Role of stimulated intrarenal angiotensinogen in hypertension

Activation of AT1 receptors by elevated intrarenal Ang II levels in Ang II dependent hypertension

Ang II exerts its pathophysiological actions by binding to two major receptors, type 1 (AT1) and type 2 (AT2) angiotensin receptors.  The cardinal role of AT1 receptors in mediating Ang II dependent hypertension has been demonstrated in various studies. Experiments using AT1a receptor knockout mice demonstrated that unilateral renal arterial clipping failed to elicit any increases in systemic arterial pressure in the absence of AT1a receptors in contrast to the robust responses seen in wild type mice [Cervenka et al. 2002, 2008]. In addition, the magnitude of the hypertension was not altered by treatment with AT2 receptor blockers or agonists. Similarly, the hypertension elicited with chronic Ang II infusions is also completely prevented or reversed by treatment with AT1 receptor antagonists [Zou et al. 1996b, 1998].  The AT1 receptors in the kidney appear to be particularly important in maintaining the hypertension caused by chronic Ang II infusions [Crowley et al. 2006]. This study used a kidney cross-transplantation strategy to show that when AT1 receptors are eliminated from the kidneys, the pressor and cardiovascular responses to the chronic Ang II infusions were markedly attenuated.

Ang II activates both AT1 and AT2 receptors. However, the hypertensinogenic effects of Ang II are mediated primarily by the AT1 receptor which is expressed in almost all cell types within the kidneys [Harrison-Bernard et al. 1997; Carey, 2005]. The AT1 receptor is a member of the G protein coupled receptor family that physically associates with Gq/11, Gi, G12 or G13 [Higuchi et al. 2007]. As shown in Figure 1, once Ang II binds to AT1 receptor, various second messengers are activated via G protein dependent pathways, resulting in the induction of vasoconstriction, generation of reactive oxygen species, changes in gene transcription and the induction of cell growth and migration [Touyz and Berry, 2002; Higuchi et al. 2007]. In the G protein dependent AT1 receptor signaling pathways, phospholipase C, A2 and D have been identified as initial mediators [Ushio-Fukai et al. 1999]. The activated phospholipases stimulate other signal transducers, including activation of Rho kinase in vascular smooth muscle cells [Higuchi et al. 2007; Mehta and Griendling, 2007; Miyata et al. 2014], leading to vasoconstriction. AT1 receptor activation also leads to substantial internalization of the ligand–receptor complex, leading to increases in intracellular Ang II [Zou et al. 1996a, 1998; Zhuo et al. 2002]. Studies using infusions of Val⁵-Ang II have shown that approximately half of the increases in intrarenal Ang II levels are due to AT1 receptor mediated uptake of circulating Ang II [Shao et al. 2009].

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Figure 1.

Proposed mechanism underlying vasoconstriction by Ang II and augmentation of proximal tubular AGT expression by Ang II and elevated cytokines during the development of hypertension.

In the kidneys, AT1 receptor expression has been detected in microdissected glomeruli, proximal convoluted and straight tubules, medullary thick ascending limbs, medullary collecting ducts, cortical collecting ducts, vasa recta bundles and arcuate arteries as well as in vascular smooth muscle cells of afferent and efferent arterioles [Harrison-Bernard et al. 1997; Navar and Harrison-Bernard, 2000]. In particular, proximal tubules exhibit high levels of AT1 receptor expression [Burns et al. 1993; Bouby et al. 1997]. Activation of luminal AT1 receptors stimulates the sodium hydrogen exchanger and increases reabsorption rate [Navar et al. 2000]. Furthermore, AT1 receptor blockade causes significant increases in glomerular filtration rate, renal blood flow and proportionately much greater increases in sodium excretion and fractional sodium excretion. Ang II blockade also leads to marked increases in the slope of the pressure natriuresis relationship [Navar et al. 2000]. These findings demonstrate the important actions of activated intrarenal AT1 receptor in regulation of renal function, electrolyte and body fluid homeostasis and blood pressure. The role of activated intrarenal AT1 receptors in the development of hypertension has also been demonstrated using kidney-specific AT1 receptor knockout mice. Chronic Ang II infusions increased blood pressure only in the early stages, and the blood pressure was subsequently decreased and sustained within normotensive levels [Crowley et al. 2006].  These results indicate that elevated Ang II contributes to blood pressure elevation by activation of systemic AT1 receptors with consequent vasoconstriction in the early stages but the activated intrarenal AT1 receptor maintains the high blood pressure by altering renal function and further stimulation of prohypertensive mechanisms.

Enhancement of intrarenal AGT in Ang II dependent hypertension

AGT expression is increased by Ang II treatments and accompanied by activation of NF-κB in both liver tissue and cultured hepatocytes [Brasier and Li, 1996; Li and Brasier, 1996], leading to augmentation of circulating AGT levels. Intrarenal AGT levels are also increased in Ang II infused rats and mice [Schunkert et al. 1992; Kobori et al. 2001; Gonzalez-Villalobos et al. 2008] and in human renin/human AGT double transgenic mice producing extra Ang II only in kidneys [Kobori et al. 2007b]. The intrarenal Ang II-AGT amplification mechanism is a pivotal mechanism that facilitates the progression of hypertension. The intrarenal AGT augmentation is attenuated by treatment with AT1 receptor blockers in patients with hypertension and experimental models [Kobori et al. 2004, 2009]. However, because physiological stimulation of the RAS in response to low-sodium diets does not cause an upregulation of intrarenal AGT expression even though intrarenal Ang II levels are quite high [Shao et al. 2013], additional factors are necessary to fully stimulate intrarenal AGT expression in pathological conditions.

During the chronic Ang II infusions, macrophage and T-cell infiltration is enhanced in the kidneys [Ozawa et al. 2007]. Administration of mycophenolate mofetil, an immunosuppressive drug, attenuates intrarenal Ang II elevation and mitigates the development of hypertension and renal injury [Bravo et al. 2007] accompanied by suppression of the immune cell infiltration in the kidney [Crowley et al. 2008]. The accumulation of immune cells in the kidneys contributes to intrarenal RAS activation and the development of hypertension and kidney injury. Activated immune cells produce many types of proinflammatory cytokines.  The RAS and proinflammatory cytokines synergize to cause hypertension [Lee et al. 2006], and there is substantial evidence of interactions between these factors. IL-6 production is strongly stimulated in activated macrophages and T cells (Th2 cells) [Recinos et al. 2007]. Indeed, IL-6 has been identified as the most abundantly expressed cytokine in isolated aorta and it exhibits the most augmented expression in response to Ang II, indicating that IL-6 is induced locally at increased levels in Ang II infused mice [Recinos et al. 2007]. Further, IL-6 levels are elevated in the kidneys of Ang II infused animals [Ruiz-Ortega et al. 2002].

As shown in Figure 1, IL-6 and the IL-6-activated JAK-STAT pathway are required for Ang II induced AGT upregulation in renal PTC [Satou et al. 2008].  This can explain previous findings that deletion of IL-6 gene mitigates increases in blood pressure and renal injury in Ang II infused animals [Lee et al. 2006; Zhang et al. 2012] and that AT1 receptor blockade attenuates intrarenal AGT augmentation accompanied by decreases in IL-6 levels in patients with diabetes mellitus [Ogawa et al. 2009]. Enhanced interferon γ (IFN-γ) formation in activated T cells, natural killer cells and macrophages during the development of Ang II associated hypertension has also been demonstrated [Crowley et al. 2008; Saha et al. 2010]. In addition to the stimulating effect of IL-6 on proximal tubular AGT expression, IFN-γ increases AGT expression in PTC [Satou et al. 2012] as well as in hepatocytes [Jain et al. 2006].  These findings suggest that AGT synthesis in proximal tubules is stimulated via Ang II induced proinflammatory factors, particularly IL-6 and IFN-γ, derived from immune cells, which may be primary mechanisms underlying elevated intrarenal AGT levels during Ang II dependent hypertension [Satou and Gonzalez-Villalobos, 2012]. Thus, future studies will be needed to elucidate the cellular mechanisms underlying the complex interactions leading to proximal tubular AGT augmentation.

AGT-mediated stimulation of endogenous intrarenal Ang II formation in Ang II dependent hypertension

Under normal conditions, AGT and Ang II concentrations in proximal tubules are greater than can be explained from the plasma concentrations [Navar et al. 2002].  Thus, it has been proposed that the high levels of proximal tubular AGT contribute to establishment of the high concentration of tubular Ang II [Navar et al. 2002]. In Ang II dependent hypertension, elevated Ang II stimulates intrarenal AGT expression as described in the section above. In vivo experiments using chronic Val⁵-Ang II infusions have shown that there is increased formation of endogenous Ileu⁵-Ang II which is the native form in rodents in chronic Ang II infused rats [Shao et al. 2010]. Chronic Val⁵-Ang II infusion increased intrarenal and urinary endogenous Ile⁵-Ang II levels, although endogenous Ile⁵-Ang II levels in plasma were lower in the Val⁵-Ang II infusion rats. These results indicate activation of intrarenal RAS leading to further Ang II production independent of systemic RAS activity in Ang II dependent hypertension. Furthermore, animal models with proximal tubule-specific AGT overexpression exhibited increased intratubular Ang II levels and the development of hypertension [Sachetelli et al. 2006; Ying et al. 2012]. Accordingly, upregulation of proximal tubular AGT expression and secretion by elevated Ang II is of major importance in facilitating intrarenal Ang II production and consequent increases in blood pressure.

Importance of intrarenal angiotensin-converting enzyme in the augmentation of intrarenal Ang II in Ang II dependent hypertension

Chronic Ang II infusion augments angiotensin-converting enzyme (ACE) levels in renal proximal tubules [Harrison-Bernard et al. 2002].  This finding is consistent with increased kidney ACE activity reported in 2K1C and chronic Ang II infused animals [Von Thun et al. 1994; Prieto et al. 2011]. Therefore, intrarenal ACE is also a key component dictating intrarenal RAS activity as well as proximal tubular AGT, renal tubular and arteriolar AT1 receptor, and collecting duct renin [Navar et al. 2011]. Furthermore, systemic administration of an ACE inhibitor ameliorates the increases in blood pressure and intrarenal Ang II content caused by Ang II infusions [Gonzalez-Villalobos et al. 2009]. A study targeting intrarenal ACE-specific function revealed that chronic Ang I infusion into mice expressing ACE only in kidney resulted in elevation of intrarenal Ang II levels with the development of slowly progressive increases in arterial pressure compared with the responses in Ang I infused wild type mice [Gonzalez-Villalobos et al. 2011]. In further studies, mice with ACE deletion in kidneys, but expressing ACE elsewhere in the body, failed to develop the same level of hypertension in response to Ang II infusions as wild type mice [Gonzalez-Villalobos et al. 2013]. These results demonstrate that the presence of ACE in the kidneys is necessary to generate sufficiently high intrarenal Ang II concentrations to stimulate several sodium transport systems.

Translational studies of augmented urinary AGT in hypertension, diabetes and kidney diseases

Since intrarenal AGT expression is stimulated in hypertension and RAS-associated diseases, urinary AGT levels have been proposed as an index of intrarenal RAS activation in hypertension, diabetic nephropathy and other cardiovascular and kidney diseases [Navar, 2013]. As shown in Figure 2, elevated urinary AGT levels were observed in hypertensive animal models including Ang II infused rats with or without high-salt diet [Kobori et al. 2003b; Lara et al. 2012], spontaneous hypertensive rats with high-salt diet [Susic et al. 2011], Dahl salt rats [Kobori et al. 2003a], and mRenin2 transgenic rats [Milani et al. 2010]. Although these animal models exhibited over threefold higher urinary AGT excretion levels compared with their respective controls, DOCA-induced hypertensive rats did not show higher urinary AGT levels [Kobori et al. 2003b], supporting the generally held concept that desoxycorticosterone acetate (DOCA)-induced hypertension is developed in a RAS-independent manner and that the urinary AGT levels can be dissociated from just elevated arterial pressure. Some animal models, including rats receiving the combination of chronic Ang II infusion plus high-salt diet, exhibited over 100-fold higher urinary AGT levels compared with their respective controls (Figure 2). These very high urinary AGT excretion rates are associated with an increased proteinuria, indicating an increased glomerular permeability to macromolecules which allows increased filtration of circulating plasma AGT [Pohl et al. 2010; Matsusaka et al. 2014]. Under these conditions, it is likely that the very high levels of urinary AGT in these models reflects a contribution from filtered AGT due to glomerular dysfunction as well as elevated intrarenal AGT production.  The relative contributions of elevated intrarenal AGT formation and filtered AGT in hypertension have not been determined and will be an important subject in future studies. A study using human subjects with hypertension established clinical relevance by showing that urinary AGT levels in patients with hypertension are higher than in control subjects and that treatment with either AT1 receptor blockers or ACE inhibitors normalized the elevated urinary AGT levels [Kobori et al. 2009].

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Figure 2.

Urinary AGT levels in rat and mouse disease models.

Augmented urinary AGT levels have also been demonstrated in nonhypertensive diseases. Diabetic animal models and patients with diabetes mellitus exhibited greater urinary AGT levels [Saito et al. 2009; Park et al. 2010; Kamiyama et al. 2012; Kim et al. 2012; Lo et al. 2012]. Importantly, urinary AGT augmentation was detected before the development of proteinuria in diabetic mice [Kamiyama et al. 2012], suggesting that an elevated urinary AGT may precede the increased proteinuria and may predict the development of diabetic nephropathy. AGT levels are increased in urine collected from patients with chronic kidney disease [Mills et al. 2012], glomerulonephritis [Urushihara et al. 2010], and immunoglobulin A nephropathy [Nishiyama et al. 2011]. Although these findings indicate that urinary AGT levels may serve as an index of augmented intrarenal RAS [Navar 2013], larger clinical studies are needed in human subjects to determine if treatment of hypertensive and diabetic subjects with AT1 receptor blockers or ACE inhibitors can reduce urinary AGT levels, thus indicating successful reduction in intrarenal RAS activity which hopefully can reduce or stop the progression of renal injury [Navar 2013].