Review article

(Pro)renin and its receptors: pathophysiological implications

Wendy W. Batenburg, A. H. Jan Danser


Tissue angiotensin generation depends on the uptake of circulating (kidney-derived) renin and/or its precursor prorenin [together denoted as (pro)renin]. Since tissue renin levels are usually somewhat higher than expected based upon the amount of (renin-containing) blood in tissue, an active uptake mechanism has been proposed. Several candidates have been evaluated in the past three decades, including a renin-binding protein, the mannose 6-phosphate/insulin-like growth factor II receptor and the (pro)renin receptor. Although the latter seemed the most promising, its nanomolar affinity for renin and prorenin is several orders of magnitude above their actual (picomolar) levels in blood, raising doubt on whether (pro)renin–(pro)renin receptor interaction will ever occur in vivo. A wide range of in vitro studies have now demonstrated (pro)renin-receptor-induced effects at nanomolar renin and prorenin concentrations, resulting in a profibrotic phenotype. In addition, beneficial in vivo effects of the putative (pro)renin receptor blocker HRP (handle region peptide) have been observed, particularly in diabetic animal models. Despite these encouraging results, many other studies have reported either no or even contrasting effects of HRP, and (pro)renin-receptor-knockout studies revealed lethal consequences that are (pro)renin-independent, most probably due to the fact that the (pro)renin receptor co-localizes with vacuolar H+-ATPase and possibly determines the stability of this vital enzyme. The present review summarizes all of the recent findings on the (pro)renin receptor and its blockade, and critically compares it with the other candidates that have been proposed to mediate (pro)renin uptake from blood. It ends with the conclusion that the (pro)renin–(pro)renin receptor interaction, if it occurs in vivo, is limited to (pro)renin-synthesizing organs such as the kidney.

  • handle region peptide
  • mannose 6-phosphate/insulin-like growth factor II receptor
  • prorenin
  • (pro)renin receptor
  • renin
  • renin-binding protein
  • vacuolar H+-ATPase


Renin is formed from an inactive precursor, prorenin, by cleavage of a 43-amino-acid N-terminal pro-segment exclusively in the juxtaglomerular cells of the kidney and secreted into the circulation. Prorenin is secreted constitutively, mainly from the kidney, but also to a lesser extent from other organs, including the reproductive organs, eye and adrenal glands [1]. Plasma prorenin concentrations are higher, sometimes up to 100-fold, than plasma renin concentrations [2].

The beneficial effects of RAS (renin–angiotensin system) blockers in the heart, kidney and other organs are independent, at least in part, of the BP (blood pressure)-lowering effect of these drugs, and it is therefore well-accepted that AngII (angiotensin II) is generated not only in the circulation, but also locally at tissue sites. Indeed, studies quantifying tissue AngII generation, using infusions of radiolabelled angiotensins to correct for uptake from plasma, revealed that AngII in the heart, kidney and adrenal largely [35], if not completely, originates from local synthesis, both under normal and pathological conditions [4,6,7]. Originally, it was thought that all components required to generate AngII locally at tissue sites [i.e. renin, angiotensinogen and ACE (angiotensin-converting enzyme)] are also synthesized at tissue sites. For instance, mast cells have been identified as a potential source of renin in the heart [8]. However, a wide range of studies have shown that this is not the case, at least for renin. Renin activity can no longer be demonstrated in cardiac tissue following a bilateral nephrectomy [9,10], nor does the isolated perfused rat Langendorff heart release renin [11,12]. The latter is also not the case following a myocardial infarction, despite the fact that this greatly up-regulates the cardiac mast cell content [13]. Moreover, the cardiac tissue levels of renin closely correlate with the plasma levels of renin, both under normal and pathological conditions [6,10,14], and cultured neonatal and adult rat cardiac myocytes and fibroblasts do not release renin or its inactive precursor, prorenin, into the medium [15,16]. Similar observations have been made in the vessel wall [17]. Taken together, therefore, it appears that the renin required for tissue angiotensin generation is taken up from the circulation, i.e. is kidney-derived. In addition, circulating prorenin may contribute to tissue AngII generation, as evidenced from studies showing elevated cardiac angiotensin levels in transgenic mice overexpressing human prorenin in the liver and human angiotensinogen in the heart [18]. In these studies, it should be taken into account that, owing to species differences, human renin does not react with rat angiotensinogen and vice versa. Angiotensin generation by non-renin enzymes is unlikely, in view of the undetectable tissue levels of both AngI (angiotensin I) and AngII following a bilateral nephrectomy, despite the elevated angiotensinogen concentrations under these circumstances [10,19].


Cardiac, adrenal and brain cells are thought to express a renin transcript lacking exon 1, encoding for a non-secretory intracellular renin [2022]. The function of intracellular renin is so far unknown. Although elevated levels of adrenal (cytosolic) renin have been reported to occur in conjunction with elevated plasma levels of aldosterone [23], there is no evidence that this intracellular renin displays AngI-generating activity, and, if so, to what degree it has access to angiotensinogen in adrenal cells. Xu et al. [21] generated secreted renin-specific-knockout mice (i.e. mice lacking renal renin expression), which still expressed intracellular renin, e.g. in the brain. The results suggested that intracellular renin expression could not compensate for the loss of secreted renin, i.e. the animals exhibit the same renal lesions, low haematocrit, inability to generate concentrated urine, decreased arterial pressure and impaired aortic constriction as renin-null mutants lacking the entire renin gene. Moreover, studies in AT-receptor (AngII receptor)-knockout mice revealed that, in the absence of AT receptors, there was no intracellular angiotensin [24]. Thus angiotensin generation cannot occur intracellularly, and all intracellular AngII is derived from AT-receptor-mediated internalization. Clearly, therefore, tissue angiotensin generation occurs extracellularly only, either in interstitial fluid or on the cell surface, and intracellular renin does not contribute to tissue angiotensin generation.


Tissue renin levels (expressed per g of wet weight), e.g. in the heart, are too high to be explained based upon the amount of (renin- and prorenin-containing) blood plasma (≈5%) in tissue, suggesting that circulating renin and/or prorenin have somehow been actively sequestered [9,10]. This would require their diffusion into the interstitial space and/or binding to ‘(pro)renin receptors’. Diffusion is supported by studies in a modified version of the isolated perfused rat Langendorff heart, allowing separate collection of coronary effluent and interstitital transudate. During perfusion of this heart preparation with renin, renin was found to diffuse slowly into the interstitial space, reaching steady-state levels that were equal to the renin levels in coronary effluent [11]. Renin measurements in rat cardiac tissue are in full agreement with the concept that renin is present in cardiac interstitial fluid in concentrations that are as high as those in blood plasma [14]. In addition, studies in rat and pigs have shown that part of tissue renin is membrane-associated [10,25]. Moreover, isolated perfused hearts of rats transgenic for human angiotensinogen release AngI during renin infusion and this release continues after stopping the renin infusion [26]. These results strongly support the idea that circulating renin binds to a renin-binding protein/receptor and that bound renin is catalytically active. It would be of even greater interest if such receptors would also bind prorenin, particularly if this would result in prorenin activation. Three potential renin/prorenin receptors have been described in the past three decades: a RnBP (renin-binding protein), the M6P (mannose 6-phosphate)/IGFII (insulin-like growth factor II) receptor and the (P)RR [(pro)renin receptor] (Figure 1). Each of these receptors is described in the following sections.

Figure 1 Schematic overview of the putative (pro)renin-related functions of RnBP, the M6P/IGFII receptor and the (P)RR

See the text for further explanation. Please note that the only renin potentially capable of interacting with the RnBP intracellularly (i.e. in the cytosol) is derived from a renin transcript lacking exon 1, encoding for a non-secretory cytosolic renin. Aog, angiotensinogen; Ang, angiotensin; AT1R, AT1 receptor; AT2R, AT2 receptor. Adapted by permission from Macmillan Publishers Ltd: Kidney International (Sihn, G., Rousselle, A., Vilianovitch, L., Burckle, C. and Bader, M. Physiology of the (pro)renin receptor: Wnt of change?, vol. 78, pp. 246–256), copyright (2010).


An intracellular RnBP was discovered almost 30 years ago in humans, rats and pigs [2729]. Binding of renin to this RnBP strongly inhibited its capacity to convert angiotensinogen into AngI (Figure 1), suggesting a role as an in vivo renin inhibitor. However, neither RnBP nor renin-bound RnBP are normally found in plasma [27]. Subsequently, two vascular RnBPs were identified in membranes from rat mesenteric arteries or cultured rat aortic smooth muscle cells [25]. Interestingly, binding to these RnBPs was inhibited by a specific active site-directed renin inhibitor, suggesting that the active site of the renin molecule might be involved in the binding process. Later it was discovered that RnBP is the enzyme N-acyl-D-glucosamine 2-epimerase, with Cys380 as the essential residue for the activity of RnBP [30,31]. In vitro studies have subsequently showed that RnBP not only inhibited renin, but that the reverse (N-acyl-D-glucosamine 2-epimerase activity inhibition by renin) is also true [32]. However, the cytoplasmic location of this enzyme does not normally allow its interaction with full-length renin stored in vesicles [32,33]. At most, RnBP might interfere with the intracellular (non-secreted) renin variant which does occur in the cytoplasm [23] (Figure 1).

Although an intronic T/C polymorphism in the RnBP gene is associated with plasma prorenin levels in males (C allele carriers displaying lower prorenin levels) [34], mice lacking the RnBP were normotensive and did not display any major alteration in their circulating or renal RAS [35]. Moreover, the RnBP was located in the collecting duct and tubules, as opposed to the juxtaglomerular location of renin, and this situation did not change following renin induction in 2K1C (two kidney, one clip) rats [36]. Taken together, therefore, a role for the RnBP in the regulation of renin/prorenin and/or RAS activity in plasma or kidney seems unlikely. A more likely role for RnBP concerns its contribution to (renal) carbohydrate metabolism [37].

M6P/IGFII receptor

Lysosomal enzymes, following their synthesis in the ER (endoplasmic reticulum), are targeted to their destination via a mechanism involving phosphomannose residues. These residues are recognized by so-called M6P receptors. The secretory granules of the juxtaglomerular cells resemble lysosomes, and renin transport in these cells involves the M6P receptor [38].

M6P receptors cycle constitutively among the Golgi, endosomes and the plasma membrane, and the majority (90%) is located in a late endosomal/pre-lysosomal compartment. Extracellular lysosomal enzymes which bind to cell-surface M6P receptors are internalized via clathrin-coated pits. They dissociate from the receptor in acidified endosomal compartments and are subsequently delivered to lysosomes. The receptor is then re-utilized.

Two structurally distinct M6P receptors have been identified: a large M6P receptor, which binds ligands independent of bivalent cations (cation-independent M6P receptor), and a small M6P receptor, which requires bivalent cations for optimal binding (cation-dependent M6P receptor) [39]. Similarities between the M6P receptor and the IGFII receptor became apparent 25 years ago [40]. The molecular cloning of both receptors subsequently confirmed that both receptors are identical, and this receptor is now known as the M6P/IGFII receptor [41,42].

The M6P/IGFII receptor is a single-transmembrane glycoprotein, which mediates the binding and internalization of a number of extracellular growth factors and peptides, including IGFII, M6P-containing molecules (such as pro-cathepsin D, proliferin and leukaemia inhibitory factor) and non-M6P-containing retinoic acid [4349]. It is also believed to facilitate the activation of latent TGF (transforming growth factor) β1 [50].

Interestingly, the M6P/IGFII receptor mediates biological effects in response to IGFII and M6P-containing agonists, such as Na+/H+ exchange in canine kidney cells [51], exocytosis of insulin from pancreatic cells [52], proliferation of neuronal precursor cells [53], endothelial chemotaxis [54] and activation of sphingosine kinase leading to the production of S1P (sphingosine 1-phosphate), the ligand for G-protein-coupled S1P receptors [55,56]. The effects of M6P/IGFII receptors are pertussis-toxin-sensitive and are mediated, among others, by a decrease in adenylate cyclase activity [57], increased IP3 (inositol 1,4,5-trisphosphate) production [51] or stimulation of PKC (protein kinase C) activity [52]. Therefore it has been suggested that the M6P/IGFII receptor, in addition to its involvement in lysosomal targeting, also mediates transmembrane signalling via a G-protein-coupled mechanism.

A large percentage of plasma renin and prorenin is phosphomannosylated, and this allows them to bind to the M6P/IGFII receptor with high affinity (Kd≈1nmol/l). It should be noted that the phosphomannosylation/glycosylation pattern of extrarenally synthesized (pro)renin is not necessarily the same as that of kidney-derived renin and prorenin, e.g. amniotic prorenin in humans and Renin-2 in mice do not carry the M6P signal [1,58,59]. M6P/IGFII receptor binding of phosphomannosylated renin and prorenin is followed by their internalization in a variety of cells, including cardiomyocytes, fibroblasts, VSMCs (vascular smooth muscle cells) and endothelial cells [5863]. Moreover, prorenin is converted into renin by an as-yet-unidentified enzyme after internalization [59]. Thus, at least theoretically, M6P/IGFII receptors might contribute to the uptake of M6P-containing (pro)renin from the circulation at tissue sites, as well as the local activation of this prorenin. However, evidence for the release of internalized renin and prorenin into the medium could not be obtained; instead, intracellular degradation of renin, presumably in lysosomes, occurred [59,61,62].

To finally settle whether prorenin binding to M6P/IGFII receptors is functionally relevant, i.e. results in intracellular signalling, either directly or by facilitating AngII formation, we quantified [3H]thymidine incorporation and AngII generation by neonatal rat cardiomyocytes following their incubation with prorenin with or without angiotensinogen [64]. Only with angiotensinogen did prorenin enhance [3H]thymidine incorporation. Remarkably, the effect of prorenin plus angiotensinogen on DNA synthesis was comparable with the effect of 100 nmol/l AngII on [3H]thymidine incorporation, although the medium AngII levels during combined prorenin plus angiotensinogen application were <1 nmol/l. Moreover, cellular AngII was virtually undetectable during exposure to prorenin plus angiotensinogen, and the AT1 receptor blocker eprosartan (but not the M6P/IGFII receptor blocker M6P) blocked the effects of both AngII and prorenin plus angiotensinogen. As extracellularly applied receptor antagonists do not internalize in significant amounts, these findings suggest that the prorenin-induced myocyte proliferation depends on cell-surface AT1 receptor activation by extracellularly generated AngII. M6P/IGFII receptors apparently do not contribute to this process, and thus the extracellular angiotensin generation occurs independently of these receptors. It may still occur on the cell surface, particularly in view of the low medium AngII levels at which proliferation occurred during prorenin plus angiotensinogen exposure.

In summary, the M6P/IGFII-receptor-mediated prorenin and renin internalization represents (pro)renin clearance rather than a mechanism allowing tissue angiotensin generation. (Pro)renin-induced signalling via this receptor seems unlikely, and the activation of prorenin following internalization most likely is the first step towards degradation.


The (P)RR is a 350-amino-acid protein that can bind both renin and prorenin [6567]. It was first described 10 years ago. (P)RR binding is supposed to induce a conformational change in prorenin by which the pro-segment is moved out of the catalytic cleft and the active site is exposed, leading to full non-proteolytic activation of prorenin. The (P)RR also directly activates signalling pathways, independent from the formation of AngII. When comparing renin and prorenin in the same assay, most studies have revealed that the affinity of prorenin for the human (P)RR is 3–4-fold higher than that of renin [68]. Therefore it seems reasonable to assume that the pro-segment facilitates binding. A peptidic antagonist has been designed based on the idea that the pro-segment of prorenin contains a ‘handle region’ (10P-19P), which binds to the receptor, allowing prorenin to become catalytically active [69]. This so-called HRP (‘handle region’ peptide) mimics the handle region and will thus bind competitively to the receptor, thereby preventing receptor-mediated prorenin activation and reducing tissue RAS activity. Because prorenin is highly species-specific, different HRPs exist for humans, rats and mice. Since the (P)RR discovery a decade ago [65], a wide range of studies have investigated the direct effects of renin/prorenin via this receptor in isolated cells, the in vivo (P)RR regulation and the consequences of HRP infusion. These findings are summarized below.

Kidney and diabetes

Ubiquitous overexpression of the human (P)RR in rats resulted in proteinuria, glomerulosclerosis [70] and COX-2 (cyclo-oxygenase-2) up-regulation [71]. The human (P)RR binds, but does not activate, rat prorenin [70], and thus it is not surprising that the plasma and tissue angiotensin levels of transgenic rats overexpressing the human (P)RR were unaltered. However, these animals displayed increased levels of aldosterone in blood plasma. This was not due to (pro)renin-induced (P)RR stimulation, as neither renin nor prorenin affected aldosterone synthesis in the human adrenocortical cell lines H295R and HAC15 [72]. HRP did prevent the development of glomerulosclerosis in human (P)RR transgenic rats, but whether this truly involved blockade of the rat (pro)renin–human (P)RR interaction was not investigated [70].

In diabetic rats, HRP infusion (0.1 mg/kg of body weight per 28 days) normalized the elevated renal angiotensin content [73], without affecting BP. Concomitantly, the development of diabetic nephropathy was prevented. Surprisingly, this HRP effect also occurred in AT1A-receptor-deficient mice [74]. Since such mice no longer display the normal (constrictor) response to AngII [75], the effect of HRP in these mice cannot be explained on the basis of the suppression of local angiotensin generation. Interestingly, ERK1/2 (extracellular-signal-regulated kinase 1 and 2) as well as phospho-p38 MAPK (mitogen-activated protein kinase) and phospho-JNK (c-Jun N-terminal kinase) MAPK were up-regulated in the diabetic kidney, both in wild-type mice and in AT1A-receptor-deficient mice, and HRP (but not ACE inhibition) fully normalized this increased phosphorylation [74]. Similar results were obtained following HRP infusion (1 mg/kg of body weight per day) in db/db mice (a model for Type 2 diabetes mellitus) [76]. Moreover, in diabetic SHRsp [stroke-prone SHR (spontaneously hypertensive rats)] on a high-salt diet, the beneficial effects of HRP (0.1 mg/kg of body weight per day) and ACE inhibition on protein excretion and heart weight (but not on renal and cardiac AngII content) were additive [77]. HRP (0.1 mg/kg of body weight per day) also normalized the elevated fasting plasma triacyglycerols (triglycerides), total cholesterol and insulin levels in rats fed high fructose, without affecting the elevated BP in these animals [78]. Taken together, these findings suggest that HRP exerts beneficial effects in a variety of (diabetic) models in an AngII-independent manner. Whether its effects are truly due to interference of the (pro)renin–(P)RR interaction in vivo remains to be proven.

In support of the renin–(P)RR interaction in vitro, renin induced ERK1/2 activation in mesangial cells in the presence of renin inhibitors, ACE inhibitors and/or AT1 receptor antagonists. This activation resulted in TGFβ1 release and the subsequent up-regulation of genes coding for profibrotic molecules such as PAI-1 (plasminogen-activator inhibitor-1), fibronectin and collagens [65,79]. Effects started to occur at renin levels below 1 nmol/l, and could be fully prevented by (P)RR siRNA (small interfering RNA). Similar results were observed in HEK (human embryonic kidney) cells with both renin and prorenin, although now concentrations of 100 nmol/l were required [80]. Since both (P)RR siRNA and Nox4 siRNA blocked the up-regulation of TGFβ1, fibronectin and PAI-1 in HEK cells, it appears that (P)RR-induced Nox4 expression underlies these profibrotic effects. Remarkably, in podocytes, 2 nmol/l human prorenin increased ERK1/2 without affecting TGFβ1 or PAI-1 [81]. (P)RR suppression with siRNA, but not RAS blockade, prevented this effect. Apparently, therefore, the (P)RR–ERK1/2-mediated activation of profibrotic pathways does not occur in all cells. Interestingly, in cultured nephritic glomeruli from rats with glomerulonephritis, ACE inhibition and (P)RR siRNA together suppressed TGFβ1 and PAI-1 expression more strongly than each separately. The authors attributed this to the fact that ACE inhibition increased renin mRNA, thereby counteracting its own effect by stimulating the (P)RR [82].

Melnyk et al. [83], by performing a gene expression profiling study in human mesangial cells, found that 20 nmol/l renin and 50 nmol/l prorenin (in the presence of 10 mmol/l M6P to block M6P/IGFII receptors) activated pathways implicated in organ damage. Among others, COX-2 up-regulation was observed. Although the effects of renin were larger, there was extensive overlap between the gene signatures, indicating that both renin and prorenin acted through similar pathways. These involved in part the TGFβ1/PAI-1 pathway, but possibly also phospho-SAPK (stress-activated protein kinase)/JNK signalling. Since (P)RR siRNA could prevent approximately one-third of the changes, non-(P)RR-mediated mechanisms appeared to have played a role as well.

Cheng et al. [84] observed that COX-2 overexpression in podocytes exacerbates diabetic nephropathy by increasing (P)RR expression. Thus not only does (P)RR stimulation result in COX-2 up-regulation, but the reverse is also true, possibly because COX-2 metabolites promote (P)RR activation. Indeed, Huang et al. [85] confirmed this positive-feedback loop. First, they demonstrated that high glucose in rat mesangial cells up-regulates (P)RR (via PKC, ERK1/2 and JNK [86]) and (pro)renin, thereby facilitating AngII generation. This AngII subsequently induced COX-2 and IL-1β (interleukin-1β). AngII-independent COX-2 up-regulation could be excluded, since no additional effect of (P)RR knockdown and AT1 receptor blockade on COX-2 expression was observed. Secondly, COX-2 inhibition reduced the glucose-induced (P)RR up-regulation, suggesting that COX-2 itself up-regulates (P)RR. In vivo, increased renal (P)RR expression in diabetic rats occurred in glomeruli and tubules, possibly through enhanced AT1 receptor and NADPH oxidase activity [87]. However, in apparent contrast with the above in vitro results on glucose-induced (P)RR up-regulation in mesangial cells, HRP (0.2 mg/kg of body weight per day) did not suppress the elevated renal interstitial AngII in diabetic rats, nor normalized the increased (P)RR expression in this model [88]. In fact, the lack of effect of HRP on (P)RR expression appears to be a general phenomenon in in vivo studies applying this drug [76,78,8991]. AT1 receptor blockade with valsartan did normalize (P)RR expression, and both HRP and valsartan suppressed the up-regulated TGFβ1 levels in the diabetic rat kidney [92]. The effects of HRP and valsartan on TGFβ1 were additive. In summary, although various studies support a link between the (P)RR and COX-2, this does not necessarily involve AngII.

Moreover, the diabetes-induced increases in renal (P)RR expression are not a uniform finding. He et al. [93] actually observed decreased (P)RR expression in the rat kidney at 1 and 3 weeks after streptozotocin injection. They proposed that this decrease is due to AT2 receptor stimulation, since in cultured mesangial cells AngII decreased (P)RR expression via AT2 receptor stimulation, and similar decreases were observed following high glucose exposure. Simultaneously, these authors observed that both (P)RR siRNA and 1 μmol/l HRP decreased ERK1/2, TGFβ1 and proliferation, and increased MMP-2 (matrix metalloproteinase-2) in rat mesangial cells, despite the virtual absence of renin in the medium of these cells [94].

In agreement with the stimulatory effects of AT1 receptors on (P)RR expression [87], RAS blockade reduced (P)RR expression in the diabetic rat kidney [95], as well as in arterial fragments obtained during surgery from diabetic and non-diabetic patients with kidney failure [96]. However, in a Goldblatt (high AngII) model of hypertension, parallel increases in (P)RR and renin were observed [97], and (P)RR up-regulation also occurred in the remnant kidney of 5/6 nephrectomized rats (particularly in tubular cells), in kidneys (and hearts) of rats with heart failure post-myocardial infarction and in kidneys of patients with end-stage kidney diseases due to diabetic nephropathy (mainly in tubular cells and collecting duct) [98100]. Schulman et al. [101] observed (P)RR up-regulation in middle-aged rats, i.e. at a stage preceding the development of renal fibrosis. To explain these contrasting findings, the negative effects of AngII (via AT2 receptors) on (P)RR expression should be taken into consideration, as well as the possibility that (P)RR expression is regulated in a RAS-independent manner, e.g. via NADPH-dependent ROS (reactive oxygen species) generation [80,87].

HRP (3.5 μg/kg of body weight per day during 14 days) did not affect hypertensive nephrosclerosis in Goldblatt (2K1C) rats [102]. Ryuzaki et al. [91], when applying HRP at 1 mg/kg of body weight per day in 2K1C rats, confirmed this finding, but simultaneously demonstrated that HRP after 12 weeks of infusion reduced the glomerulosclerosis index, tubulointerstitial damage, TGFβ1 expression and AngII levels in the non-clipped kidney (but not in the clipped kidney). Under no condition did HRP affect BP or proteinuria. Krebs et al. [103] also did not observe effects of HRP (0.1 mg/kg of body weight per day) in the clipped kidney of 2K1C rats. Since damage in the clipped kidney usually occurs acutely (in a renin-dependent manner), these findings suggest that HRP is unable to interfere with the renin–(P)RR interaction in the clipped kidney. The mechanism of its long-term effects in the non-clipped kidney remains to be determined.

Vessel wall and heart

Overexpression of the human (P)RR in rat VSMCs resulted in hypertension [104], without altering the levels of RAS components. HRP (0.1 mg/kg of body weight per 28 days) diminished cardiac fibrosis in high-salt-fed SHRsp [89], without affecting BP or the circulating RAS. HRP also decreased left ventricular mass and left ventricular fibrosis in SHR receiving a high-salt diet when given at a dose of 0.1 mg/kg of body weight per day [105]. No such effects occurred in SHR fed on a normal salt diet [105]. Unexpectedly, in SHR on a normal salt diet treated with the renin inhibitor aliskiren, HRP (1 mg/kg of body weight per day) diminished both the BP-lowering effect of aliskiren and its beneficial effects on coronary endothelial function and cardiomyocyte area [106]. These latter effects of HRP occurred in an angiotensin-independent manner.

These findings raise doubt on whether HRP truly acts as an inhibitor of the (pro)renin–(P)RR interaction outside the kidney. In fact, Leckie and Bottrill [107] were unable to find a binding site for HRP in human endothelial cells, and HRP even bound to embryonic stem cells lacking the transmembrane region of the (P)RR [108]. Moreover, Feldt et al. [108] observed increased ERK1/2 phosphorylation in U937 monocytes following exposure to 10 nmol/l human renin or 2 nmol/l human prorenin, but 10 μmol/l human HRP did not block this phenomenon. In contrast, mouse HRP blocked the mouse prorenin (2 nmol/l)-induced ERK1/2 phosphorylation in mouse endothelial cells at concentrations of 10 and 100 μmol/l [109], i.e. far above the levels obtained in vivo [110]. To explain these discrepancies, it has been proposed that, in certain cells, and possibly particularly in vitro, the (P)RR is not or only scarcely located on the cell surface, thus not allowing an easy access for HRP [109].

Indeed, Yoshikawa et al. [111], like Batenburg et al. [66], observed that the majority of (P)RR in VSMCs is located in the ER and the Golgi, and not on the cell membrane. Similar observations have been made in neonatal rat cardiomyocytes [112]. Nevertheless, renin and/or prorenin, when applied at high concentrations (up to 100 nmol/l), did bind to VSMCs in a (P)RR-dependent manner [113] and induced ERK1/2 and Akt phosphorylation, DNA and protein synthesis, and PAI-1 release [113115]. (P)RR knockdown with siRNA prevented these effects [113,116]. The (pro)renin-induced effects on PAI-1 release were additive to those of AngII [113,115]. In neonatal rat cardiomyocytes, 2 nmol/l prorenin-induced p38 MAPK activation resulted in HSP27 (heat-shock protein 27) phosphorylation and an alteration in actin filament dynamics [112], and implanting Matrigel plugs containing 100 nmol/l human prorenin in mouse flanks facilitated neovascularization, possibly because prorenin induced ERK1/2 activation in endothelial cells at such high concentrations [117].

Taken together, these findings do support the (pro)renin–(P)RR interaction at exceptionally high (pro)renin levels in vitro. However, the conflicting results obtained with HRP in vivo and in vitro argue against the idea that this drug truly blocks the (pro)renin–(P)RR interaction in the intact animal.

Eye and brain

HRP blocked ischaemia-induced retinal neovascularization and ocular inflammation in endotoxin-induced uveitis [118,119]. Moreover, it reduced retinal inflammation [as shown by a reduction in the retinal protein levels of VEGF (vascular endothelial growth factor) and ICAM (intercellular adhesion molecule)-1] in diabetic rats, when applied at a dose of 1 mg/kg of body weight per day [109]. As the latter also occurred in diabetic AT1-receptor-deficient mice, this effect could not be attributed to AngII suppression [109]. Wilkinson-Berka et al. [110] confirmed the protective effects of HRP on VEGF and ICAM-1 in rats with oxygen-induced retinopathy, but simultaneously observed that HRP injured neuro-glia [110]. The protective and deleterious effects of HRP suggest that the drug may act as a partial agonist [68]. Interestingly, (P)RR and type 1 collagen staining were higher in donor eyes with dry age-related macular degeneration and hypertension compared with donor eyes with no eye disease [120]. Human retinal pigment epithelial cells were found to express the (P)RR, and 1 pmol/l prorenin induced ERK1/2 phosphorylation and type 1 collagen up-regulation [120]. A higher concentration of prorenin down-regulated the (P)RR, in agreement with the findings by Schefe et al. [121,122]. These authors identified the transcription factor PLZF (promyelocytic leukaemia zinc finger protein) as a direct protein interaction partner of the C-terminal domain of the (P)RR by yeast two-hybrid screening and co-immunoprecipitation. A similar observation has been made for the AT2 receptor [123]. Importantly, on activation of the receptor by (pro)renin, PLZF is translocated to the nucleus and repressed the transcription of the (P)RR itself, thereby creating a short negative-feedback loop. In other words: high (pro)renin levels, as occurring during RAS blockade, will suppress (P)RR expression, thus preventing excessive receptor activation.

The (P)RR is widely expressed in the human brain [124], with the highest expression found in the pituitary and frontal lobe. It co-localized with vasopressin and oxytocin in the paraventriuclar and supraoptic nuclei, raising the possibility that the (P)RR is related to the central control of water-electrolyte metabolism and/or BP. Indeed, adeno-associated virus-mediated overexpression of human (P)RR in the supraoptic nucleus of normal rats resulted in increases in plasma and urinary vasopressin, and decreases in water intake and urine output, without any effect on BP or heart rate. Vice versa, knockdown in SHR attenuated the age-dependent increases in BP and decreased heart rate and plasma vasopressin [125]. Neurons from SHR displayed a 50% greater ERK1/2 response than neurons from WKY (Wistar–Kyoto) rats to 20 nmol/l human renin. However, given the virtual absence of (pro)renin the brain, such concentrations are unlikely to ever occur in vivo.

Renin synthesis does occur in the eye [126], although its levels in ocular fluid were not higher than those in plasma [127]. Taken together, these findings do support an important role for the (P)RR in the eye and brain, but whether this truly depends on (P)RR activation by (pro)renin remains controversial.


Despite three decades of research, none of the currently proposed receptors fulfils the ideal concept of a (pro)renin receptor, i.e. a receptor that sequesters circulating renin and/or prorenin with high affinity (low picomolar range) at tissue sites and subsequently facilitates angiotensin generation. The RnBP most probably is unrelated to the RAS, and the M6P/IGFII receptor may, at most, function as a clearance receptor for renin and prorenin. This leaves the (P)RR as the most promising candidate. However, as outlined above, 10 years of research have not resulted in a clear picture of the (pro)renin–(P)RR interaction in vivo. Confusingly, renin and prorenin may even act as agonists of this receptor, exerting angiotensin-independent effects. Importantly, the affinity of the receptor for renin and prorenin is in the high nanomolar range (possibly even 20 nmol/l for renin [68]), which is difficult to reconcile with the picomolar levels of renin (≈0.5 pmol/l) and prorenin (≈5 pmol/l) in extracellular fluid [2]. It is, however, in full agreement with the observations that (P)RR overexpression in rats did not result in changes in RAS component levels [71,104] and that, in most in vitro studies investigating the (pro)renin–(P)RR interaction, high nanomolar (up to 100 nmol/l [80]) (pro)renin concentrations were required to observe the effects. As shown in Figure 2, when studying human prorenin-dependent effects (on DNA synthesis, ERK1/2 phosphorylation and PAI-1 release) in rat VSMCs that overexpress the human (P)RR, it became clear that AngII-dependent effects started to occur at prorenin levels in order of 4 nmol/l, whereas direct (AngII-independent) effects occurred at levels of 20 nmol/l.

Figure 2 DNA synthesis, ERK1/2 phosphorylation and PAI-1 release in rat aortic VSMCs overexpressing the human (pro)renin receptor following incubation with (A) 4 nmol/l prorenin±150 nmol/l angiotensinogen or (B) 20 nmol/l prorenin alone

DNA synthesis was quantified on the basis of [3H]thymidine incorporation. Results are represented as a percentage of baseline as means±S.E.M from data taken from Batenburg et al. [113]. *P<0.05 compared with 100%. Aog, angiotensinogen; PR, prorenin.

Moreover, prorenin overexpression, elevating plasma prorenin levels up to 400-fold in rodents, did raise BP in an angiotensin-dependent manner [128130]. However, it did not result in the fibrosis and/or glomerulosclerosis that were expected based upon in vitro studies with renin and prorenin. Possibly, for such direct effects prorenin increases of >1000-fold are required (Figure 3), and renin increases of >100000-fold. However, prorenin and renin levels up to three and five orders of magnitude respectively, above their normal levels are unlikely to ever occur in vivo, at least in non-(pro)renin-synthesizing organs. Whether they do occur in (pro)renin-synthesizing tissues (e.g. in renal interstitial fluid) remains to be proven. The maximum plasma (pro)renin increases that have been described in humans (e.g. in severe heart failure or during RAS blockade) are approximately 50–100-fold for renin (but usually well below 10-fold), and 2–3-fold for prorenin [2,6,131]. Consequently, although the 400-fold increase in circulating prorenin in prorenin-overexpressing rodents may have resulted in (P)RR-dependent AngII formation (thus explaining the AngII-dependent phenotype in these animals) [128130], this observation is unlikely to reflect normal physiology.

Figure 3 Prorenin concentrations in vivo under normal and pathological situations and in transgenic animal models overexpressing prorenin compared with the prorenin concentrations required to induce AngII-mediated and direct effects

In vivo and in vitro prorenin concentrations have been taken from values reported in [2,6,128130,135] and [113] respectively. TGR, transgenic.

Therefore the phenotype that develops in response to (P)RR overexpression must represent (P)RR effects that are RAS-independent [132,133]. In fact, it has recently been reported that the (P)RR co-localizes with V-ATPase (vacuolar H+-ATPase) in the kidney [134]. This may relate to the observation that the 8.9 kDa accessory protein ATP6AP2 (V-ATPase lysosomal accessory protein 2) of V-ATPase is a post-translationally truncated version of the (P)RR, resembling its C-terminal domain [68]. V-ATPases play an important role in the acidification of subcellular compartments. The (P)RR is indispensable for V-ATPase integrity, as in cardiomyocyte-specific (P)RR-knockout mice, the abundance of several V-ATPase subunits is decreased in cardiomyocytes, resulting in the development of heart failure due to defective autophagy and ultimately cell death [132]. Moreover, the (P)RR functions as an adaptor between V-ATPase and receptors for members of the Wnt family of signalling molecules [133]. These findings clearly indicate the importance of the (P)RR beyond prorenin binding, and raise doubt on the (P)RR as a (pro)renin binder/activator in vivo. This does not rule out that the (P)RR is an important player in tissue damage and/or that RAS blockade exerts beneficial effects by reducing its expression. The current findings with HRP are inconclusive, mainly because no in vivo study convincingly shows that this drug blocks the (P)RR–(pro)renin interaction. It is even possible that HRP is a partial (P)RR agonist, independently of renin/prorenin, thus explaining some of its conflicting results. (P)RR blockade itself, given its interference with V-ATPase, might well be detrimental.

Future studies should now rigorously address whether the (pro)renin–(P)RR interaction truly occurs in vivo and, if so, where. Clearly, such interaction seems impossible in organs not synthesizing (pro)renin, such as the heart and vessel wall, where the (pro)renin levels (per g of tissue), at most, are comparable with those in plasma (per ml of plasma). An interaction is also unlikely in organs where (pro)renin is barely detectable, such as the brain. This leaves the kidney and extrarenal prorenin-synthesizing organs, such as the ovary, as the most likely sites of interaction. Alternatively, if the (pro)renin–(P)RR interaction cannot be demonstrated in vivo, the search for a (pro)renin receptor will continue.


Our own work was performed within the framework of Dutch Top Institute Pharma project ‘Renin–angiotensin system blockade beyond angiotensin II’ [project number T2-301].

Abbreviations: ACE, angiotensin-converting enzyme; AngI, angiotensin I; AngII, angiotensin II; AT receptor, AngII receptor; BP, blood pressure; COX-2, cyclo-oxygenase-2; ER, endoplasmic reticulum; ERK1/2, extracellular-signal-regulated kinase 1 and 2; HRP, ‘handle region’ peptide; ICAM, intercellular adhesion molecule; IGFII, insulin-like growth factor II; JNK, c-Jun N-terminal kinase; M6P, mannose 6-phosphate; MAPK, mitogen-activated protein kinase; PAI-1, plasminogen-activator inhibitor-1; PKC, protein kinase C; PLZF, promyelocytic leukaemia zinc finger protein; (P)RR, (pro)renin receptor; RAS, renin–angiotensin system; RnBP, renin-binding protein; S1P, sphingosine 1-phosphate; SHR, spontaneously hypertensive rats; SHRsp, stroke-prone SHR; siRNA, small interfering RNA; TGF, transforming growth factor; 2K1C, two kidney, one clip; V-ATPase, vacuolar H+-ATPase; VEGF, vascular endothelial growth factor; VSMC, vascular smooth muscle cell


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