Introduction

The renin–angiotensin–aldosterone system (RAAS) is an ancient pathway1 that has evolved into a central mechanism by which mammalian blood pressure (BP) and fluid homeostasis are regulated. Research in this field started with the discovery in 1898 by Tigerstedt and Berman2 that a renal extract when injected into rabbits induced a rapid increase in systemic BP. Subsequent work over many years established the RAAS as a pivotal contributor to cardio-renal diseases. Hence, inhibitors of the middle and distal portions of the RAAS pathway, angiotensin-converting enzyme (ACE) inhibitors (ACEI) and AT1 receptor blockers, respectively, have become mainstay therapies for treating hypertension. In 2007, aliskiren, the first direct renin inhibitor (DRI), was approved for the treatment of hypertension. This heralded the therapeutic control of BP by inhibiting the RAAS at its first and rate-limiting step. However, RAAS blockade is also effective for treating renal disease3, 4, 5, 6 and the efficacy of aliskiren for this purpose is currently being investigated. Accordingly, the subject of this review is to summarize the preclinical evidence for renoprotection by aliskiren, and to discuss recently published information on the possible mechanism(s) for these benefits. As it is well recognized that there is a tissue as well as circulating RAAS,7 the preclinical actions of aliskiren will be discussed in the context of the intrarenal RAAS and the potential for its inhibition by aliskiren.

Intrarenal RAAS

Goormaghtigh8 first proposed that renin was produced just outside of the glomerulus in juxtaglomerular (JG) cells. However, work over the past several decades indicates a broader pattern of intra-glomerular RAAS expression. Indeed, the kidney possesses a fully functional tissue RAAS9, 10, 11 with expression along virtually the entire nephron.

Gene and protein expression of angiotensinogen (Aogen), renin and ACE has been reported in cultured glomerular mesangial cells12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 and podocytes.25, 26, 27, 28 Mesangial expression of Aogen and ACE has also been documented in renal tissue sections.19, 23

Mesangial cells synthesize Ang II22, 24 and aldosterone, the latter apparently through an Ang II-dependent mechanism.29 Podocytes also produce Ang II25, 26, 28 and aldosterone.30 Angiotensin type 1 receptors are expressed on mesangial cells19, 24, 31 and podocytes26, 28, 32 and mineralocorticoid receptors are also expressed by both cell types.29, 33, 34

The likely importance of the local glomerular RAAS relates to the pathogenesis of glomeruloscleorsis and the loss of glomerular permselectivity observed in many glomerular diseases. In cultured mesangial cells, Ang II35, 36, 37 and aldosterone38, 39 provoke synthesis of extracellular matrix (ECM) proteins that accumulate in glomerulosclerosis. In podocytes, Ang II has been shown to induce apoptosis,40, 41 cytoskeletal rearrangement42 and nephrin loss43 in podocytes, alterations that have been linked to albuminuria.44 Aldosterone can also damage podocytes by inducing apoptosis and reducing nephrin gene expression.30, 33, 45 Thus, through autocrine or endocrine mechanisms, Ang II and aldosterone may induce damage and/or pro-fibrotic pathways in mesangial cells or podocytes, and thus contribute to glomerular damage.

Renal tubular cells express all of the components of the RAAS. Renin mRNA and protein are expressed in the proximal and connecting tubules, as well as the collecting ducts.19, 46, 47, 48, 49, 50, 51, 52, 53, 54 Interestingly, mechanisms for regulating tubular renin seem to be independent of those in the JG cell.47, 48, 50, 52 Proximal tubular cells express mRNA54, 55, 56 and protein53, 57 for ACE and Aogen.58, 59, 60, 61 In vivo evidence that proximal tubular cells may synthesize Ang II has also been reported.62

The above studies indicate the presence of a functional renal tubular RAAS, which likely has an important function in regulating sodium and fluid balance, and hence BP. However, the tubular RAAS may also be linked to the pathogenesis of tubulo-interstitial fibrosis. For instance, Ang II induces epithelial-mesenchymal transformation in tubular cells,63 a process thought to presage renal fibrosis.64

Renoprotective effects of renin inhibition with aliskiren

Aliskiren is a competitive inhibitor of renin; it binds to the active site of the enzyme and thereby prevents access to its substrate, Aogen. In turn, activation of the RAAS cascade is blocked. Given the above evidence that the primary and damaging effector molecule (Ang II) of the RAAS is made at many sites throughout the kidney, and that inhibition of renin blocks the RAAS at the first and rate-limiting step in Ang II formation, it is reasonable to propose that a DRI may provide distinct renoprotective benefits in a variety of renal diseases. The ensuing discussion will outline the evidence that the DRI aliskiren ameliorates renal damage in experimental renal disease. However, let us first consider some aspects of aliskiren that bear on its use in experimental settings.

Testing aliskiren in animal models

Certain important characteristics of aliskiren impact its use in animal models. First, because aliskiren is a human renin inhibitor, it is a less potent inhibitor of rodent renin than human renin65 (Table 1). Thus, although rodent models of renal disease are very commonly used, models that express rat renin are not ideal for testing aliskiren. This issue can be circumvented by using a transgenic rat model with an activated RAAS. The so-called double transgenic rat66 expresses the human genes for renin and Aogen, and thereby provides a model in which a human DRI can inhibit human renin in a rodent. A second model, the TG(mRen-2)27 (mRen-2) rat expresses the mouse ren-2 gene,67 and takes advantage of the capacity of mouse renin to cleave rat Aogen. Aliskiren is effective in this model as the IC50 of the drug against mouse renin is relatively low (Table 1). The organ damage seen in dTGR68 and mRen-2 rats69 is ameliorated by RAAS blockade, giving relevance to testing aliskiren in these models.

Table 1 Inhibitory activity of aliskiren against human and rodent renin
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A second relevant property of aliskiren is its low oral bioavailability in rodents, in which oral dosing of this DRI does not result in good efficacy. Rather, consistent efficacy is seen when aliskiren is administered to rodents by subcutaneously implanted osmotic minipumps.

Aliskiren protects against experimental renal disease

Hypertensive renal disease

The first evidence for renoprotection by aliskiren was reported in dTGR. The activated RAAS in these animals leads to severe hypertension with renal damage, including albuminuria, elevated serum creatinine, renal infiltration of macrophages, and ECM deposition.68, 70 In this model, aliskiren (0.3 or 3 mg kg–1 per day) normalized the BP and reversed existing renal damage, evidenced by normalizing albuminuria and serum creatinine levels. Furthermore, glomerular collagen IV accumulation and renal macrophage and lymphocyte contents were lowered.71 This paper also showed a reduction in renal cortical Ang I and Ang II levels in aliskiren-treated dTGR, thus documenting an inhibitory effect of this DRI on the intrarenal RAAS. Moreover, Ang I-forming capacity of the sera from these rats was also significantly reduced. A subsequent report by this group72 showed that treatment with aliskiren inhibited complement activation and cellular infiltration in kidneys of dTGR. These studies showed the efficacy of aliskiren in high Ang II-induced renal injury and suggested a beneficial effect of the drug in complement-dependent renal disease. Notably, an earlier study of dTGR showed that when the renin inhibitor remikiren and a non-RAAS blocking anti-hypertensive therapy were compared at comparable BP-lowering doses, remikiren showed stronger renoprotection,70 suggesting BP-independent effects of renin inhibition.

In both the above studies,71, 72 AT1 receptor blockers were included as positive controls; they were not intended to be comparator agents. These drugs were administered at different dosages and by different routes of administration than aliskiren. Although comparable renoprotective effects were observed with aliskiren and its comparator in each of these studies, conclusions on comparative efficacy vs. aliskiren could not be made.

In this regard, we tested in mRen-2 rats the effects of 4 weeks of treatment with aliskiren or enalapril, both administered through osmotic minipumps to achieve constant plasma levels of each drug, at doses that achieved comparable tail cuff BP control (Figure 1a) (Avigdor, Hu, Jen, and Feldman, unpublished data). Compared with vehicle controls, albuminuria was reduced similarly by both therapies (Figure 1b), as were plasma levels of Ang II and aldosterone (Table 2) at the end of the experiment. This study indicated that when drug delivery and BP lowering were similar, aliskiren induced comparable reductions in albuminuria and blockade of the circulating RAAS, including lowering plasma aldosterone.

Figure 1
figure 1

Effect of aliskiren and enalapril on blood pressure (a) and albumin excretion (b) in mRen-2 rats. mRen-2 rats were treated with aliskiren or enalapril by subcutaneous osmotic minipumps. Blood pressure was measured by the tail cuff method and urinary albumin levels were measured by ELISA. *P<0.05 vs. vehicle.

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Table 2 Effect of aliskiren enalapril on plasma Ang II and aldosterone levels in mRen-2 rats
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Endothelial nitric oxide synthase (eNOS) regulates renal hemodynamics and renal function.73 Deficiencies in eNOS lead to accelerated renal damage in models of renal disease,74 highlighting the importance of nitric oxide in renal health. Recently, aliskiren was shown to inhibit the renal disease that develops in eNOS−/− mice.75 Increases in albuminuria, glomerulosclerosis and renal macrophage infiltration observed in the vehicle-treated knockout mice were significantly reduced by aliskiren treatment (25 mg kg–1), as were levels of glomerular superoxide and renal NADPH oxidase. Importantly, hydralazine given at a dose that lowered tail cuff BP similarly to aliskiren did not show these beneficial effects. This work showed that the renoprotective actions of aliskiren do not require eNOS-mediated pathways. Moreover, this protection seems to result at least partially from effects that are beyond BP control, possibly by inhibiting Ang II-induced damaging effects in the kidney.

The anti-inflammatory effect of aliskiren observed in the kidneys of dTGR71 and eNOS−/− 75 mice is not a renal-specific effect. Recently, Ino and coworkers76 reported that at a non-BP-lowering dose, aliskiren reduced leukocyte adhesion in a murine vascular injury model. Furthermore, this effect of aliskiren was associated with a reduction in the injury-induced up-regulation of adhesion molecules on the vascular endothelium, pointing toward a possible mechanism for the reductions in inflammatory infiltrates noted above in the kidneys of dTGR71 and mice.75

Diabetic nephropathy

Diabetic nephropathy (DN) is the leading cause of end-stage renal disease in the developed world.77 The RAAS is thought to have a central function in the pathogenesis of this disease, evidenced by numerous studies showing that RAAS blockade by ACEIs and AT1 receptor blockers slows the progression of DN.3, 4, 5, 6 However, these treatments do not halt the progression of this disease. Thus, the underlying mechanisms for progression of DN continue even in patients treated with accepted therapies. The intrarenal RAAS is thought to be activated during diabetes, based on in vitro and in vivo evidence.21, 22, 24, 25, 48, 54, 55, 69, 78 Therefore, the effect of renin inhibition in diabetic conditions is of great interest.

In vitro effects of aliskiren in high glucose conditions

Albuminuria is the first clinically detectable change in renal function in DN, resulting from a reduction in permselectivity of the glomerular capillary wall. The glomerular permeability barrier consists of three layers, the outer most glomerular visceral epithelial cells (podocytes) that cover the (middle) glomerular basement membrane, which is lined on its luminal side by the inner layer, the fenestrated endothelium. Podocytes seem to have a key function in preventing the escape of plasma proteins across the glomerular capillary wall into the urine. Their involvement in the development of albuminuria and glomerulosclerosis in DN is increasingly accepted.44, 79

During diabetes, podocytes are exposed in vivo to high glucose levels and stretch forces, the latter because of glomerular hypertrophy and hyperfiltration. When these conditions are modeled in vitro in mouse podocytes, the RAAS becomes activated. Indeed, podocytes subjected to mechanical stretch show Ang II-mediated apoptosis.41 Aliskiren was tested for its effects on cultured mouse podocytes exposed to high glucose levels.25 In such podocytes, high glucose induced increased expression of renin mRNA and protein, and Ang II formation was detected. The latter was not inhibited by the ACEI captopril, but it was suppressed by aliskiren and the non-selective chymase inhibitor chymostatin. These findings suggest that high glucose conditions activated the podocyte RAAS at the level of renin, leading to increased Ang II formation. If we can assume that captopril actually entered the podocytes in this study, the results further indicate that Ang II formation in these cells was mediated primarily by non-ACE pathways (that is, chymase). To the extent that the up-regulation of glomerular chymase in diabetic kidneys18 may suggest a function for ACE-independent Ang II formation in DN, renin inhibition may have a protective advantage over ACEI in this disease. Thus, these in vitro results may be an example in which blocking the RAAS at the top of the cascade (that is, at renin) may achieve different outcomes than by inhibiting elsewhere in the pathway.

The loss of podocytes from the glomerular capillary wall is associated with the development of albuminuria80 and glomerulosclerosis,81 and apoptosis of podocytes has been suggested as a mechanism for such loss.44 Phillips and coworkers82 reported that aliskiren attenuated the high glucose-induced increase in cleaved caspase-3 in cultured mouse podocytes, suggesting a protective effect of aliskiren against podocyte apoptosis in a high glucose milieu. Moreover, these authors showed that aliskiren inhibited the high glucose-induced increase in fibronectin mRNA and protein in these cells.

The above two in vitro studies in podocytes have potential clinical significance because they suggest that aliskiren can induce effects that relate directly to protecting the glomerular filtration barrier. Aliskiren has a molecular weight (551.8) small enough to be filtered by the glomerulus, and indeed the drug appears in the urine.83 Thus, in vivo exposure of podocytes to aliskiren is predicted. As podocytes express a functional RAAS,25, 26, 27, 28 renin inhibition in these cells by aliskiren may provide a cellular mechanism (that is, podocyte protection) for the anti-albuminuric effect observed in human beings84, 85 as well as animals.71, 75, 86, 87, 88

In vivo effects of aliskiren in experimental DN

Transgenic mRen-2 rats with superimposed STZ-induced diabetes develop albuminuria, glomerulosclerosis and tubulo-interstitial fibrosis,69 similar features as in human DN. Aliskiren (10 mg kg–1 per day) and the ACEI perindopril (0.2 mg kg–1 per day) were compared for their renoprotective effects in this model.88 The chosen dose of perindopril lowered tail cuff BP 36 mm Hg more than in aliskiren-treated STZ-mRen-2 rats. However, despite this difference, aliskiren attenuated albuminuria and glomerulosclerosis to similar degrees as perindopril. Interestingly, levels of albuminuria correlated well with BP in vehicle- and perindopril-treated mRen-2 rats, but not in aliskiren-treated rats. Moreover, histological evaluation showed that aliskiren conferred significantly greater protection against tubulo-interstitial fibrosis than perindopril. These two differential effects of aliskiren vs. perindopril may reflect a BP-independent renoprotective effect of aliskiren, possibly by inhibiting the intrarenal RAAS to a greater extent than the ACEI.

In another study with diabetic mRen-2 rats in which BP was monitored continuously by telemetry, aliskiren at 10, 30 and 60 mg kg–1 per day dose dependently reversed existing hypertension and prevented the development of albuminuria over the 10 weeks study.87 At the end of this study, renal cortical gene expression of collagen I and the pro-fibrotic growth factor, transforming growth factor-β (TGF-β), in the mRen-2 rats were significantly reduced in the aliskiren vs. the vehicle group.

In this experiment, treatment of diabetic mRen-2 rats with the high-dose (60 mg kg–1 per day) aliskiren was discontinued about 3 weeks into the study, but the animals were monitored for the remainder of the 10 weeks experiment (Webb, Zhou, Feldman, unpublished data). Withdrawal of aliskiren treatment was accompanied by an increase in BP and the development of albuminuria. The rise in BP was gradual, taking about 10 days to reach vehicle-control levels. Moreover, at the end of 10 weeks, gene expression of renal cortical TGF-β was still suppressed, despite stopping treatment with aliskiren about 7 weeks earlier. It is unknown whether this suppression of TGF-β reflects a continued presence or persistent effect of aliskiren in the kidneys, or whether RAAS inhibition early in the development of the hypertensive phenotype of this model confers long-term effects on growth factor expression.

Aliskiren has also shown renoprotective effects in db/db mice, a model of type 2 diabetes.86 In this study, treatment with aliskiren ameliorated the albuminuria and glomerulosclerosis seen in the vehicle-treated mice. These benefits were accompanied by reductions in expression of TGF-β, collagen IV and nephrin, as well as p22phox and NADPH oxidase activity.

Collectively, the above in vitro and in vivo studies suggest that the functional and structural renoprotection conferred by aliskiren in experimental renal disease may be explained by inhibition of the intrarenal RAAS, which may result in BP-independent renoprotective mechanisms (for example, anti-inflammatory, anti-oxidant) that reduce ECM accumulation and possibly mitigate against cellular (for example podocyte) damage. These studies also provide hints of persistent renoprotective effects of this DRI.

Potential novel mechanisms of aliskiren's renoprotective actions

Given that aliskiren blocks renin, it is not surprising that this drug has renoprotective effects in the experimental settings described above. Rather, the intriguing question is: ‘does a DRI, by blocking the RAAS at the first and rate-limiting step, impart a specific renoprotective mechanism?’ New evidence is emerging that suggests this may be the case.

Aliskiren localizes in the kidney

The kidney is the main source of renin; this organ contains a fully functional RAAS distributed over virtually all regions of the nephron, as described earlier. Consequently, as this pathway has been linked closely with renal damage, a distinct renoprotective advantage is expected from blocking the intrarenal RAAS. For a drug to inhibit the intrarenal RAAS and protect against organ damage, an obvious requirement is that it partitions to an appropriate renal compartment. On the basis of the experiments discussed below, this requirement seems to be met by aliskiren.

Sprague–Dawley rats were treated with 10 mg kg–1 per day aliskiren for 2 weeks by osmotic minipumps, and their plasmas and kidneys were studied for the presence of the drug. Renal levels of aliskiren were 46-fold over plasma levels, indicating that renal localization of aliskiren occurred, but was not due simply to equilibration from the plasma.87 Other renin inhibitors have shown a similar renophilic property.89 Although this study showed that aliskiren partitions to the kidney, it did not indicate the renal compartment in which aliskiren localized.

Therefore, in another experiment, the renal topographical localization of aliskiren was explored by administering 14C-aliskiren intravenously to normotensive rats and harvesting their kidneys 2 h afterward. By light microscopy, cryostat sections revealed heavy autoradiographic labeling in 100% of glomeruli on each renal section, indicating extensive partitioning of aliskiren throughout these structures87 (Figure 2a). Moreover, images suggestive of the presence of aliskiren in JG cells of the afferent arterioles were obtained, although such localization was not identified conclusively in these relatively thick cryostat sections. However, the possibility that aliskiren can enter vascular structures was shown clearly by the presence of label in larger caliber intrarenal arteries in these kidneys (Figure 2b), and preliminary evidence from recent studies suggests that in fact, aliskiren may localize in the afferent arterioles (Feldman et al., paper in preparation). However, in contrast to glomeruli, autoradiographic evidence for the presence of aliskiren in the tubulo-interstitium was not observed within the short duration of exposure in this experiment.87

Figure 2
figure 2

Autoradiographic localization of aliskiren in rat kidneys. Hanover–Wistar rats were injected intravenously with 14C-aliskiren and 2 h later the kidneys were removed for autoradiography. (a) Two glomeruli (G) show extensive autoradiographic labeling, indicating the presence of aliskiren. (b) A renal cortical artery (arrow) shows extensive autoradiographic labeling in vascular wall. (Reproduced with permission from Lippincott Williams & Wilkins.87)

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The above studies show that aliskiren partitions to the kidneys and localizes in the renal vasculature, possibly in structures known to contain renin. Moreover, extensive partitioning of aliskiren to the glomeruli suggests that it can access mesangial cells and podocytes, potentially inhibiting the RAAS in these cells and conferring structural and functional renoprotection observed in rodents.71, 75, 86, 87, 88 Light microscopic studies with labeled aliskiren have not been conducted in disease models in which changes in (extraglomerular) vascular permeability may facilitate permeation of the drug to the tubulo-interstitium, with potential access to tubular cells and fibroblasts. However, it seems reasonable to speculate that this may occur, and it may have relevance to the extent that enhanced expression of tubular renin may be linked to the development of tubulo-interstitial fibrosis.47

Not only does aliskiren partition to the kidneys in rats, its renal presence is prolonged. Two lines of evidence support this concept. First, dTGR were treated with aliskiren (3 mg kg–1 per day) for 2 weeks, during which time BP (tail cuff) and albuminuria were significantly reduced vs. vehicle controls. After a 3-week washout period, renal and plasma levels of aliskiren were measured. Aliskiren was not detectable in the plasma from these rats, but drug levels in the kidney were still above the IC50 for inhibiting human renin.90 Second, Vaneckova and coworkers91 treated mRen-2 rats with aliskiren (10 mg kg–1 per day) for 4 weeks, during which time BP (telemetry) was normalized. At the end of this treatment period, plasma and renal Ang II levels were lowered to those in non-hypertensive vehicle controls, confirming the observations in dTGR71 that treatment with aliskiren reduces renal Ang II content. At this time, aliskiren treatment of mRen-2 rats was stopped for 12 days washout. Importantly, after the washout period, plasma and renal Ang II levels were still reduced to vehicle-control levels.

Taken together, the above studies showed that aliskiren partitions to the kidney and exhibits a prolonged renal residence. These studies also suggest that aliskiren inhibits the intrarenal RAAS, and that such inhibition is enduring. In view of the growing evidence that 24-h BP control predicts better outcomes for target organ protection,92, 93, 94 such long-acting inhibition of the intrarenal RAAS may be especially important in conditions in which this pathway is activated, such as believed to occur in DN.78

Aliskiren may bind to intrarenal renin

The autoradiographic images discussed above87 provoke questions regarding the intracellular fate of aliskiren. Whether labeled aliskiren found in the kidneys of rats represents renin-bound inhibitor is an important issue. It seems doubtful that all of the labeled aliskiren observed in glomeruli in these studies was bound to renin: it is highly unlikely that so much renin was present in the glomeruli of the normotensive rats used in this study.69, 95, 96 Moreover, it seems similarly implausible that exposure to aliskiren for 2 h in normotensive rats could have induced recruitment of such high levels of glomerular renin, as has been shown in pre-glomerular vessels after chronic RAAS blockade,97 especially as aliskiren is a relatively weak inhibitor of rat renin (Table 1). However, some of the label seen in glomeruli may have reflected aliskiren bound to renin that possibly was present in the mesangial matrix. In addition, as aliskiren can penetrate cultured cardiomyocytes,98 the drug may have entered mesangial cells and podocytes, both of which express renin20, 24, 25, 26 to which aliskiren could bind. Furthermore, the unequivocal evidence for labeled aliskiren in the walls of renal vessels prompts the proposal that this DRI can enter JG cells, and possibly incorporate into forming or formed renin granules. Recent in vitro studies provide evidence for the latter possibility.

Cultured JG cells do not store renin,99 making them unsuitable for studying the intracellular renin-binding capacity of aliskiren. However, a human mast cell line synthesizes and secretes (pro)renin.100 Krop and coworkers101 used these cells to ask whether aliskiren can incorporate into renin granules. They found that mast cells that had been incubated for 7 days with aliskiren secreted renin that displayed inhibited enzyme activity (that is, inhibited Ang I-forming capacity). These data show that aliskiren can access intracellular renin, bind or incorporate into renin granules, and remain associated with the enzyme through the secretory process. Similar results were seen with prorenin, which is constitutively released from HMC-1 cells.102 This study lays the groundwork for understanding the intracellular fate of aliskiren. Moreover, the implications from this work and the autoradiographic data are that aliskiren may provide an unusual example in which an inhibitor incorporates into its (still intracellular) target even before the latter is secreted. In this case, (pro)renin would be secreted from JG cells to the plasma or the tubulo-interstitium103 in an already inhibited state. The renin activity in this (pro)renin-aliskiren complex would be inhibited: this renin (or prorenin, which may undergo activation at tissue sites) could not cleave Aogen, and the tissue and systemic RAAS cascades would not be activated. As this mechanism should operate independently of plasma drug levels,101 it may help to explain the prolonged BP benefits observed in rats discussed earlier, and in patients in whom aliskiren treatment has been stopped.104

Aliskiren, prorenin and the (pro)renin receptor

The recent discovery of a receptor for renin and prorenin, the (P)RR,105 has added an important new dimension to the tissue RAAS.106, 107, 108 In the kidney, gene and/or protein expression of the (P)RR has been reported in the glomerular mesangium,109 vascular wall87, 109 and tubular cells.87, 110 Gene and protein expression for the (P)RR has also been reported in podocytes.111, 112, 113

Stimulation of the (P)RR initiates Ang II-dependent and -independent effects. The former include a gain in catalytic activity for renin bound to the (P)RR (vs. unbound renin). This effect amplifies the formation of Ang II at the cell surface.109 Furthermore, on binding to the (P)RR, prorenin, which is normally inactive, becomes catalytically active (it can cleave Aogen to form Ang I), possibly because of a conformational change induced by the binding.109, 114, 115, 116 The Ang II-independent effects of (P)RR stimulation include activation of extracellular signal-regulated kinase1/2,109, 117, 118 and heat shock protein 27119 and phosphatidylinositol-3 kinase p85α120 pathways. The production of TGF-β and plasminogen-activator-1 is also increased, as are gene expression and production of collagen I and fibronectin, respectively.117, 121 Thus, through the (P)RR, renin and prorenin can induce direct effects on cells. Continuing work on this receptor has led to the isolation and characterization of a soluble form of the (P)RR,111 the significance of which remains to be elucidated.

As the (P)RR can activate prorenin, it has been suggested that this receptor may have an important function in the activation of prorenin at tissue sites, with consequent Ang II-mediated tissue damage.122 Prorenin is inactive because a 43 amino-acid prosegment covers the enzyme's active site. Incubation of prorenin with a renin inhibitor under the appropriate conditions permits access of the inhibitor to the active site of prorenin, while the prosegment, in equilibrium between ‘covering’ and ‘lifted’ conformations, is lifted from its position covering the active site.123 In vitro studies showed that when incubated with prorenin, aliskiren binds to the active site of the enzyme87 and can inhibit Ang I formation that would normally occur from non-proteolytic [(P)RR-mediated] activation of prorenin.116 This finding is significant because activation of prorenin at tissue sites might occur by (P)RR-mediated mechanisms or by proteolytic cleavage of the prosegment. The latter may occur in tissues in which inflammatory or dead/dying parenchymal cells may release proteases such as cathepsin G or elastase,124 which are capable of cleaving the prosegment. In either case, the Ang I-forming capacity of prorenin and the subsequent formation of Ang II (in the presence of ACE or chymase) would be blocked by bound aliskiren. The ability of aliskiren to inhibit prorenin activation has additional relevance because circulating levels of prorenin are about 10-fold those of renin.125 This represents high potential renin activity. In addition, plasma prorenin levels can be substantially elevated in diabetes126 and are strongly associated with microvascular complications of this disease.127 Thus, to the extent that activation of prorenin at tissue sites may contribute to local Ang II formation and tissue damage, aliskiren may provide tissue-protective effects. However, it must be noted that the concept of prorenin-induced tissue injury is not fully established; in two models of prorenin over-expressing mice, cardio-renal damage was not observed.128, 129

The potential clinical significance of the (P)RR is highlighted in a set of studies by Ichihara and coworkers (reviewed elsewhere130) that suggest a central function for this receptor in the pathogenesis of DN. This body of work is particularly provocative because the authors have reported dramatic benefits in STZ-diabetic rats with a putative (P)RR blocker. However, attempts by other investigators to reproduce the cellular effects of the blocker, and its in vivo benefits (although in non-diabetic models of renal damage), have largely been unsuccessful.115, 118, 131, 132 Replication by others of the experimental designs used by Ichihara and coworkers may clarify some of the questions on the function of the (P)RR in DN and in tissue damage in general.

What is the effect of aliskiren on the function of the renal (P)RR? Aliskiren does not inhibit binding of (pro)renin to the (P)RR,87, 118 nor does it prevent (pro)renin-induced cell signaling.87, 118, 120 However, in STZ-diabetic mRen-2 rats treated with aliskiren for 10 weeks, in situ hybridization on renal sections revealed a clear reduction in gene expression of the (P)RR in the glomeruli and tubular cells compared with vehicle-control diabetic mRen-2 rats.87 Interestingly, in vitro studies showed that aliskiren did not affect gene expression of (P)RR in mesangial cells. These data suggest that aliskiren can suppress mesangial cell gene expression of the (P)RR in vivo, but probably not through a direct cellular effect. Regardless, if this reduced gene expression also leads to lower (P)RR density at the cell surface, a reduction in Ang II-dependent and -independent effects could be expected from administration of aliskiren. This topic deserves more investigation.

In addition to suppressing gene expression of the (P)RR, treatment of diabetic mRen-2 rats with aliskiren also led to a reduction in renal cortical mRNA abundance for TGF-β and collagen I.87 It is tempting to invoke a causative relationship between reductions in gene expression of the (P)RR, TGF-β and collagen I in the above study. Indeed, elegant in vitro experiments by Huang and coworkers18 established a strong link between renin-induced production of TGF-β and ECM production in cultured mesangial cells, whereas blockade of the (P)RR with siRNA significantly inhibited this response. However, a similar causative conclusion from the in vivo study87 awaits a more robust proof.

Working model of renoprotective mechanism of action of aliskiren

From the data presented heretofore, it is possible to propose a working model for the mechanism(s) by which aliskiren protects the kidneys in experimental settings (Figure 3). Although this model is necessarily speculative, it provides testable hypotheses.

Figure 3
figure 3

In this working model of the renoprotective actions of aliskiren, outcomes for which in vitro or in vivo evidence exists are referenced (see References); no reference indicates a logical, but still speculative outcome. Solid arrows indicate the existence of in vitro or in vivo evidence to support a causal link between the events. Dotted lines denote a logical, but as yet, unproven causal link between events. * indicates Feldman DL et al., manuscript in preparation. Once aliskiren localizes in the kidney (for example, JG cells), it may incorporate into renin granules or prorenin and stay in these cells until released with (pro)renin. This renal localization would lead to a longer renal residence time than if unbound, and ultimately would lead to prolonged local RAAS blockade. Aliskiren that deposits in the glomeruli may inhibit mesangial and podocyte renin, inhibiting Ang II-mediated glomerular ECM accretion and loss of permselectivity. Renin or prorenin bound to aliskiren in JG cells would be released as inhibited enzymes and would theoretically have no abilities to cleave Aogen. The formation of Ang II would be inhibited, maintaining a quiescent local (tissue) and systemic RAAS. If aliskiren reduces synthesis of the (P)RR as a result of suppressing its gene expression, lower receptor density on the cell surface would be expected, reducing the opportunity for (pro)renin to bind to diminished numbers of receptors. Once released from JG cells, inhibited renin could still bind to the (P)RR, but could not gain the amplified catalytic activity that has been described after such binding. Similarly, prorenin released as bound to aliskiren could not become activated by proteolytic or non-proteolytic mechanisms. These actions on renin and prorenin would reduce Ang II formation at the cell surface. Extrarenal tissues that take up already inhibited renin would also theoretically derive protection from these mechanisms. Finally, in addition to the mechanisms described here, renoprotection would also derive from aliskiren binding to (pro)renin in the plasma.

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Summary

Although the idea to inhibit renin as a means of blocking the RAAS originated from studies that were conducted over 100 years ago,2 it is only recently that the effects of inhibiting this enzyme have been studied in depth. There is still much to learn about the mechanism of action of renin inhibition, and how it differs from those of ACEI and AT1 receptor blockers. The possibility that aliskiren binds to intracellular (pro)renin, leading to release of already inhibited enzyme seems to be a novel mechanism. However, it still must be documented that this occurs in vivo, and in fact that such a mechanism confers a benefit for organ protection. Toward this end, preclinical studies offer the means by which aliskiren's mode of action can be revealed more fully, thus generating hypotheses that can be tested in patients.