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Department of Physiology and Hypertension and Renal Center of Excellence, Tulane University Health Sciences Center, New Orleans, Louisiana (H.K., L.G.N., A.N.); Division of Nephrology and Endocrinology, University of Tokyo School of Medicine, Tokyo, Japan (M.N.); and Department of Pharmacology and Hypertension and Kidney Disease Research Center, Kagawa University Medical School, Kagawa, Japan (A.N.)
Abstract I. Introduction II. Physiological Actions of Angiotensin II in the Kidney A. Role of Angiotensin II in the Regulation of Renal Hemodynamics B. Role of Angiotensin II in the Regulation of Tubular Function 1. Proximal Tubules. 2. Distal Tubules. 3. Collecting Ducts. III. Regulation of Circulating Renin-Angiotensin System—Classic Renin-Angiotensin System Pathways IV. Mechanisms Responsible for Independent Regulation of Intrarenal Renin-Angiotensin System A. Angiotensinogen B. Renin and Prorenin C. Angiotensin-Converting Enzyme D. Angiotensin II Receptors E. Intrarenal Angiotensin II 1. Interstitial and Tubular Angiotensin II. 2. Intracellular Angiotensin II. F. Alternative Enzyme Pathways G. Other Factors 1. Renal Development and Aging. 2. Gender Differences. V. Augmentation of the Intrarenal Renin-Angiotensin System during Progression of Hypertension and Renal Injury A. Animal Studies 1. Angiotensin II-Dependent Hypertensive Models. a. Angiotensin II-infused hypertensive animals. b. Renovascular hypertensive animals. c. Transgenic animals. 2. Other Hypertensive Models. a. Dahl salt-sensitive rats. b. Spontaneously hypertensive rats. 3. Diabetic Animals. 4. Other Kidney Disease Models. 5. Cardiovascular Implications of Renal-Specific Regulation of the Renin-Angiotensin System. B. Clinical Studies 1. Hypertensive Patients. a. Renovascular hypertension. b. Other hypertension. 2. Patients with Renal Injury. a. Chronic kidney diseases. b. Diabetes. c. Dialysis patients. d. Other kidney diseases. VI. Effects of Pharmacological Intervention with Antihypertensive Agents on the Intrarenal Renin-Angiotensin System A. Angiotensin-Converting Enzyme Inhibitors B. Angiotensin Receptor Blockers C. beta-Blockers D. Calcium Blockers E. Renin Inhibitors and Chymase Inhibitors VII. Conclusions
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I. Introduction |
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; Paul et al., 2006). In addition to its physiological roles, locally produced Ang II induces inflammation, cell growth, mitogenesis, apoptosis, migration, and differentiation, regulates the gene expression of bioactive substances, and activates multiple intracellular signaling pathways, all of which might contribute to tissue injury. Clinical and preclinical studies on the effects of pharmacological investigations with ACEIs and ARBs support the notion that Ang II exerts a cardinal role in the pathogenesis of hypertension and renal injury via activation of AT1 receptors when inappropriately activated (Timmermans et al., 1993; Navar et al., 2000). Importantly, because the kidney plays a crucial role in the development of hypertension, hypertension is both a cause and consequence of renal disease (Navar, 1997, 2005; Paul et al., 2006). Accordingly, the Seventh Report of the Joint National Committee (JNC7), the European Society of Hypertension/European Society of Cardiology (2003 ESH-ESC), and the Japanese Society of Hypertension (JSH2004) recommended that ACEIs and ARBs be used in concert with diuretics as first-line therapy to reduce blood pressure in patients with hypertension and renal disease (Chobanian et al., 2003; Cifkova et al., 2003; Ikeda et al., 2006).
Recent attention has been focused on findings that local Ang II levels are differentially regulated in the kidney. Because there often is not clear evidence for markedly elevated circulating renin or Ang II concentrations, identification of local RAS activity is essential for understanding the mechanisms mediating pathophysiological functions. In particular, the Ang II contents in renal tissues are much higher than can be explained on the basis of equilibration with the circulating concentrations (Navar et al., 1997, 1999a,b; Navar and Nishiyama, 2004). Furthermore, the demonstration of much higher concentrations of Ang II in specific regions and compartments within the kidney indicates selective local regulation of intrarenal Ang II (Navar and Nishiyama, 2001, 2004; Ichihara et al., 2004b; Pendergrass et al., 2006). Thus, it is now apparent that intrarenal Ang II levels are regulated in a manner distinct from circulating Ang II concentrations. It has also been revealed that Ang II produced locally in the kidney exerts an important regulatory influence on renal hemodynamics and functions as a paracrine factor (Navar et al., 2000; Paul et al., 2006). Further studies demonstrate that reduced renal function and its structural changes are associated with inappropriate activation of the intrarenal Ang II, leading to the development of hypertension and renal injury (Navar et al., 2003; Navar, 2005).
In this review, we will briefly summarize the paracrine roles of intrarenal Ang II and review recent findings related to its independent regulation with special emphasis on roles in the pathogenesis of hypertension and renal injury. We will also discuss evidence regarding the effects of pharmacological intervention with antihypertensive agents on intrarenal Ang II. The molecular mechanisms responsible for Ang II-induced cell injury have been reviewed by Kim and Iwao (2000) and Touyz and Schiffrin (2000) and will not be discussed in detail in this review.
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II. Physiological Actions of Angiotensin II in the Kidney |
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Exogenous administration of Ang II elicits dose-dependent decreases in renal blood flow and glomerular filtration rate (Yamamoto et al., 2001; Paul et al., 2006). Although there is agreement that Ang II exerts substantial direct effects on the renal microvasculature and glomerular mesangium, there remains controversy regarding the intensity of actions at various sites and the relative contribution of systemically and intrarenally formed Ang II to the overall regulation of renal hemodynamics. The observation that Ang II increases the filtration fraction has frequently been used to support the notion that Ang II predominantly constricts the postglomerular arterioles (Schor et al., 1980; Heller and Horacek, 1986; Alberola et al., 1994). It should be emphasized, however, that this misconception is based on the failure to recognize that an increase in filtration fraction can occur as a consequence of parallel increases in both pre- and postglomerular arteriolar resistances (Navar and Rosivall, 1984; Rosivall et al., 1984; Carmines et al., 1987). Indeed, in vivo micropuncture studies in rats have clearly demonstrated that Ang II elicits reductions in single nephron glomerular filtration rate and glomerular plasma flow and increases in both afferent and efferent arteriolar resistance (Blantz et al., 1976; Baylis and Brenner, 1978; Schor et al., 1980; Rosivall and Navar, 1983). The decreases in glomerular filtration rate are also attributed to the effects of Ang II to reduce the glomerular filtration coefficient, which is thought to be due to changes in contractility of mesangial cells (Blantz et al., 1976; Baylis and Brenner, 1978; Schor et al., 1980; Paul et al., 2006). Because both AT1 and AT2 receptors are expressed in mesangial cells (Sharma et al., 1998), these may influence the glomerular filtration coefficient. However, the exact mechanism by which Ang II regulates the glomerular filtration coefficient remains to be clarified.
Although it was originally reported that Ang II did not constrict isolated rabbit afferent arterioles, there are many reports demonstrating that Ang II constricts both afferent and efferent arterioles (Carmines et al., 1986; Mitchell and Navar, 1988; Loutzenhiser et al., 1991; Ichihara et al., 1997; Yamamoto et al., 2001). Ito et al. (1991, 1993), and Yoshida et al. (1994) showed that inhibition of nitric oxide synthesis markedly augmented the afferent arteriolar responses to Ang II, indicating that high levels of nitric oxide may be present in the dissected afferent arterioles perfused with cell-free solutions. Studies using the in vitro blood-perfused juxtamedullary nephron preparation (Carmines et al., 1986; Ichihara et al., 1997), renal tissue transplantation into hamster cheek pouch (Click et al., 1979), and hydronephrotic rat kidneys (Steinhausen et al., 1987; Dietrich et al., 1991; Loutzenhiser et al., 1991; Inman et al., 1995) also showed similar results. Yamamoto et al. (2001) used an intravital tapered-tip lens-probe video-microscopy system and demonstrated that intrarenal infusion of Ang II constricts both afferent and efferent arterioles in anesthetized dogs. These collective observations indicate that, rather than predominantly constricting efferent arterioles, Ang II elicits vasoconstrictor actions on both pre- and postglomerular resistance vessels; however, the experimental circumstances may influence the reactivity of the afferent more than of the efferent arterioles.
It should be recognized that Ang II elicits the glomerular hemodynamic changes described above without causing significant proteinuria. In both animals and humans, acute Ang II infusion sufficient to change renal hemodynamics does not elicit proteinuria (Loon et al., 1989; Pagtalunan et al., 1995). These observations are in agreement with the prediction based on the mathematical modeling that alterations in glomerular pressure can cause less change in macromolecule filtration if the capillary wall structure is not altered (Bohrer et al., 1977). However, sustained elevation of intrarenal Ang II induces proteinuria accompanied by progressive injury of the glomerular filtration barrier, which is composed of the glomerular endothelium, glomerular basement membrane, and podocytes (glomerular visceral epithelial cell) (Miller et al., 1991; Hoffmann et al., 2004; Whaley-Connell et al., 2006). Locally produced Ang II directly induces podocyte injury via activation of AT1 receptors, independent of hemodynamic changes (Durvasula et al., 2004; Liang et al., 2006; Liebau et al., 2006). Therefore, pharmacological interventions of these effects of Ang II are useful for reducing proteinuria in patients with renal injury.
The overall renal hemodynamic responses to Ang II blockade with ACEIs and ARBs have been quite variable because of the counteracting influences of the associated decreases in systemic arterial pressure. If arterial pressure remains within the renal autoregulatory range, renal blood flow is generally increased by Ang II blockade (Navar et al., 1996; Paul et al., 2006); however, the glomerular filtration rate responses have been much more variable, either increased (Kimbrough et al., 1977; Rosivall et al., 1986; Tamaki et al., 1993), unchanged (Omoro et al., 2000), or decreased (Hall et al., 1979b). In vivo micropuncture studies showed that Ang II blockade increases single nephron filtration rate as well as single nephron plasma flow when arterial pressure is not markedly reduced (Kon et al., 1993; Cervenka et al., 1998; Cervenka and Navar, 1999; Paul et al., 2006). Similarly, intrarenal infusion of subpressor doses of ARBs significantly increased both whole kidney renal blood flow and glomerular filtration rate (Nishiyama et al., 1992; Tamaki et al., 1993), suggesting that Ang II blockade increases the glomerular filtration coefficient. Most clinical studies also show that the glomerular filtration rate remains stable when Ang II blockade is instituted (Andersen et al., 2000; Fridman et al., 2000; Agodoa et al., 2001). The most direct way to explain increases in renal blood flow without changes in glomerular filtration rate is by combined decreases in both pre- and postglomerular arteriolar resistance. In some studies, glomerular filtration rate has been shown to be increased slightly in response to treatment with ACEIs and ARBs (Fridman et al., 1998; Pechère-Bertschi et al., 1998). However, a significant reduction in the glomerular filtration rate has often been seen in patients with renal disease (Hansen et al., 1995; Apperloo et al., 1997). Decreases in arterial pressure in response to Ang II blockade are pronounced during sodium-depleted states (Navar et al., 1996; Paul et al., 2006). Usually, in hypertensive patients with renal disease, ACEIs and ARBs are often added to other drugs, including diuretics, under the conditions where intake of sodium is restricted. Thus, it seems likely that Ang II blockade with ACEIs and ARBs causes a marked reduction in blood pressure, leading to decreases in glomerular filtration rate when extracellular fluid volume is low. In addition, in patients with established glomerular disease, it may be difficult to maintain the glomerular filtration rate by sufficient increases in glomerular filtration coefficient when glomerular pressure is reduced by treatment with ACEIs and ARBs. In patients with more severe renal disease, the afferent arterioles may also become less responsive to ACEIs and ARBs.
In addition to its direct constrictor effects on glomerular arterioles and mesangium, Ang II also regulates renal hemodynamics by exerting a modulatory influence on the sensitivity of the tubuloglomerular feedback mechanism (Navar et al., 1996; Paul et al., 2006). This mechanism provides a balance between the reabsorption capabilities of the tubules and the filtered load by regulating the glomerular filtration rate (Nishiyama et al., 2004a). When flow-dependent changes in the tubular fluid solute concentration at the level of the macula densa in the terminal part of the loop of Henle are sensed, signals are transmitted to the afferent arterioles and glomerular mesangium to constrict or dilate to maintain stability of the filtered load (Navar et al., 1996; Paul et al., 2006). The tubuloglomerular feedback mechanism also participates in autoregulatory responses of renal vascular resistance and glomerular filtration rate (Nishiyama et al., 2004a; Paul et al., 2006). Although it was demonstrated that Ang II does not directly mediate the tubuloglomerular feedback response, its level of activity exerts an important modulatory influence on the sensitivity of the vascular and mesangial elements that respond to signals from the macula densa cells (Ploth, 1983; Schnermann and Briggs, 1986; Mitchell et al., 1992; Braam et al., 1995; Schnermann et al., 1997; Traynor et al., 1999). The tubuloglomerular feedback responsiveness is enhanced during either systemic or peritubular capillary infusion of exogenous Ang II (Schnermann and Briggs, 1986; Mitchell et al., 1992). Furthermore, Ang II blockade with ACEIs and ARBs markedly attenuates the tubuloglomerular feedback responsiveness as assessed by stop-flow pressure feedback responses to increases in distal nephron perfusion rate (Ploth, 1983; Braam et al., 1995). Similarly, both AT1 receptor knockout and ACE-deficient mice have markedly attenuated tubuloglomerular feedback responses to increases in distal nephron perfusion rate (Schnermann et al., 1997; Traynor et al., 1999). Collectively, these findings indicate that Ang II enhances the sensitivity of the vascular and mesangial elements that mediate tubuloglomerular feedback-induced alterations in single nephron function. These effects probably are mediated by direct actions on the vascular smooth muscle cells and mesangial cells as well as by modulating the Na+/H+ exchange activity of the macula densa cells (Peti-Peterdi and Bell, 1998; Kovács et al., 2002). A modulatory influence of Ang II on tubuloglomerular feedback responsiveness shifts the operating point of the system and allows the nephron filtration rate to be maintained at a lower distal nephron volume delivery (Navar et al., 1996; Paul et al., 2006). During conditions of elevated intrarenal Ang II levels, the modulatory influence of Ang II on tubuloglomerular feedback responsiveness is of pivotal importance in maintaining the Ang II-mediated stimulation of proximal tubular reabsorption and the consequent decrease in distal nephron volume delivery. In this manner, the interactive effects of increased Ang II levels to enhance both proximal tubular reabsorption rate and sensitivity of the tubuloglomerular feedback mechanism elicit sustained decreases in distal nephron volume delivery and, thus, urinary sodium excretion.
Enhanced preglomerular vascular tone and blunted microvascular autoregulatory responsiveness to changes in perfusion pressure are observed in Ang II-dependent hypertensive models (Ichihara et al., 1997; Inscho et al., 1999). The blunted autoregulatory responsiveness of the afferent arteriole in Ang II-dependent hypertension apparently results from chronic elevation of Ang II levels because acute exposure to 10-fold greater concentrations of Ang II does not affect autoregulatory behavior (Inscho et al., 1996). Chronic treatment with ARBs prevents the deterioration of renal autoregulatory responsiveness in Ang II-infused rats (Inscho et al., 1999). However, Ang II blockade does not affect renal autoregulatory behavior in normal animals (Navar et al., 1986; Persson et al., 1988).
B. Role of Angiotensin II in the Regulation of Tubular Function
Ang II is one of the most powerful sodium-retaining hormones in the body. The direct intrarenal actions of Ang II that contribute to increased tubular reabsorption are complex, including constriction of glomerular arterioles, which alter peritubular capillary dynamics and renal medullary blood flow, and direct actions on tubular epithelial cell transport. Although the quantitative contribution of each of these hemodynamic and tubular actions may vary in different physiological circumstances, high intrarenal Ang II levels contribute to salt and water retention through direct actions on renal tubular transport function when inappropriately stimulated (Navar and Nishiyama, 2004).
Ang II is also one of the body's most important regulators of aldosterone, which stimulates sodium reabsorption, primarily through the mineralocorticoid receptors in the connecting and cortical segments of the collecting tubule. Furthermore, Ang II directly enhances urinary concentration in the collecting tubule and collecting ducts.
Because all of the components of the RAS are found in the kidney and significant amounts of Ang II can be formed locally, considerable interest has focused on the possibility that intrarenally formed Ang II may be more important than circulating Ang II in controlling renal function. Several studies demonstrated the fact that intrarenal infusion of ARBs or ACEIs, at rates that produced no changes in plasma aldosterone concentration and minimal effects on systemic hemodynamics, increased sodium excretion (Kimbrough et al., 1977; Hall et al., 1979a; Klag et al., 1996; Cervenka et al., 1998). Intrarenal infusion of Ang I, to stimulate local formation of Ang II, also reduced sodium excretion (Rosivall and Navar, 1983). These results emphasize the contribution of intrarenally formed Ang II in regulating sodium excretion.
In addition to maintaining fluid and electrolyte homeostasis, Ang II participates in a variety of tubular functions, including induction of cellular hypertrophy and oxidative stress. Details of biological function specific for each tubular segment are described below.
1. Proximal Tubules.
Normally, the potent antinatriuretic effects of Ang II are due primarily to increased tubular reabsorption rather than to reductions in glomerular filtration rate (Hall et al., 1986; Mitchell et al., 1992). In vivo perfusion of rat proximal tubules with an ultrafiltrate-like solution containing either ACEIs or ARBs decreased the volume reabsorption, suggesting modification of proximal tubule transport by locally produced Ang II independent from the systemic RAS (Quan and Baum, 1996).
Microperfusion studies of isolated proximal tubules have shown that the Ang II effect on proximal tubule sodium transport is bimodal; Ang II at physiological concentrations (picomoles per liter) significantly stimulates proximal tubule sodium reabsorption, whereas pharmacological micromole per liter concentrations inhibit transport (Harris and Young, 1977; Schuster et al., 1984). Reabsorption of sodium by Ang II in proximal tubules is coupled with bicarbonate reabsorption, which is mediated by inhibition of adenylate cyclase (Liu and Cogan, 1989). Using in vivo microperfusion in the Munich-Wistar rat, Liu and Cogan (1987) showed that administration of luminal Ang II increased proximal tubule bicarbonate reabsorption. These findings were confirmed using electrophysiological methods in isolated perfused rabbit renal proximal tubules (Coppola and Fromter, 1994a,b). Perfusion of rabbit proximal tubules with luminal Ang II after treatment with ACEIs increased volume and bicarbonate reabsorption (Baum et al., 1997), supporting the role of intrarenally produced Ang II to stimulate proximal tubule volume and bicarbonate transport.
Molecular mechanisms of direct stimulation of fluid reabsorption by Ang II within the proximal tubule involve increased transcellular sodium and bicarbonate reabsorption via activation of apical Na+/H+ exchange, basolateral Na+-HCO3- cotransport, and basolateral Na+/K+-ATPase and via insertion of H+-ATPase into the apical membrane (Liu and Cogan, 1988; Garvin, 1991; Mitchell et al., 1992; Eiam-Ong et al., 1993; Wang and Giebisch, 1996). Stimulation of Na+-HCO3- cotransport by Ang II is mediated by diverse signaling pathways, including activation of the Src family of tyrosine kinase and the classic mitogen-activated protein kinase pathway (Espiritu et al., 2002; Robey et al., 2002).
In view of these observations that the intratubular activation of the RAS stimulated proximal fluid reabsorption, recent analysis of tissue-specific ACE knockout mice using micropuncture techniques gave unexpected results (Hashimoto et al., 2005). In this unique model, tissue ACE is deleted, but ACE is selectively expressed in the liver (Cole et al., 2003). Whereas disruption of ACE often causes low blood pressure, which complicates renal functional studies, this model is able to maintain sufficient plasma levels of ACE and subsequently normal blood pressure. Proximal tubular fluid reabsorption of these genetically altered mice was comparable with that observed in wild-type mice despite the essentially complete absence of tissue ACE. These findings are in contrast with the previous findings that an acute reduction in local Ang II formation exerted a profound inhibitory effect on fluid reabsorption. The discrepancy may lie in the chronicity of Ang II blockade, and chronic ACE deficiency is apparently associated with compensatory events that normalize fluid reabsorption along the proximal tubule. It is also possible that proximal tubular Ang II is formed through alternative pathways not requiring ACE.
In addition to regulation of fluid and electrolyte balance, Ang II plays an important role in hypertrophy of proximal tubular cells. In rat proximal tubular epithelial cells, Ang II induces cellular hypertrophy and activates relevant downstream signal transduction pathways (Wolf et al., 1993; Hannken et al., 1998, 2000; Guo et al., 2004). The Ang II-induced tubular cell hypertrophy is inhibited by ARBs, suggesting that the AT1 receptor contributes to the tubular cell hypertrophy (Chatterjee et al., 1997). Cells undergoing hypertrophy are arrested in the G1 phase of the cell cycle, and p27Kip1, an inhibitor of cyclin-dependent kinases, is required for Ang II-induced hypertrophy of proximal tubular cells (Wolf and Stahl, 1996; Terada et al., 1999; Wolf et al., 2001, 2003).
Transfection of AT1 receptors into a renal proximal tubular cell line LLCPKcl4, which does not express endogenous Ang II receptors, increased protein synthesis without DNA synthesis in response to Ang II, as indicated by increased [3H]leucine incorporation without increases in [3H]thymidine incorporation (Burns and Harris, 1995). The stimulation of protein synthesis and cell hypertrophy without increasing cell number was mediated by activation of the epidermal growth factor receptor (Chen et al., 2006). Recent studies also demonstrated involvement of connective tissue growth factor in mediating Ang II-induced tubular cell hypertrophy (Liu et al., 2006). In cultured proximal tubular cells, Ang II stimulated the expression of connective tissue growth factor and increased the total protein content as well as cell size, which were markedly inhibited by cotreatment with an antisense oligonucelotide for connective tissue growth factor.
With blockade of transforming growth factor-
receptor, Ang II-mediated hypertrophy can be converted into cell proliferation. Rats that received Ang II infusion had an increased number of proliferating cell nuclear antigen- and transferase dUTP nick-end labeling-positive cells in proximal tubules with a possible involvement of AT2 receptors (Cao et al., 2000), suggesting that Ang II also triggers both proliferation and apoptosis in tubular epithelial cells under certain circumstances.
A role for Ang II in induction of oxidative stress in the kidney has been extensively studied. Treatment of Wistar-Kyoto rats with subcutaneous Ang II infusions from osmotic minipumps induced oxidative stress in association with increased expression of the p22phox component of NADPH oxidase and decreased expression of extracellular superoxide dismutase in the renal cortex (Welch et al., 2005). These effects were mediated via AT1 receptors and were offset by protective effects of AT2 receptors (Chabrashvili et al., 2003). Measurement of PO2 in the lumen of proximal tubules and distal tubules gave low values, which can be ascribed to inefficient utilization of O2 due to oxidative stress. Therefore, it is likely that Ang II induced oxidative stress in both proximal and distal tubules.
Another function of renal proximal tubule cells regulated by Ang II is endocytosis of urinary protein components. Ang II at physiological concentrations as low as 1 nM increased albumin endocytosis through AT2 receptors located on the luminal side and triggered the activation of protein kinase B in a porcine proximal tubular cell line (Caruso-Neves et al., 2005). This report clearly indicates that Ang II is also involved in the regulation of endocytosis of urinary protein in renal proximal tubule cells under physiological conditions.
2. Distal Tubules.
Ang II infusion increased distal fractional sodium reabsorption (Olsen et al., 1985). Intravenous infusion of Ang II stimulated distal bicarbonate reabsorption during microperfusion experiments (Levine et al., 1994). Ang II also regulated distal bicarbonate reabsorption during modifications of food intake in the rat (Levine et al., 1996). Studies of separate perfusions of early and late segments of cortical distal tubule showed that Ang II stimulated early distal bicarbonate reabsorption, whereas the late distal effect was mostly on amiloride-sensitive sodium reabsorption, i.e., on sodium channels (Wang and Giebisch, 1996). Ang II acts to stimulate Na+/H+ exchange in both early and late distal segments via activation of AT1 receptors and the vacuolar H+-ATPase in late distal segments (Barreto-Chaves and Mello-Aires, 1996). Experiments performed in distal tubules of nephrectomized rats indicated that AT1 receptor blockade caused marked reduction of synthesis and insertion of apical H+-ATPase in A-type intercalated cells (Levine et al., 2000). The effects of Ang II on sodium reabsorption in distal tubular segments further enhance and amplify the effects in proximal tubules, leading to much greater overall efficiency of sodium conservation.
3. Collecting Ducts.
In the proximal and distal tubules, Na+ serves as a counterion for H+ secretion. Thus, Ang II augments H+ secretion and Na+ absorption in these tubular segments. Despite a dramatic up-regulation of H+ secretion in the proximal and distal tubules by Ang II, infusion of Ang II does not produce a metabolic alkalosis, suggesting a compensatory regulation of acid secretion in other segments of the nephron. To support this notion, Ang II decreased H+ secretion in the perfused rat outer medullary collecting ducts (Weiner et al., 1995; Wall et al., 2003). This can be explained by a reduction in H+-ATPase activity (Tojo et al., 1994; Valles and Manucha, 2000). However, different results regarding the effect of Ang II on H+ secretion have been reported. Whereas selective aldosterone deficiency created by adrenalectomy with glucocorticoid replacement resulted in down-regulation in the expression of the H+-ATPase B1 subunit in medullary collecting ducts, Ang II increased the expression of the B1 subunit of H+-ATPase in the medullary collecting ducts and thus may up-regulate H+ secretion in this tubular segment of these animals (Valles et al., 2005).
Ang II also plays an important role in regulation of the sodium channel in the collecting ducts via a mechanism that is not dependent on circulating aldosterone. In isolated perfused rabbit cortical collecting ducts, Ang II directly stimulated apical membrane epithelial sodium channel activity (Peti-Peterdi et al., 2002). With low-salt diets, associated with activation of the RAS, the expression of the 
-epithelial sodium channel was markedly decreased in AT1a receptor knockout mice (Brooks et al., 2002).
The inner medullary collecting ducts are responsible for the final concentration of the urine. Mice with gene deletion of the AT1a receptor exhibit defects in urinary concentrating ability (Oliverio et al., 2000). The effects of RAS activation in the inner medullary collecting ducts may be mediated by stimulation of urea transport, which maintains the medullary interstitial osmotic gradient. In rat terminal inner medullary collecting ducts, low concentrations of basolateral Ang II increases vasopressin-stimulated urea permeability and induces phosphorylation of the urea transporter (Kato et al., 2000). These data suggest that Ang II stimulates the urinary concentrating mechanism, leading to increased water reabsorption.
In addition to direct effects, Ang II regulates function of the collecting ducts via aldosterone. Ang II stimulates the zona glomerulosa of the adrenal cortex to produce the sodium-retaining hormone, aldosterone. Aldosterone stimulates ionic transport in the principal cells by increasing the number of open sodium and potassium channels in the luminal membrane and the activity of Na+/K+-ATPase pump in the basolateral membrane. Thus, aldosterone promotes sodium chloride reabsorption and potassium secretion in the principal cells of the cortical collecting tubular segment of the nephron. It further stimulates H+ secretion in the intercalated cells of the cortex and tubular cells in the outer medulla (Navar et al., 1996; Paul et al., 2006).
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III. Regulation of Circulating Renin-Angiotensin System—Classic Renin-Angiotensin System Pathways |
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; Schnermann et al., 1997). Although circulating active renin and prorenin are released mainly from the kidney, other tissues also secrete prorenin into the circulation, and prorenin can be converted to renin by limited proteolysis such as that with trypsin activation in the circulation (Sealey et al., 1986). Angiotensinogen is primarily formed and constitutively secreted by hepatic cells into the circulation, thus allowing systemic formation of Ang II throughout the circulation (Brasier and Li, 1996). On release into the circulation, renin cleaves angiotensinogen at the N terminus to form the decapeptide, Ang I (Navar et al., 1997). The circulating concentrations of angiotensinogen are abundant, being more than 1000 times greater than the plasma Ang I and Ang II concentrations (Navar and Nishiyama, 2001). Although some species variation exists, changes in renin activity thus determine the rate of Ang I formation in the plasma from the huge stores of circulating angiotensinogen (Ichihara et al., 2004b; Paul et al., 2006). Figure 1 shows the representative plasma angiotensinogen concentrations measured in anesthetized rats and expressed as nanomoles per liter; the Ang I and Ang II concentrations are expressed as picomoles per liter, indicating that the active Ang II concentration in the plasma is a small fraction of the available Ang II in the form of angiotensinogen. Therefore, even small relative changes in the rates of Ang I and Ang II generation may make large absolute differences in the circulating concentrations. As is well known, renin is synthesized and stored in substantial quantities in the granules of juxtaglomerular cells and is released in response to various stimuli (Schweda and Kurtz, 2004; Paul et al., 2006). Thus, large changes in plasma renin levels can occur rapidly, leading to changes in Ang I generation. The concentrations of angiotensinogen in the plasma are close to the Michaelis-Menten constant of the proteolytic activity of renin such that changes in substrate concentrations can also influence the Ang I generation rate; however, changes in angiotensinogen synthesis occur slowly and thus are less responsible for the dynamic regulation of plasma Ang I and Ang II than renin (Deschepper, 1994; Brasier and Li, 1996). Ang I is easily converted to Ang II, due not only to the circulating dipeptidyl carboxypeptidase, ACE, but also to the widespread presence of ACE on endothelial cells of many vascular beds including the lung (Navar et al., 1997; Ichihara et al., 2004b; Paul et al., 2006). Although other pathways for Ang II formation have been identified in certain tissues (Fig. 1), the circulating levels of Ang II reflect primarily the consequences of the renin and ACE enzymatic cascade on angiotensinogen (Erdös, 1990; Johnston, 1994). The resultant increases in plasma Ang II exert powerful actions throughout the body through activation of AT1 receptors (Timmermans et al., 1993; Paul et al., 2006). Several angiotensinases and peptidases are then able to metabolize Ang II further (Reudelhuber, 2005). It is recognized that several of the smaller peptides, including Ang III, Ang IV, and Ang 1-7, have biological activity, but their plasma levels (except for Ang1-7) are much lower than those of Ang II (Haulica et al., 2005; Pendergrass et al., 2006).
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IV. Mechanisms Responsible for Independent Regulation of Intrarenal Renin-Angiotensin System |
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; Re, 2003; Kobori et al., 2006; Re and Cook, 2006; Suzaki et al., 2006b), and, thus, it has been difficult to delineate the quantitative contributions of systemically delivered versus locally formed Ang peptides to the levels existing in any given tissue. Emerging evidence suggests that local formation is of major significance in the regulation of the Ang levels in many organs and tissues. For example, there is substantial evidence that the Ang peptide levels in the brain are regulated in an autonomous manner (Baltatu et al., 2000). Although every organ system in the body has elements of the RAS, the kidney is unique in having every component of the RAS with compartmentalization in the tubular and interstitial networks as well as intracellular accumulation. Recent attention has been focused on the existence of unique RASs in various organ systems. Various studies have demonstrated the importance of the tissue RAS in the brain, heart, adrenal glands, and vasculature as well as in the kidney (Mitchell and Navar, 1995; Navar et al., 2006). In this regard, the kidneys, as well as the adrenal glands, are unique in terms of the tissue concentrations of Ang II, which are much greater than can be explained by the concentrations delivered by the arterial blood flow (Ingert et al., 2002a). There is substantial evidence that the major fraction of Ang II present in renal tissues is generated locally from angiotensinogen delivered to the kidney as well as from angiotensinogen locally produced by proximal tubule cells. Ang I delivered to the kidney can also be converted to Ang II (Rosivall and Navar, 1983; Komlosi et al., 2003). Renin secreted by the juxtaglomerular apparatus cells and delivered to the renal interstitium and vascular compartment also provides a pathway for the local generation of Ang I (Hackenthal et al., 1990; Schnermann et al., 1997). ACE is abundant in the rat kidney and has been located in the proximal and distal tubules, the collecting ducts, and renal endothelial cells (Casarini et al., 1997). Therefore, all of the components necessary to generate intrarenal Ang II are present along the nephron.
Although most of the circulating angiotensinogen is produced and secreted by the liver, the kidneys also produce angiotensinogen (Kobori et al., 2006). Intrarenal angiotensinogen mRNA and protein have been localized to proximal tubule cells, indicating that the intratubular Ang II could be derived from locally formed and secreted angiotensinogen (Darby and Sernia, 1995). The angiotensinogen produced in proximal tubule cells seems to be secreted directly into the tubular lumen in addition to producing its metabolites intracellularly and secreting them into the tubule lumen (Lantelme et al., 2002). Proximal tubule angiotensinogen concentrations in anesthetized rats have been reported in the range of 300 to 600 nM, which greatly exceed the free Ang I and Ang II tubular fluid concentrations (Navar et al., 2001). Because of its molecular size, it seems unlikely that much of the plasma angiotensinogen filters across the glomerular membrane, further supporting the concept that proximal tubule cells secrete angiotensinogen directly into the tubule (Rohrwasser et al., 1999). To determine whether circulating angiotensinogen is a source of urinary angiotensinogen, Kobori et al. (2003b) infused human angiotensinogen into normotensive rats; however, circulating angiotensinogen was not detectable in the urine. The failure to detect human angiotensinogen in the urine indicates limited glomerular permeability and/or tubular degradation. These findings support the hypothesis that urinary angiotensinogen originates from the angiotensinogen that is formed and secreted by the proximal tubules and not from plasma in rats (Kobori et al., 2003b). Formation of Ang I and Ang II in the tubular lumen subsequent to angiotensinogen secretion may be possible because some renin is filtered and/or secreted from juxtaglomerular apparatus cells. The identification of renin in distal nephron segments may also provide a possible pathway for Ang I generation from proximally delivered angiotensinogen. Intact angiotensinogen in urine indicates its presence throughout the nephron and, to the extent that renin and ACE are available along the nephron, substrate availability supports continued Ang I generation and Ang II conversion in distal segments (Ding et al., 1997; Davisson et al., 1999). Once Ang I is formed, conversion readily occurs because there are abundant amounts of ACE associated with the proximal tubule brush border. Casarini et al. (1997) found that ACE activity is present in tubular fluid throughout the nephron except in the late distal tubule. They demonstrated that the ACE activity is higher at the initial portion of the proximal tubule but then decreases to the distal nephron and increases again in the urine. This evidence suggests proximal ACE secretion, degradation, and/or reabsorption associated with secretion in the collecting ducts. Therefore, intratubular Ang II formation may occur not only in the proximal tubule but also beyond the connecting tubule (Fig. 2). Thus, renal tissue ACE activity is critical to maintain the steady-state Ang II levels in the kidney. Indeed, Modrall et al. (2004) demonstrated that knockout mice that do not exhibit bound tissue ACE in the kidney have 80% lower intrarenal Ang II levels compared with wild-type mice. In addition to the marked reduction of intrarenal Ang II levels, this tissue ACE knockout mouse showed significant depletion of its immediate precursor Ang I in renal tissue, which supports the concept that Ang II exerts a positive feedback loop on proximal angiotensinogen (Ingelfinger et al., 1999; Kobori et al., 2001a,b, 2002).
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The proximally formed angiotensinogen that is secreted into the tubular fluid flows into the distal nephron, allowing intraluminal Ang II formation to continue throughout the length of the nephron with the residual angiotensinogen appearing in the urine (Ding et al., 1997; Rohrwasser et al., 1999). Ding et al. (1997) demonstrated in mice harboring the gene for human angiotensinogen fused to the kidney-specific androgen-regulated protein promoter that human angiotensinogen was localized primarily to proximal tubule cells (see section V.A.1.c.). They found abundant human angiotensinogen in the urine but only slight traces in the systemic circulation. This finding suggests that most of the angiotensinogen formed in proximal tubule cells is destined for secretion into the lumen. Rohrwasser et al. (1999) demonstrated luminal localization of angiotensinogen in proximal tubular cells in vivo and showed, in monolayer proximal tubule cell cultures, that most of the angiotensinogen was detected near the apical membrane. They also reported that angiotensinogen was detected at low nanomoles per liter concentrations in urine from mice and human volunteers. Kobori et al. (2002) evaluated the changes in urinary angiotensinogen excretion rates in Ang II-infused rats maintained on high-salt diets to suppress basal levels and observed an approximately 4-fold increase with Ang II infusion (80 ng/min) in urinary angiotensinogen excretion rates. Angiotensinogen was measured using both Western blot analysis and radioimmunoassay determination of generated Ang I after incubation with excess renin, thus demonstrating the fact that urinary angiotensinogen contained intact active angiotensinogen. They extended these results further to show that chronic Ang II infusions to normal rats significantly increased the urinary excretion rate of angiotensinogen in a time- and dose-dependent manner that was associated with elevations in systolic blood pressure and kidney Ang II levels but not with plasma Ang II concentrations (Kobori et al., 2003b). To determine whether the increase in urinary angiotensinogen excretion was simply a nonspecific consequence of the proteinuria and hypertension, further studies were done in rats made hypertensive with DOCA salt plus a high-salt diet. Although urinary protein excretion in DOCA salt-induced volume-dependent hypertensive rats was increased to the same or to a greater extent, urinary angiotensinogen was significantly lower in volume-dependent hypertensive rats than in Ang II-dependent hypertensive rats and was not greater than in control rats. This study also demonstrated that there was a significant relationship between urinary angiotensinogen and kidney Ang II content in rats given different doses of Ang II to achieve different levels of hypertension. These results provide further evidence that urinary angiotensinogen may be a useful index of intrarenal Ang II activity (Kobori et al., 2002, 2003b, 2004) (Fig. 2). Recently, two independent groups have developed an enzyme-linked immunosorbent assay system to measure angiotensinogen directly (Lantelme et al., 2005; Suzaki et al., 2006a). Outcomes of clinical studies are expected in the near future.
Strictly speaking, renin is not a hormone; however, it can be considered as such because of its role in determining Ang I generation and because it is subject to tight control. Hence, the plasma renin concentration or activity is often used as a measure of the overall activity of the RAS. In most species, renin synthesized by the juxtaglomerular apparatus cells is the primary source of both circulating and intrarenal renin levels. However, some strains of mice also produce substantial amounts of renin in the submandibular and submaxillary glands as an expression of the duplicated renin gene, Ren2 (Catanzaro et al., 1983).
The secreted active form of renin contains 339 to 343 amino acid residues after proteolytic removal of the 43-amino acid residue at the N terminus of prorenin. Circulating active renin and prorenin are released mainly from the kidney, but other tissues also secrete prorenin into the circulation (Sealey et al., 1986). Besides serving as the precursor for active renin, it has been suggested that circulating prorenin is taken up by some tissues where it may contribute to the local synthesis of Ang peptides (Prescott et al., 2002). In the heart under normal conditions, renin is not produced and its transcript is undetectable or extremely low (Ekker et al., 1989). Nevertheless, transgenic rats expressing the Ren2 renin gene exhibit high circulating prorenin levels in the absence of the cardiac transgene, prorenin internalization into cardiomyocytes with generation of Ang, and cardiac damage (Peters et al., 2002). These effects suggest that uptake of circulating prorenin but not active renin may play an important role in cardiac hypertrophy.
Although there have been suggestions that renin itself or perhaps prorenin may directly elicit cellular effects, independent of the generation of Ang II, the well established role of renin is to act on angiotensinogen, a protein with a glycosylated weight of 52 to 64 kDa and synthesized primarily by the liver to form the decapeptide Ang I. However, the (pro)renin receptor may also initiate intracellular signaling to activate extracellular signal-regulated kinases 1/2 (Nguyen et al., 2002) and p38 mitogen-activated protein kinase (Saris et al., 2006). In the heart and kidney, the recently described renin receptor (Nguyen et al., 1996) binds renin and prorenin, leading to an increase in the catalytic efficiency of Ang I formation from angiotensinogen. It has also been reported recently that the binding of prorenin to an intrinsic prorenin-binding receptor plays a pivotal role in the development of diabetic nephropathy by a mechanism that involves the receptor-associated prorenin system (Ichihara et al., 2004a,b).
It should be recognized that juxtaglomerular apparatus cells are not the only intrarenal structures in which renin has been localized. Kidneys from rats treated chronically with ACEIs also exhibit renin immunoreactivity of the afferent arteriole extending well beyond the juxtaglomerular apparatus loci up toward the interlobular artery, suggesting that ACE inhibition induces a recruitment of cells that in the basal state were not expressing the renin gene (Gomez et al., 1988). Positive renin immunoreaction has been observed in cells of glomeruli and of proximal and distal nephron segments as well as its mRNA (Moe et al., 1993). In addition, renin mRNA and protein expression have been also reported in proximal and distal nephron segments (Rohrwasser et al., 1999; Lantelme et al., 2002; Prieto-Carrasquero et al., 2004). Using immunoblotting, Rohrwasser et al. (1999) found that renin was secreted by microdissected arcades of connecting tubule cells, indicating that renin is probably secreted into the distal tubular fluid. They also demonstrated that renin activity was observed in excreted urine (Rohrwasser et al., 2003).
C. Angiotensin-Converting Enzyme
Ang I is rapidly converted into the major effector of this system, Ang II, by ACE, which is located on endothelial cells in many vascular beds and on membranes of various other cells including brush border membranes of proximal tubules (Schulz et al., 1988; Mezzano et al., 2003ab). The localization of ACE within the kidney in various species has been well characterized. However, there are some important differences between humans and commonly used experimental animals (Metzger et al., 1999). Indeed, Metzger et al. (1999) reported that kidneys from normal human subjects predominantly expressed ACE in the brush border of proximal tubular segments, and very little ACE expression was observed on vascular endothelial cells. ACE was not detectable in the vasculature of the glomerular tuft or even in the basolateral membranes of epithelial cells. In contrast, there was intense labeling on the endothelial cells of almost all of the renal microvasculature of rats. However, kidneys from human subjects with non-neoplastic diseases manifested increased expression on vascular endothelial cells (Metzger et al., 1999). These data indicating much lower endothelial expression in renal vascular endothelial cells in humans help explain the much lower Ang I to Ang II conversion rates that have been reported for human kidneys compared with those of other species (Danser et al., 1998). Danser et al. (1998) reported that less than 10% of arterially delivered Ang I is converted to Ang II, which is much lower than the amount reported for dogs (Rosivall et al., 1983). The reduced ACE expression on renal vascular endothelial cells in humans implies that the influence of intrarenal Ang II formed from circulating precursors may not be of major significance.
Most of the actions of Ang II on renal function are the consequence of activation of Ang II receptors, which are widely distributed in various regions and cell types of the kidney. Two major categories of Ang II receptors, type 1 (subtypes 1a and 1b) and type 2, have been described, pharmacologically characterized, and cloned (Murphy et al., 1991; Sasamura et al., 1992; Nakajima et al., 1993). However, most of the Ang II hypertensinogenic actions are generally attributed to the AT1 receptors (Ito et al., 1995). AT1 receptor transcript has been localized to proximal tubules, the thick ascending limb of the loop of Henle, glomeruli, arterial vasculature, vasa recta, arcuate arteries, and juxtaglomerular cells (Tufro-McReddie et al., 1993b). In rodents, there are two AT1 receptor subtypes, with type 1a being the predominant subtype in all nephron segments, whereas type 1b is more abundant than type 1a only in the glomerulus (Bouby et al., 1997). In mature kidneys, type 1a receptors have been localized to the luminal and basolateral membranes of several segments of the nephron, as well as on the renal microvasculature in both cortex and medulla, smooth muscle cells of afferent and efferent arterioles, epithelial cells of the thick ascending limb of Henle, proximal tubular apical and basolateral membranes, mesangial cells, distal tubules, collecting ducts, and macula densa cells (Paxton et al., 1993; Harrison-Bernard et al., 1997; Miyata et al., 1999). This evidence is consistent with the localization of the transcript for the AT1 receptor subtypes in all of the renal tubular and vascular segments (Miyata et al., 1999). Nevertheless, renal microvascular functional studies obtained from mice lacking the type 1a receptor gene have shown that the afferent arteriole has both type 1a and type 1b receptors, whereas the efferent arteriole only expresses type 1a receptors (Harrison-Bernard et al., 2003).
The regulation of intrarenal Ang II receptors in hypertensive conditions is complex because vascular and tubular receptors respond differently during high Ang II states (Navar et al., 2002). In general, high Ang II levels associated with a low-salt diet decrease glomerular AT1 receptor expression but increase tubular AT1 receptor levels (Cheng et al., 1995). Studies in two-kidney, one-clip Goldblatt hypertensive rats demonstrated that glomerular AT1 receptors were decreased by 2 weeks after clipping, but vascular receptors were not decreased until 16 weeks (Amiri and Garcia, 1997). However, glomerular AT1 receptor density was not increased in the one-kidney-one-clip model, although vascular AT1 receptor density was increased (Amiri et al., 1999). In the Ang II-infused rat model of hypertension, total kidney AT1 receptor mRNA levels and protein levels were not suppressed but were maintained by 2 weeks of Ang II infusion sufficient to cause marked hypertension (Harrison-Bernard et al., 1999). However, Wang et al. (1999) reported that type 1 receptor protein was reduced in both ischemic and contralateral kidneys of two-kidney, one-clip Goldblatt and two-kidney, one-wrap Grollman hypertensive models and in kidneys of Ang II-infused rats. AT2 receptors were down-regulated only in ischemic kidneys. In transgenic rats harboring the mouse Ren2 renin gene, Zhuo et al. (1999) found increased AT1 receptor binding in vascular smooth muscle of afferent and efferent arterioles, juxtaglomerular apparatus, glomerular mesangial cells, proximal tubular cells, and renomedullary interstitial cells. It was suggested that up-regulation of AT1 receptors in multiple renal cells may contribute to the pathogenesis of hypertension in these rats. Harrison-Bernard et al. (2002) extended the analysis in Ang II-infused rats with in vitro autoradiography and showed differential responses with significant decreases in glomeruli and inner stripe but not in proximal tubules. Furthermore, ACE binding was significantly increased in proximal tubules of Ang II-infused rats. Thus, vascular and glomerular AT1 receptors are down-regulated, but the proximal tubular receptors are either up-regulated or not sign
