Renin Inhibition: What Are the Therapeutic Opportunities?

Home , Captopril, Enalaprilat, Enzyme inhibitor, Lisinopril, Pepstatin, Remikiren

Special Commentary

Naomi D.L. Fisher and Norman K. Hollenberg Departments of Medicine and Radiology, Brigham and Women’s Hospital and Harvard Medical School, Boston, Massachusetts

Blockade of the renin-angiotensin system with angiotensin-converting enzyme inhibitors or angiotensin receptor blockers has become a crucial element in cardiovascular and renal medicine. This review evaluates the potential of renin inhibition as an adjunct to therapies that depend on renin system interruption. J Am Soc Nephrol 16: 592–599, 2005. doi: 10.1681/ASN.2004100874

hree years ago, we responded to an invitation from Pharmacologic Interruption of the Renin- Expert Opinion in Investigative Drugs to address the cur- Angiotensin System T rent status of renin inhibition (1). We bemoaned that all There are a number of steps (Figure 1) at which the renin development of renin inhibitors seemed to have come to an system can be interrupted (1). ␤-Adrenergic blocking agents end. The decision to close renin inhibitor development pro- reduce renin release. ACE inhibitors block the conversion of grams reflected the cost of their synthesis, continuing problems Ang I to Ang II downstream from the renin step. ARB interfere with bioavailability, and the remarkable success of a major with the interaction of the hormone Ang II with a specific AT1 competitor: the angiotensin II (Ang II) antagonist class. We receptor. Renin inhibition interferes with the first and rate- pointed out the possibility that alternative approaches based on limiting step in the cascade, interaction of renin with its sub- x-ray crystallography and reconstruction of the structure of the strate angiotensinogen (AGT). No pharmacologist would have active site would lead to novel agents. The opportunity pro- chosen the ACE step for inhibition. As pointed out by Skeggs, vided by this approach is attractive because renin is a target the rate-limiting step, blocking the catalytic effect of renin on its that is actually soluble and amenable to x-ray crystallographic substrate, would have been the logical first choice. ACE inhi- studies (2). Unbeknownst to us at that time and clearly unre- bition developed first as an unintended consequence of the lated to our call for action, there were at least four programs search for an explanation for the drop in BP induced by the aiming at novel renin inhibitors based on crystallography, as venom of a pit viper (6). The venom was found to contain a reviewed below. This is a stage in the field where information peptide, which acted as a “bradykinin-potentiating factor”: from patent applications is often more useful than the biomed- ical literature (2). Pharmacologic consideration suggested that an inhibitor of bra- The current development of candidate agents provided the dykinin metabolism might also be an inhibitor of ACE, as both primary rationale for this update. Recent advances in our un- substrates are cleaved by a peptidyl-dipeptide hydrolase. That derstanding of pathophysiology provide additional reasons for reasoning proved to be correct (6). interest. One such evolution, involving the possibility that a The renin step is a very attractive target for another reason, renin binding site might translate the effect of prorenin from a the remarkable specificity of renin for its substrate (1). In Figure bystander into a contributor to disease, is immediately relevant 2, the species specificity of a renin inhibitor for human and to the renin inhibitor story (3). Also, recent interest in the marmoset plasma renin is contrasted with the inhibition of dog efficacy of combinations of angiotensin-converting enzyme and rat renin. The marmoset is a primate. In the same figure, (ACE) and angiotensin receptor blocker (ARB) to therapeutics the inhibition of human renin is contrasted with inhibition of is relevant (4). If a combination of agents is to be used for two other aspartic proteinases, pepsin and cathepsin-D, both of maximal efficacy, then it would make good therapeutic sense which are far less responsive to the renin inhibitor. This inher- for one of the agents to be acting at the level of the rate-limiting ent specificity has clinical implications, as it reduces the likeli- step (2). Indeed, this field can trace its origins to this consider- hood of unwanted additional interaction and thus side effects. ation. Skeggs et al. (5) argued in 1957 that “since renin is the A third consideration involves the state of the various ele- initial and rate-limiting substance in the renin-angiotensin sys- ments of the renin-angiotensin cascade on blockade. Both ACE tem, it would seem that renin inhibition would be [ellip] the inhibitors and ARB lead to a reactive renin rise and thus to an most likely to succeed.” increase in the angiotensin peptides, both Ang I and Ang II in the case of ARB and Ang I in the case of the ACE inhibitors.

Published online ahead of print. Publication date available at www.jasn.org. Only renin inhibition will render the renin-angiotensin system (RAS) quiescent, suppressing all of the Ang I–derived prod- Address correspondence to: Dr. Norman K. Hollenberg, Brigham and Women’s Hospital, Harvard Medical School, 75 Francis Street, Boston, MA 02115. Phone: ucts. Whether that is an advantage, only time and future stud- 617-732-6682; Fax: 617-232-2869; E-mail: [email protected] ies can tell.

concentration, the most widely used index of RAS activity, is not increased—indeed, it is suppressed. The mechanism that is responsible for the increase in plasma prorenin remains ob- scure. Equally mysterious is the mechanism of the possible contribution of prorenin to the pathogenesis of the microvascular injury. In the case of the eye, a fascinating series of experiments identified prorenin in the human and bovine eye (12–14): Prorenin was elevated in the vitreous humor in association with proliferative retinopathy. The relation of prorenin to total renin is different in the patient with diabetes and in other patients in whom the renin system has been activated through the use of oral contracep- tives, renovascular hypertension, or treatment with an ACE inhibitor. In the patient with diabetes, the increase in prorenin exceeds substantially the increase in prorenin in the other conditions (26). Although the increase in prorenin in plasma has Figure 1. The key steps by which pharmacologic antagonists or been attributed to a range of factors, including glycation of a blockers interfere with the renin-angiotensin system are shown responsible processing enzyme or autonomic dysfunction (7), together with representative plasma concentrations from rats. neither factor would account directly for the association with Reproduced from reference 48 with permission. both retinopathy and nephropathy, and they are not directly compatible with the documented suppression of renin release in the same patients. We are reasonably confident that these issues, as interesting as they are, have never been featured in an article dealing with renin inhibition. Why have we done so? The answer involves a fascinating, possibly relevant, and potentially very important study by Nguyen et al. (3). This group reported the identification of a renin receptor in the human kidney. This receptor is localized primarily in the mesangium of glomeruli but also in the subendothelium of arteries of the heart and kidney. Its function is fascinating. First, the binding of renin to this receptor induced a four- to five-fold increase in the catalytic efficiency of conversion of AGT to Ang I. Second, prorenin was found to bind the renin receptor, and when prorenin was bound, it was as active as renin in inducing a biologic response. Finally, receptor occupation by renin or prorenin led to an intracellular signaling process associated with activation of mitogen-activated protein kinases, ERK-1 and ERK-2, without involvement of angiotensin generation. If these actions of prorenin contribute to pathophysiology, as seems likely, and if the renin inhibitors interrupt this sequence, as seems possible, then Figure 2. Species specificity of the renin inhibitor CGP 29287. renin inhibitors would bring to the treatment of the patient with Note that the two primate sources of renin, human and mar- diabetic nephropathy or retinopathy an opportunity that far sur- moset, were blocked with low concentrations of the inhibitor, whereas much higher concentrations were required to block passes what an ACE inhibitor or an Ang II antagonist can do. canine and rat renin. Below the blockade of human renin with this agent is contrasted with the very poor efficacy on pepsin Opportunities Created by Combination and cathepsin-D, other aspartic proteinases. Reproduced from Therapy reference 26 with permission. Is blockade induced by an ACE inhibitor “complete”? A related term is “break through,” in which plasma Ang II concentration begins to rise toward the pretreatment level. ACE Opportunities Related to Prorenin and the inhibition and ARB, via the short feedback loop, lead to RAS Intrarenal Renin Receptor activation. The ACE inhibitors are competitive antagonists and, Multiple studies have shown that the onset of microvascular thus, compete with Ang I for the receptor site on the enzyme. disease in the patient with diabetes, both nephropathy and Do Ang I levels rise as a consequence of the renin response to retinopathy, coincides with elevated concentrations of prorenin blockade, rise to levels that lead to a return of Ang II formation? in plasma (7–15). The onset of microvascular disease and the The answer is that they often do. Can we overcome that by increase in prorenin are not necessarily associated with hyper- increasing the ACE inhibitor dose? Of course we can, except the tension (10). Moreover, in this setting, plasma active renin side effects tend to become intolerable. 594 Journal of the American Society of Nephrology J Am Soc Nephrol 16: 592–599, 2005

For related reasons, a substantial number of reports have Thus, findings raise a crucial question for combination therapy: described the influence of a combination of an ACE inhibitor or “How important, quantitatively, is the reactive renin response to an ARB, typically with proteinuria as the end point (4,16–21). blockade with an ACE inhibitor or ARB?” The effects on protein- Without exception, these studies report that the combination uria of a combination of an ACE inhibitor and an ARB suggest was more effective in reducing proteinuria than either agent that the reactive response is quantitatively important. We believe alone. Such studies, to be truly informative, must deal effec- that there are powerful reasons for performing an experiment in tively with the issue of dose. To our way of thinking, none has which an ACE or an ARB is combined with a renin inhibitor in the done so. As ARB are far better tolerated than ACE inhibitors, an patient with proteinuria. Blockade at the most sensitive step is optimal study would involve assessing the influence of the likely to provide the best possible therapy. ARB at the top of its dose-response relationship for proteinuria reduction and then ascertaining whether adding an ACE inhib- Renin Inhibitors itor improves the therapeutic response. Renin belongs to the aspartic proteinase family, which in- Because to date no study has been designed to meet that cludes pepsin, cathepsin-D, and chymosin (26). This class of demanding criterion, the interpretation of the available studies enzyme is named because of a crucial feature: Each enzyme has must be limited. What is clear, however, is that increasing the two aspartic acid residues in their active site that are necessary degree of renin-system blockade at any step improves the ther- for catalytic activity. Three-dimensional models of human re- apeutic response. Our next challenge is to work out how best to nin, based on x-ray crystal structure of other aspartic protein- achieve that goal. In combination therapy, it seems intuitively ases, reveal co-planar, symmetrically arranged ␤-carbonyl obvious that one of the steps blocked should be the rate- groups of the aspartic acid residues at 38 and 226, held in an limiting step. intricate web of hydrogen bonds. The active site appears as a long, deep cleft that can accommodate seven amino acid units Opportunities Created at the Rate-Limiting of substrate. The enzyme also contains a mobile flap that, when Step closed, lies across the cleft and probably holds substrate in the Most of us carry around a notion of the rate-limiting step active site (27). Renin has a similar bilobal structure, consistent from the course in biochemistry that we took many years ago. with the hypothesis that aspartic proteinases, including renin, Enzyme-catalyzed reactions often consist of several component arose from a common ancestral gene (28). Their common cata- steps. These different controlling steps may limit the rate of the lytic sequence involves hydrolysis: Water stereospecifically at- overall reaction to different extents. Each step is characterized tacks the carbonyl of the scissile amide bond (29). by a rate constant. Most of us were taught that the step with the Proof of principle that blocking the renin step would produce lowest rate constant is rate limiting or the determinant of the a biologically important response first came from an immuno- rate of the overall reaction. However, as often pointed out logic approach. Although initially antisera produced against (22–24), this definition can be misleading. As conditions crude renin extracts were used, these were followed by poly- change, the different specific activity for different enzymatic clonal and monoclonal antibodies against purified renin for reactions that determine reaction velocity can vary. As an al- multiple species (30). Indeed, the most potent inhibitor of hu- 11 ternative, it has been argued that for steady state, the rate- man renin described so far had an IC50 of 10 M (26). limiting step is the “most sensitive” step, i.e., the step that, if Pepstatin, a peptide derived from microbial metabolism, acts perturbed, causes the largest change in overall velocity (23). By as a competitive inhibitor of most aspartic proteinases, with an classical criteria, the renin-AGT interaction is the rate-limiting IC50 of 0.05 nmol for pepsin, for example. Unfortunately, it step for the renin cascade. Figure 1 provides the necessary proved to be a weak inhibitor of renin with an IC50 of 10 nmol information. Navar et al. (48) recorded the plasma concentra- (31). Statin, an unusual amino acid found in pepstatin, is tion of the various elements of the cascade in the rat. AGT thought to act as a “transition state” mimic, resembling the concentration is in the mid to high pmol range. Ang I, con- tetrahedral conformation of the natural substrate at the scissile versely, is in a low fmol range—more than three orders of bond (32). This amino acid proved to be an important part of magnitude lower. Ang II concentration is slightly lower. Thus, the structure: Action evolution later. in the comparison of the renin and ACE steps, the renin step The approach that followed for the early development of provides the largest step down in concentration. By the newer renin inhibitors involved two different pathways (33), as sum- and more flexible definition, it is not yet clear whether the renin marized in Figure 3. One was based on the hypothesis that the step is always rate-limiting—although compelling data that do prosegment of prorenin is capable of inhibiting enzyme activity so indicate have been presented (25). Despite a remarkable by preventing access to the substrate. This pathway developed increase in plasma renin mass after administration of the renin only weak inhibitors. The other approach was to synthesize inhibitor aliskiren, plasma Ang I and Ang II concentration peptic analogs of the N-terminal amino acid sequence of the remained suppressed. Taken in all, the available data suggest substrate AGT. Substitution of the amino acid at the AGT that in the plasma compartment, the renin step is always rate scissile bond (positions 10 and 11) produced octapeptide ana- limiting. Whether that extends to tissue compartments remains logs that were active competitive inhibitors. The most effective, open. There may be circumstances in which the substrate con- known as “renin inhibitory peptide,” had an inhibitory potency centration is very much lower and in which the ACE step or the against human renin in the micromolar range and was suffi- number of AT1 receptors becomes rate limiting. ciently active to be useful for research (33). Over time, the J Am Soc Nephrol 16: 592–599, 2005 Renin Inhibition and the Kidney 595

Opportunities Created by the Development of New Agents Most of the early renin inhibitors involved variation on the original peptide structure. The possibility that alternative approaches to the formulation of renin inhibitors based on x-ray crystallography and reconstruction of the structure of the active site would lead to novel agents has recently found remarkable success. Four groups have developed novel agents on the basis of this principle (Figure 4). The most advanced agent, aliskiren, was developed by Speedel and Novartis. Although Figure 4 lists an activity of 0.6 nM, in fact the activity might be substantially greater. It has been tested in humans (25,35) and is enter- ing Phase III evaluation. The agents developed by Actelion-Merck and by Pfizer (Fig- ure 4) are much earlier in development. The agents developed by these two groups show some similarities in structure but a different level of activity in early reports. Concurrent Pharma- ceuticals has also developed renin inhibitors of two major classes with sufficient activity to be of substantial interest. No information on the bioavailability of the last three groups of agents in humans has yet been reported.

Efficacy in BP Reduction A number of studies on BP in various animal models have shown either equivalence of ACE and renin inhibition or a small advantage of one or the other (26). The principle that blocking renin would reduce BP was established early in humans (34). The most instructive studies have been those in which responses to an ACE inhibitor and to a renin inhibitor were assessed in the same patients. Jeunemaitre et al. (36) compared CJP-38560A, a renin inhibitor, with captopril, both at what were considered to be maximal doses: 0.25 mg/kg of the renin inhibitor and 50 mg of captopril. Salt intake was unrestricted, presumably fairly high in a French Figure 3. Structure and potency of renin inhibitors developed population. The patients had a marked fall in mean BP after approximately in sequence. Reproduced from reference 33 with captopril (15.3 Ϯ 1.5 mmHg) but showed only a limited (6.4 Ϯ permission. 1.2 mmHg) mean BP fall after the renin inhibitor. Thus, if this renin inhibitor was actually used at the maximum dose, then peptide scissile bond was replaced by other transition state mi- captopril had an additional action unrelated to renin in these metics, including a reduced peptide bond or hydroxy-ethylene selected patients. group (26). These synthetics produced a surfeit of potent. The Neutel et al. (37) reported precisely opposite findings in a result was inhibitors with potency in the nM range (Figure 3). comparison of intravenous placebo enalaprilate (1.25 mg intra- Having achieved sufficient potency, emphasis shifted from the venously) and enalkiren (in a dose range from 0.03 to 1 mg/kg) development of agents that would retain potency to those that in hypertensive patients who were hydrochlorothiazide treated would also be bioavailable (Figure 3). The best of these agents, to activate the renin system. The acute decrease in systolic BP of remikiren and enalkiren, were very potent and active on paren- 18.5 Ϯ 0.4 mmHg during enalkiren was substantially greater teral administration but showed oral bioavailability that was sub- than the 12.6 Ϯ 0.7 mmHg during enalaprilate (P Ͻ 0.01). The stantially less than 10%, more often in the 1 to 2% range (33). fall in diastolic BP during enalkiren (11.9 Ϯ 0.4 mmHg) was also Over time, progress was being made in dealing with the slightly greater than during enalaprilate (9.2 Ϯ 0.4 mmHg; P Ͻ bioavailability problem. The last three agents shown in Figure 0.01). In the patients in whom the plasma renin activity rose 3, FK906, zankiren, and A-74273, all showed some improve- most, a similar pattern emerged, but the differences were non- ment in oral bioavailability and efficacy in animal studies and significant in the low or normal renin group. This analysis in humans (33). Unfortunately, at this crucial stage, all of the might explain the negative findings described above by Jeun- programs were canceled for commercial reasons (27). The rapid emaitre et al. (18) in individuals on a liberal salt intake. advance of the Ang II antagonists was thought by the decision Kiowiski et al. (38) compared another renin inhibitor, R0–42- makers to make ultimate commercial success unlikely. 5892, with captopril (50 mg). The renin inhibitor was given by 596 Journal of the American Society of Nephrology J Am Soc Nephrol 16: 592–599, 2005

Figure 4. Structure, activity, and stage of development of renin inhibitors in current evaluation. All were developed on the basis of x-ray crystallography and active site modeling.

mouth in a 600-mg dose. In these normal individuals, captopril were testing when we first assessed the renal hemodynamic re- decreased diastolic BP from 60 Ϯ 5to51Ϯ 7 mmHg, and the sponse to a renin inhibitor was straightforward (42,45): To the renin inhibitor did not. Salt intake was described as normal. extent that ACE inhibitors reduce kinin degradation and thereby RO-42-5892 was thought to be poorly bioavailable. induce vasodilator prostaglandin formation or activation of endo- Stanton et al. (35) compared the BP response to aliskiren, dose thelial nitric oxide release, the renal vasodilator response to ACE range 37.5 to 300 mg/d, with losartan at a dose of 100 mg/d. inhibition should exceed the response to renin inhibition. Indeed, Aliskiren induced a dose-related fall in daytime ambulatory BP we thought that the difference in magnitude of the renal vascular averaging 11.0 mmHg for the highest dose: The daytime sys- response would provide a measure of the magnitude of the con- tolic BP fall with 100 mg of losartan was an essentially identical tribution of other vasodilators compared with reduced Ang II 10.9 mmHg. formation. To our surprise, the renal vasodilator response to the In none of the studies was the opportunity taken to add the renin inhibitor enalkiren exceeded expectations from our experi- ACE inhibitor to the renin inhibitor or the renin inhibitor to the ence with ACE inhibitors (42). In a more rigorous follow-up, a ACE inhibitor when a nadir in BP had been reached. Taken in three-arm design was used to compare placebo; the same renin all, the conclusion would be that the more renin-dependent the inhibitor, enalkiren; and captopril in random order and double BP level, the larger the fall induced by renin inhibition. As there blind. The placebo did nothing. Captopril and enalkiren both led are no studies that go beyond very brief intervals of drug to striking renal vasodilation (43). The response to enalkiren again administration, the ultimate implications of these studies for was significantly larger than the response to captopril. The mag- therapeutics are limited. nitude of the response to the renin inhibitor in this study con- firmed our earlier observation as did a more recent study with Efficacy: Renal Responses another renin inhibitor, zankiren, that induced a larger renal va- Ang II plays a crucial role in the normal control of the renal sodilator response than could have been anticipated from the ACE circulation and renal function. Moreover, the remarkable effi- inhibitor experience (44). cacy of ACE inhibition in limiting renal injury in patients with The renal vascular response to renin inhibition at the top of diabetes and other forms of nephropathy placed a high priority the dose-response curve in these three studies is summarized in on examining the influence of renin inhibitors on the renal Figure 5 in a meta-analysis format (28). All studies were per- blood supply and kidney function. When our studies in hu- formed in healthy men who were Ͻ40 yr of age and who were mans began, there was already substantial experience in animal in balance on a 10-mEq sodium intake to activate the renin models to suggest that renin inhibitors would induce a robust system. All were studied by the same group of physicians, renal vasodilator response (39–41). The hypothesis that we nurses, dietitians, and technicians on the same metabolic ward J Am Soc Nephrol 16: 592–599, 2005 Renin Inhibition and the Kidney 597

with species, renin inhibitors display species specificity: The guinea pig was selected because R042-5892, a renin inhibitor developed for primates, is also effective in the guinea pig. Doses of the agents were adjusted to induce an identical, small, but unambiguous fall in BP and were administered coded and in a random sequence. This careful, elegant study design re- vealed remarkable differences in the overall response to the three classes of agent. Despite an identical fall in BP, the renin inhibitor induced a substantially larger increase in renal plasma flow, GFR, diuresis, and natriuresis—as in the humans (46). The authors suggested that the potentiated response to renin inhibition could reflect greater lipophilicity and tissue penetration, leading to a local, intrarenal action at the site of Ang II production. As an alternative, the guinea pig might share with humans substantial non–ACE-dependent conversion of Ang I to Ang II, perhaps via chymase (47). Taken in all, the data suggest strongly that in the case of the kidney, renin inhibition is far more effective than ACE inhibition in blocking Ang II formation. These observations clearly could have clinical significance.

What Is the Future for Renin Inhibition? Although for a time it seemed that all renin inhibitor development had been closed, it is now clear that the search for an orally bioavailable renin inhibitor continues (49–51). We believe that the potential of renin inhibition in human therapy has been underestimated and indeed shows substantial promise. Should clues concerning microvascular disease at the level of Figure 5. Change in renal plasma flow in response to angioten- the kidney and the eye and the possible influence of a renin sin-converting enzyme (ACE) inhibitors and renin inhibitors, inhibitor prove to be correct, then this pathway will have led to each at the top of the relation between dose and renal vascular truly important advances in treatment. Diabetic nephropathy response. Note that the three ACE inhibitors induced a maxi- and diabetic retinopathy are, respectively, the leading cause of 2 mal response in the 90- to 100-ml/1.73 m per min range, ESRD and blindness in the United States and much of the rest whereas the renin inhibitors induced a significantly larger re- of the world. sponse. using the same techniques for measurement of p-aminohippu- References rate clearance as the index of renal perfusion. Also shown in 1. Fisher NDL, Hollenberg NK: Is there a future for renin inhibitors? Expert Opin Investig Drugs 10: 417–426, 2001 Figure 5 are responses to three ACE inhibitors, each study 2. Maibaum J, Feldman DL: Renin inhibitors as novel treat- performed at the top of the dose-response curve of renal blood ments for cardiovascular disease. Expert Opin Ther Patients flow. All were performed over the same time interval and 13: 589–603, 2003 under the same conditions. In the special case of the captopril: 3. Nguyen G, Delarue F, Burckle C, Bouzhir L, Giller T, Sraer enalkiren comparison, the same subjects were involved. By J-D: Pivotal role of the renin/prorenin receptor in angio- ANOVA, there was no statistical difference identified among tensin II production and cellular responses to renin. J Clin the three ACE inhibitors or between the two renin inhibitors, Invest 10: 1417–1427, 2002 but a striking difference between the ACE and renin inhibitors 4. Segura J, Christiansen H, Campo C, Ruilope LM: How to was highly significant (P Ͻ 0.001). The renal vasodilator re- titrate ACE inhibitors and angiotensin receptor blockers in sponse to renin inhibition in healthy humans under these con- renal patients: According to blood pressure or proteinuria? ditions, in the range of 140 to 150 ml/min per 1.73 m2, exceeded Curr Hypertens Rep 5: 426–429, 2003 5. Skeggs LT, Kahn JR, Lentz KE, Shumway NP: Preparation, by Ͼ0% the response to ACE inhibitors, which averaged ap- purification, and amino acid sequence of a polypeptide proximately 90 to 100 ml/min per 1.73 m2—when theory said it renin substrate. J Exp Med 106: 439–453, 1957 should be less. 6. Ondetti MA: Design of specific inhibitors of angiotensin- In a subsequent study, El-Amrani et al. (46) compared sys- converting enzyme: New class of orally active antihyper- tematically the renal vascular response to a renin inhibitor tensive agents. Science 196: 441–444, 1977 (R042-5892), an Ang II antagonist (losartan), and an ACE inhib- 7. Skinner SL: The pathophysiology of prorenin. In: The Re- itor (lisinopril) in guinea pigs. Because renin structure varies nin-Angiotensin System, edited by Ian J, Robertson S, Ni- 598 Journal of the American Society of Nephrology J Am Soc Nephrol 16: 592–599, 2005

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