obtained to date indicate that increases in circulating or local Ang II concentrations elicit a positive augmentation of intrarenal AGT mRNA and protein leading to increased secretion of AGT into the tubular fluid. Together with the sustained or increased tubular ACE levels, the augmented AGT increases intratubular Ang II which further augments sodium transport via stimulation of AT1 receptors. The augmented AGT production and secretion increase AGT delivered to the distal nephron segments which can interact with renin and ACE produced by principal cells of CNT and CD cells to form more Ang II and stimulate distal transport activity. Nevertheless, in a pathophysiologic environment, inappropriate stimulation of the intratubular RAS may be an important contributor to the development and maintenance of hypertension and associated renal injury112. While this positive augmentation of intrarenal angiotensin by Ang II appears to be counter-intuitive to normal feedback regulation, the process is primarily a local amplification mechanism to increase intratubular Ang II thus effecting rapid homeostatic regulation of sodium reabsorption without equivalent increases in circulating Ang II. Furthermroe, there are “brakes” in the system as described earlier to prevent uncontrolled positive feedback51.
The authors thank Debbie Olavarrieta for preparing the manuscript and figures.
Sources of Funding:
Research support to the authors include grants from NIH (RO1HL-26371, RO1DK-072408, 2K99DK-083455 and P20RR-017659) and from the American Heart Association (09BGIA2280440 and 10GRNT3020018).
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