The (pro)renin receptor – An emerging player in hypertension and metabolic syndrome

Abstract

The (pro)renin receptor (PRR) is a multifunctional protein that is expressed in multiple organs. Binding of prorenin/renin to the PRR activates angiotensin-II dependent and independent pathways. The PRR is also involved in autophagy and Wnt/ß catenin signaling, functions that are not contingent on prorenin binding. Emerging evidence suggests that the PRR plays an important role in blood pressure regulation and glucose and lipid metabolism. Herein, we review PRR function in health and disease, with particular emphasis on hypertension and the metabolic syndrome.

The renin angiotensin system (RAS) plays a vital role in the maintenance of blood pressure (BP) and sodium homeostasis. In this system, circulating angiotensinogen is cleaved sequentially by renin and angiotensin converting enzyme to generate angiotensin-II (Ang-II) which then modulates BP through a multitude of effects including vasoconstriction, activation of the sympathetic nervous system, increased aldosterone synthesis, and antinatriuresis ¹. A number of organs contain their own RAS, wherein Ang-II can exert highly localized effects ². Additional components of the RAS have now been identified ³; amongst these, the (pro)renin receptor (PRR) has received much attention ⁴. Substantial efforts have been made to understand the localization, regulation and function of the PRR both at a molecular and system level. Further, the recent development of PRR antagonists ^5,6 has advanced our understanding of the complex functions of this pleiotropic protein. Herein, we review current knowledge on the biological roles of the PRR, focusing on its involvement in hypertension and the metabolic syndrome.

Basic biology of the (pro)renin receptor

The PRR, encoded by the ATP6AP2 gene on the X chromosome, was first cloned in 2002 ⁴ and is highly conserved across species ⁷. The full-length 350-amino acid protein localizes to the plasma membrane and encompasses a large extracellular domain that can be cleaved to form the soluble PRR (sPRR) and a smaller transmembrane and cytoplasmic domain (M8.9) ⁸ (Figure 1). The PRR has been localized to several organs including the kidney, heart, vascular smooth, brain, adipose tissue, liver, eye and placenta ⁴.

Figure 1.

Basic biology of the (pro)renin receptor (PRR). (A) Simplified schematic of the PRR protein; (B) Prorenin/renin dependent PRR function; and (C) Prorenin/renin independent PRR function. Binding of the prorenin to the full length or sPRR induces non-proteolytic activation of prorenin to cleave angiotensinogen while binding of renin to full length or sPRR increases catalytic efficiency. Independent of Ang-II, binding of prorenin/renin to the PRR activates intra-cellular signaling pathways. AGT – angiotensinogen; Ang-I – angiotensin-I; Ang-II – angiotensin-II; PRR – (pro)renin receptor; sPRR – soluble (pro)renin receptor; p38MAPK – mitogen activated prorenin kinase; ERK1/2 – extracellular signal-regulated kinase; PI3K – phosphoinositide 3-kinase; PLZF – promyelocytic leukemia zinc finger; Wnt/ß catenin.

The PRR serves a multitude of functions that depend, at least in part, on whether it is intact or cleaved into soluble and membrane/cytoplasmic components (Figure 1). Binding of prorenin to full-length PRR induces non-proteolytic activation of prorenin-mediated angiotensinogen cleavage, while renin bound to the PRR has four-fold higher catalytic efficiency as compared to unbound renin ⁴. The soluble fragment (sPRR) also binds and activates prorenin and renin. Independent of Ang-II generation, prorenin/renin binding to membrane-bound full-length PRR activates intra-cellular signaling pathways such as p38 MAPK and ERK1/2 ^4,9-11. PRR (M8.9) can function as an accessory subunit of the vacuolar H⁺-ATPase and is involved in lysosomal acidification ¹²; this function of the PRR (M8.9) appears to be independent of prorenin/renin binding ¹³. More recently, the PRR has been reported to be involved in the Wnt/ß catenin signaling cascade with an essential role in embryonic development, cell differentiation and metabolism ¹⁴.

Challenges in studying the (pro)renin receptor

Establishing the physiological role of the PRR has been challenging since global or cell-specific deletion of PRR often causes early lethality or organ malformation associated with abnormal lysosomal acidification ^7,15-18. Cardiomyocyte-specific PRR ablation results in lethal cardiac failure within 3 weeks after birth ¹⁵. Podocyte- specific PRR knockout (KO) mice develop severe proteinuria and die of renal failure in the first month after birth ^16,17. Collecting duct (CD) specific PRR KO mice have pronounced apoptosis, marked renal hypoplasia, and a malformed CD system ¹⁹. Loss of neuronal or adipose tissue PRR does not appear to affect organ structure or function ^20-22, suggesting that the lysosomal function of the PRR is tissue-specific. A recent study attempted to induce global PRR inactivation in adult mice using ROSA26-creERT2; although this model was not efficient in targeting brain, kidney, aorta or white adipose tissue, the inducible PRR KO mice displayed early lethality, marked weight loss, hypoglycemia and hypercholesterolemia associated with pathologic changes in the colon, bone marrow and liver ¹⁸.

Antagonists that block prorenin from the binding to the PRR have also been developed ^5,6. The first PRR blocker was directed toward the handle region peptide (HRP) and prevented the binding of prorenin to the PRR; despite initial promising results ⁵, HRP has now fallen out of favor due to partial agonistic properties ²³. A newer agent, PRO20, acts as a competitive antagonist, is identical to the first 20 amino acids of the prorenin segment and contains all of the PRR binding sites ⁶. As discussed later, several groups have validated the specificity of PRO20 in preventing prorenin-mediated PRR function ^6,24,25.

Despite the challenges using gene targeting and PRR antagonism, as discussed in the following sections, substantial progress has been made in uncovering the physiological and pathophysiological roles of the PRR.

Role of the (pro)renin receptor in blood pressure regulation

Kidney

The PRR has been localized to mesangial cells, podocytes, proximal tubule, distal convoluted tubule, the luminal membrane of intercalated cells, and principal cells with the highest renal expression in intercalated cells ^17,26,27. The CD PRR may be particularly important since prorenin is luminally secreted by the CD where it may act on the luminal PRR to stimulate sodium reabsorption and elevate blood pressure (BP) ²⁸. Relevant to this, renal medullary PRR expression is enhanced in hypertensive rats (Ang-II infusion and 2-kidney and 1 clip Goldblatt hypertension) ^29,30. Renal medullary infusion of PRO20 reduced the hypertensive response and renal injury to Ang-II infusion in rats ²⁵. Similarly, infusion of PRR shRNA in the renal medulla decreased expression of the epithelial sodium channel (ENaC) in rats (although the effect on BP was not examined) ³¹. In order to delineate the role of nephron PRR in BP regulation, we developed an inducible renal tubule PRR knockout (KO) mouse ³² to avoid the deleterious effects of PRR deletion on organ development. Renal tubule PRR KO mice had similar BP as controls under varying sodium intake but had an attenuated hypertensive response with reduced renal ENaC expression following Ang-II infusion ³³. Further, prorenin stimulated ENaC activity in acutely isolated cortical CD in control mice but not in renal tubule PRR KO mice ³³. Two other mouse models with CD specific PRR deletion recapitulated the protective effects of PRR deletion on Ang-II induced hypertension and ENaC expression ^34,35. In contrast, Trepiccione et al observed no differences in BP, sodium excretion or ENaC expression following Ang-II infusion in their nephron wide PRR KO mouse model compared to controls ³⁶. Instead, the authors observed reduced ability to excrete an acid load, blunted vacuolar H⁺-ATPase activity and expression, and increased renal medullary lysosomal protein and autophagosome markers ³⁶. One explanation for the discordant findings may be related to the high dose of Ang-II used to induce hypertension (1000 ng/kg/min in the latter study vs 300-600 ng/kg/min in others) as well as the timing of PRR deletion (prenatal in the Trepiccione study versus during adulthood in other studies). Renal tubular PRR also modulates water reabsorption through Ang-II dependent and independent mechanisms ^32,36-38. Recent studies have shown that the PRR regulates vasopressin-stimulated AQP2 expression via activation of prostaglandin EP4 receptor ³⁸ and the Wnt/β-catenin pathway ³⁷.

A fundamental question is if the intercalated and/or principal cell PRR modulates renal sodium and water reabsorption. To address this, we developed mice with principal or intercalated cell specific deletion of the PRR ³⁹. Although loss of the PRR selectively in intercalated cells resulted in lower body weight, there was no effect on renal histology, medullary ENaC or aquaporin-2 expression, urine concentrating ability, or prorenin-stimulated ENaC activity by isolated CD. In contrast, principal cell specific PRR deletion reduced ENaC and AQP2 expression in the renal medulla, decreased urine concentrating ability, and abolished prorenin-stimulated ENaC activity in isolated CD. Thus, these studies indicate that principal, but not intercalated, cell PRR modulates renal sodium and water transport.

The macula densa PRR also appears to regulate BP and systemic renin release ⁴⁰. Using a combination of cell culture and in-vivo studies, Riquier-Brison et al demonstrated that the macula densa PRR amplifies renin/prorenin-stimulated renin release via ERK1/2 signaling in a short-loop feed forward mechanism ⁴⁰. Consistent with this, mice with macula densa specific PRR ablation have reduced BP and plasma renin levels; these effects were more prominent following treatment with low salt diet and renin-angiotensin system (RAS) blockers ⁴⁰.

The renal PRR may also contribute to development of hypertension in kidney disease. Mice heterozygous for PRR deletion in nephron progenitor cells (homozygous deletion leads to early neonatal death) have fewer glomeruli, altered glomerular basement membrane ultrastructure, and develop hypertension at 2 months of age ⁴¹, suggesting that early loss of PRR may be involved in developmental programming of hypertension. Recently, Xu et al found increased renal PRR expression, activation of the intra-renal RAS, and salt sensitive hypertension in rats fed a high fructose diet; treatment with PRO20 reduced high fructose induced sodium retention, BP and intra-renal RAS activation ⁴². Polycystic kidney disease is associated with early onset of hypertension; an animal model of polycystic kidney disease had increased renin expression in cysts and mis-localization of the PRR from the luminal membrane of intercalated cells to the basolateral membrane of principal cells ⁴³. However, despite growing evidence for the renal PRR in BP regulation and renal function, some inconsistencies exist. Overexpression of the PRR under the control of cytomegalovirus early enhances/chicken ß-actin (CAG) promoter (expressed in embryonic stem cells) in mice does not alter BP or albuminuria despite a 25-80-fold increase in renal PRR expression ⁴⁴. Conversely, transgenic rats with ubiquitous human PRR overexpression remain normotensive despite proteinuria and progressive nephropathy ⁴⁵. Taken together, these studies raise the possibility that while general PRR overexpression may not affect BP, specific renal cell (particularly the CD) PRR is involved in BP regulation particularly under pathophysiological conditions (Figure 2).

Figure 2.

Organ specific functions of the (pro)renin receptor (PRR).VSMC – vascular smooth muscle cells.

Cardiovascular system

Cardiac myocyte PRR is closely linked to the vacuolar H⁺-ATPase as well as the ryanodine receptor ⁴⁶. Cardiac PRR expression is increased in diabetes ⁴⁶, myocardial infarction ⁴⁷ and heart failure ⁴⁸. In addition, high sodium intake and severe sodium restriction modulate cardiac PRR expression ⁴⁹. Rats fed a high salt diet (8.9% NaCl) had elevated BP and cardiac fibrosis associated with enhanced cardiac expression of prorenin/renin and the PRR ⁴⁹. Similarly, very low salt intake (0.01% sodium) in rats increased cardiac expression of prorenin/renin and the PRR, and accelerated cardiac and perivascular fibrosis despite normal blood pressure ⁵⁰.

The in vivo role of cardiac PRR in hypertension and cardiac function has been difficult to determine since, as mentioned above, constitutive deletion of the PRR in the cardiomyocytes leads to lethal heart failure as a result of impaired lysosomal function ¹⁵. Global or cardio-specific overexpression of the PRR did not alter BP or cardiac structure or function at baseline ^45,47 or in response to stress and ischemic injury ⁴⁷. Similarly, mice with constitutive overexpression of PRR under the control of CAG promoter had no changes in BP or cardiac function despite a 400-fold increase in cardiac PRR expression ⁴⁴. In contrast, over-expression of PRR in adult rat hearts via adenovirus mediated gene delivery for 2 weeks caused reduced left ventricular ejection fraction and resulted in Ang-II independent activation of cell signaling pathways ⁵¹. Taken together, these studies suggest that either the cardiac PRR does not play a role in BP regulation or compensatory mechanisms counter any effects exerted by the increased PRR expression (Figure 2).

The PRR is also found in vascular smooth cells where it localizes to the cell surface and interacts with the vacuolar H⁺-ATPase ⁵². Vascular smooth muscle cell specific loss of PRR in mice caused non-atherogenic sclerosis of the abdominal aorta in the face of unchanged BP ⁵². Rats with vascular smooth cell-specific overexpression of the human PRR develop hypertension and tachycardia at 6 months of age ⁵³. Vascular smooth cell PRR also mediates prorenin-stimulated smooth muscle migration in cultured aorta ⁵⁴, induction of inflammatory mediators such as plasminogen activator inhibitor- 1, and activation of ERK1/2 signaling pathways ⁵⁴. Thus, the vascular smooth cell PRR, seemingly more than the cardiac PRR, may play a role in BP regulation; such an effect occurs via both Ang-II dependent and -independent pathways (Figure 2).

Brain

The central nervous system PRR may play a vital role in the local RAS, particularly given that brain renin levels are thought to be too low to generate significant local Ang-II ⁵⁵. Within the brain, the PRR has been localized to the supraoptic nucleus, nucleus of the solitary tract, the subfornical organ (SFO) and the paraventricular nucleus, all of which are vital regulators of cardiovascular function and volume homeostasis. As in other tissues, binding of prorenin/renin to the neuronal PRR enhances Ang-II synthesis and activates intracellular signaling pathways such as ERK1/2 ⁵⁶.

The role of the brain PRR in hypertension has been studied extensively over the last several years ⁵⁶ (Figure 2). Supraoptic nuclear specific knock-down of the PRR in spontaneously hypertensive rats attenuated age-related increases in BP ⁵⁷. Intracerebroventricular (ICV) infusion of PRR shRNA in mice overexpressing both human renin and angiotensinogen reduced BP, lowered cardiac and vasomotor sympathetic tone, and improved baroreflex sensitivity ⁵⁸. These findings suggested that brain Ang-II was generated by PRR; to test this hypothesis, Li et al generated neuron-specific PRR KO mice and observed reduced brain Ang-II levels and an attenuated hypertensive response to ICV infusion of mouse prorenin ²⁰. Further, neuron-specific PRR deletion or ICV infusion of PRO20 diminished deoxycorticosterone acetate-salt-induced neuronal PRR expression, Ang-II formation and hypertension ^6,20. Post-mortem analysis of human brain tissues showed higher neuronal PRR expression in the SFO in hypertensive vs. normotensive patients, and this significantly correlated with BP ⁵⁹. Finally, recent studies have demonstrated that the neuronal PRR exerts Ang-II independent functions by stimulating sympathetic outflow via reactive oxygen species and inducible nitric oxide synthase signaling along with activation of ERK/NADPH oxidase 4 pathways ⁶⁰.

As mentioned earlier, unlike PRR deletion in other organs, neuronal PRR deletion or pharmacological blockade with PRO20 within the brain does not detectably alter lysosomal function, metabolism or survival ^6,20 since the neuronal PRR does not appear to modulate vacuolar H⁺-ATPase activity or the Wnt/β-catenin signaling pathway. This relatively unique feature of the neuronal PRR may be helpful in developing novel PRR antagonists that specifically block prorenin dependent functions.

Adipose tissue

Adipose tissue is increasingly recognized as an endocrine organ and expresses many components of the RAS, including the PRR ⁶¹. Adipose tissue PRR expression increases with obesity ^62-64 and, similar to other tissues, binding of renin/prorenin to adipocyte PRR results in Ang-II generation as well as ERK1/2 activation ^62-64. Using constitutive adipocyte-specific PRR deletion, Wu et al observed elevated systolic BP and increased plasma sPRR levels in KO compared to control mice under varying fat intake without observable differences in systemic RAS components ²¹ (Figure 2). The increase in sPRR, which may have contributed to the observed hypertension, was unexpected. Notably, these mice had aberrant glucose and lipid homeostasis, so it is possible that sPRR production by other organs or sPRR metabolism was secondarily affected. As discussed in more detail below, adipocyte PRR is also an important modulator of glucose and lipid homeostasis and may contribute to insulin resistance in obesity ^62-64.

Placenta

The placenta PRR may regulate BP although the evidence is limited. Among gestational tissues (amnion, chorion, decidua and placenta), the PRR is most highly expressed in the placenta where it is thought to play a role in prorenin-stimulated prostaglandin synthesis ⁶⁵.

The first study to describe an association between placental RAS and pre-eclampsia noted a 2-fold increase in plasma and placental prorenin levels and increased placental PRR immunostaining in a rat model of pre-eclampsia ⁶⁶. This study also reported increased plasma prorenin levels in preeclamptic women compared to normotensive pregnant women ⁶⁶. A subsequent study found higher placental PRR expression and plasma sPRR levels in preeclamptic women; systolic BP positively correlated with placental PRR but not plasma sPRR ⁶⁷. Although plasma prorenin/renin levels were not measured, these findings raise the possibility of placental PRR-mediated RAS activation in preeclampsia. Recently, Suggule et al examined maternal blood samples for prorenin, renin and sPRR in a group of preeclamptic, diabetic and healthy pregnant women ⁶⁸. While no differences were observed in plasma prorenin or renin levels among the study groups, plasma sPRR levels were increased in diabetic pregnancies but not preeclamptic pregnancies. These findings raise several questions about the role of placental PRR in altered BP regulation (Figure 2), including the source of the plasma sPRR in healthy and complicated pregnancies, whether plasma sPRR is an indicator of local RAS activation within the placenta, and the biological function of the placental PRR.

Clinical studies

Single nucleotide polymorphisms (SNPs) in the ATP6AP2 gene have been linked to BP and vascular disease. Using linkage-disequilibrium analyses, Hirose et al identified three SNPs in the ATP6AP2 gene (located in the promoter region, intron 5 and the 3’ untranslated region) and examined the association with BP in a Japanese cohort of 1100 participants ⁶⁹. T-allele carriers of the IVS5+169C > T polymorphism (found in intron 5) had higher ambulatory BP in men but not women ⁶⁹. These results were replicated in a separate study of Caucasian men where higher office and ambulatory systolic BP was observed in T-allele than C-allele carriers ⁷⁰. In contrast, the association of lacunar infarction and left ventricular hypertrophy was higher in women with the +1513A>G polymorphism (located in the 3’ translated region) with no difference in prevalence of lacunar infarction or left ventricular hypertrophy among the three SNPs in men ⁷¹. While these studies lend credence to the notion that the PRR contributes to BP regulation and cardiovascular disease, they need to be validated in larger clinical studies as well as determining the phenotypic effects of these mutations. ATP6AP2 gene mutations have also been associated with X-linked mental retardation, epilepsy and parkinsonism ^72-74 but these SNPs are limited to single families of 2-7 affected individuals.

Alterations in plasma sPRR levels may be associated with hypertension as well as other cardiovascular and renal conditions. Plasma sPRR levels did not change with age, sex, posture, hormonal status or circadian rhythm, but are 25% lower in black than white participants ⁷⁵. Plasma sPRR levels increase as the severity of kidney disease worsens ^76,77. High serum sPRR levels have been described in hemodialysis patients and are associated with atherosclerosis risk ⁷⁸. Plasma sPRR levels are elevated in patients with chronic heart failure with reduced ejection fraction and are even greater in patients with concomitant heart failure and renal dysfunction ⁷⁹. Finally, elevated plasma sPRR levels occur in pre-eclampsia ⁶⁷ and obstructive sleep apnea ⁸⁰. Thus, plasma sPRR levels may be of pathological significance and/or could be of value as a biomarker in the development and progression of kidney and cardiovascular disease. A summary of all current experimental and clinical studies of the PRR in BP regulation is described in Table 1.

Table 1.

Experimental and clinical models of the (pro)renin receptor in blood pressure regulation

Experimental studies	Species	Promoter/Cre	Blood pressure (BP)	Phenotypic characteristics	Organ specific effects	Reference
Global
Over-expression of PRR	Mice	CAG promoter (embryonic stem cells)	No change	Renal expression ↑ 25-80 fold Cardiac expression ↑ 400 fold	No proteinuria No cardiac dysfunction	44
Over-expression of human PRR	Rats	CAG promoter (embryonic stem cells)	No change	No change	Proteinuria Glomerulosclerosis	45
PRR KO	Mice	ROSA26-creERT2	Not reported	Early lethality and weight loss	Pathology in colon, bone marrow and liver	18
Kidney
Podocyte PRR KO	Mice	podocin-Cre	Not reported	Nephrotic syndrome, early post-natal lethality	Severe renal injury	16,17
HRP in 2-kidney 1-clip Goldblatt	Rat	-	No change	HRP did not change plasma renin or aldosterone levels	No change in cardiac or renal injury	107
PRO20 infusion in inner medulla	Rat	-	↓ Ang-II infused hypertension (100 ng/kg/min)	-	↓ proteinuria and glomerulosclerosis following Ang-II	25
PRR shRNA injected to renal medulla	Rat	-	No change	-	Reduced α-ENaC abundance	31
Renal tubule PRR KO	Mice	Pax8-rtTA/LC1- Cre (inducible)	↓ Ang-II infused hypertension (600 ng/kg/min)	Urinary concentration defect	Reduced α-ENaC abundance	33
CD specific PRR KO	Mice	AQP2-Cre	↓ Ang-II infused hypertension (300 ng/kg/min)	-	Reduced α-ENaC abundance	34
CD specific PRR KO	Mice	Hoxb7-Cre	↓ basal systolic BP ↓ Ang-II infused hypertension (400 ng/kg/min)	Renal hypoplasia and ↓ renal function	Reduced cleaved α-ENaC and γ-ENaC abundance	35
Renal tubule PRR KO	Mice	Pax8-rtTA/LC1- Cre (inducible)	No change in BP with Ang-II infusion (1000 ng/kg/min)	↑ lysosomal proteins and autophagosome markers	↓ ability to excrete acid load	36
CD cell specific PRR KO	Mice	AQP2-Cre Principal cell specific	No change in BP	Urinary concentration defect	Reduced α-ENaC abundance	39
CD cell specific PRR KO	Mice	B1-ATPase-Cre Intercalated cell specific	No change in BP	Lower body weight	No change in ENaC abundance	39
Macula densa specific PRR KO	Mice	nNOS/CreERT2	↓ BP at baseline and following low Na+ + RAS blocker	↓ plasma renin concentration	↓ cyclooxygenase-2 and prostaglandin E synthase	40
Nephron progenitor cells	Mice	Six2GFPCre	Hypertension at 2 months of age	Homozygous deletion – early lethality	↓ glomeruli number and thickening of basement membrane	41
Cardiovascular system
Cardiomyocyte PRR KO	Mice	α MHC-Cre	Not reported	Lethal heart failure within 3 weeks of birth	Autophagic vacuoles in cardiomyocytes	15
Cardiomyocyte specific PRR overexpression	Mice	α MHC promoter	Not reported	170-fold ↑ PRR mRNA and 5-fold ↑ PRR protein	No change in cardiac function at baseline and following injury	47
Cardiomyocyte specific PRR overexpression	Rats	Adenovirus mediated gene delivery	Not reported	2-3 fold ↑ PRR protein	↓ left ventricular ejection fraction	51
Vascular smooth muscle cell PRR KO	Mice		No change in BP	Non-atherogenic sclerosis of aorta	VSMC autophagic cell death	52
Vascular smooth muscle cell PRR overexpression	Rats	Smooth muscle cell myosin heavy chain gene	Hypertension at 6 months of age	Tachycardia	-	53
Brain
Supra-optic nucleus PRR knock-down	Rats (SHR)	Adenovirus mediated gene delivery	↓ age associated hypertension	↓ heart rate	↓ plasma vasopressin levels	57
ICV PRR shRNA infusion	Mice	Overexpressing human renin and angiotensinogen	↓ BP	↓ sympathetic tone ↑ baroreflex sensitivity	↓ Ang-II type 1 receptor and vasopressin levels in brain	58
Neuron specific PRR KO	Mice	Nefh-Cre	↓ BP in ICV infusion of prorenin/ DOCA-salt	↓ sympathetic tone ↑ baroreflex sensitivity	↓ brain Ang-II levels	20
ICV PRO20 infusion	Mice	-	↓ BP Ang-II and DOCA-salt hypertension	↓ sympathetic tone ↑ baroreflex sensitivity	↓ brain hypothalamic Ang- II levels with DOCA-salt	6
Adipose tissue
Adipose tissue PRR KO	Mice	Adiponectin-Cre	↑ BP at baseline and following high fat diet	Lipodystrophy	↑ plasma sPRR	21
Clinical Studies	Species	Controls	Blood pressure	Other pertinent findings
Pre-eclamptic women	Human	Normotensive pregnant women	↑ BP	↑ placental PRR and ↑ plasma sPRR BP correlated with placental PRR but not plasma sPRR		67
Pre-eclamptic and diabetic women	Human	Healthy pregnant controls	Not reported	↑ plasma sPRR in diabetes but not preeclamptic or healthy controls		68
Japanese cohort of 1100 participants	Human	-	↑ BP in men with IVS5+169C > T polymorphism	-		69
Caucasian cohort	Human	-	↑ BP in men with IVS5+169C > T polymorphism	-		70

KO - knock out; Ang-II – Angiotensin-II; ENaC – epithelial Na channel; RAS – renin angiotensin system blocker; ICV – intracerebroventricular; sPRR- soluble PRR; DOCA-salt – Deoxycorticosterone acetate;

Role of the (pro)renin receptor in glucose and lipid metabolism

Diabetes and insulin resistance

Soon after the discovery of the PRR, a decoy peptide termed ‘handle region peptide’ (HRP) was developed to block prorenin binding to the PRR ⁵. Early studies demonstrated that HRP markedly inhibited the development of diabetic nephropathy and reduced renal Ang-I and Ang-II levels in streptozotocin (STZ) induced Type 1 diabetic rats despite no changes in body weight, hyperglycemia and BP ⁵. Subsequently, HRP treatment was reported to be protective against development of diabetic nephropathy in Type 1 and 2 diabetic rodent models ^81-83,84,85 and in reversing established diabetic nephropathy in STZ-induced diabetes ⁸⁶. However, recent studies have shown that HRP increases inflammatory markers and cardiac fibrosis in diabetes ^23,87 and may even have partial agonistic effects, questioning the specificity of HRP with regards to PRR function ⁸⁸. Still, enhanced PRR expression is reported in whole kidney extracts in diabetic rats ^83,89 and cultured renal cell lines exposed to high glucose ^27,90,91. This is of particular significance since intrarenal, and particularly CD, prorenin/renin levels are markedly elevated in diabetes ⁹² and may contribute to the development of diabetic nephropathy via interaction with the renal PRR.

Other studies have provided indirect evidence for enhanced renal medullary PRR expression in insulin resistance. Mice with a null mutation of the carcinoembryonic antigen-related cell adhesion molecule 1 (Ceacam 1) had insulin resistance, visceral obesity and postprandial hyperglycemia associated with increased expression of medullary PRR and activation of tubular RAS components ⁹³; all of these abnormalities were exacerbated by high fat intake ⁹⁴. The same group demonstrated that high fat intake per se augments renal medullary PRR expression and Ang-II levels leading to hypertension, while renal tubular PRR KO mice were protected from high-fat diet induced hypertension ⁹⁵. Thus, the renal PRR might play a role in the development and progression of renal injury in diabetes and insulin resistance.

Several studies have examined the role of adipocyte PRR in obesity and insulin resistance. Mice fed a high fat diet for 10 weeks had increased adipose tissue PRR expression without changes in heart, kidney and liver PRR levels ⁶⁴. HRP treatment decreased weight gain, adipose tissue and circulating leptin levels, adipocyte inflammatory markers, plasma glucose and insulin, and triglyceride levels in obese mice ⁶⁴. These effects appear to be due to redistribution of fat storage by increased adipogenesis, decreased lipogenesis and activation of triglyceride/free fatty acid cycling, resulting in reduced visceral adipose tissue and increased subcutaneous fat deposition ⁹⁶. Consistent with the above studies, PRR mRNA was modestly increased in subcutaneous fat obtained from insulin resistant obese women compared to insulin sensitive obese women ⁶⁴.

Recently, adipose tissue-specific PRR KO mice have been developed which show lower body weights compared to controls ^21,22. When adipose tissue PRR is deleted using the adipocyte protein 2-Cre (which reportedly reduces PRR gene expression by 50-80%), hemizygous male KO mice have lower total fat mass, higher total lean mass and increased basal metabolic rate ²². Male KO mice and high fat-fed female KO mice had lower plasma insulin levels compared to WT mice although glucose tolerance test results were similar between KO and control mice. Further, circulating adiponectin levels were elevated in both male and female mice with no differences in circulating lipids or systemic renin levels ²². In contrast, adipocyte PRR deletion induced by adiponectin-Cre (with higher gene targeting efficiency) results in elevated plasma insulin levels likely due to hepatic steatosis and impaired insulin clearance ²¹; these mice also demonstrate improved insulin sensitivity under high fat intake ²¹. Collectively, these studies highlight the importance of adipose tissue PRR in obesity and insulin resistance. Further studies are needed to determine the molecular mechanisms involved in adipocyte PRR modulation of insulin signaling, particularly under diabetic conditions.

Finally, PRR expression is described in both human and mouse pancreatic islet cells and can modulate GLP1R signaling and insulin processing to affect insulin secretion ⁹⁷. Additionally, PRR expression was reduced in islet cells from human diabetics compared to healthy controls, raising the possibility of a role for the PRR in insulin processing and/or secretion.

Lipid homeostasis

A novel, Ang-II independent function attributed to the PRR is regulation of lipoprotein metabolism. In a series of elegantly designed studies, Lu et al identified sortilin-1 (SORT1) as a key PRR interacting protein by mapping the PRR-interactome in HEK293 cells ⁹⁸. They determined that the PRR was a post-transcriptional regulator of SORT1 that in turn modulates low-density lipoprotein (LDL) metabolism. In cultured hepatocytes, knock-down of the PRR reduced SORT1 and LDL receptor abundance leading to severely attenuated cellular LDL uptake which could be reversed by treatment with lysosomotropic agents such as bafilomycin A1 ⁹⁸, suggesting that PRR regulation of LDL receptor and SORT1 was independent of vacuolar H⁺-ATPase activity. The authors then demonstrated that mice injected with antisense oligonucleotides specifically targeting hepatic PRR had increased plasma LDL levels with an unexpected decrease in plasma triglyceride levels on a normal fat diet and during early stages of high fat intake ⁹⁹. Further, hepatic PRR knock-down attenuated diet-induced obesity and hepatosteatosis in chronic high fat diet fed mice and reduced both plasma cholesterol and triglyceride levels in LDL-receptor deficient mice regardless of fat intake ⁹⁹. These effects appear to be mediated via reduced lipid synthesis and increased fatty acid oxidation via acetyl-CoA carboxylase and pyruvate dehydrogenase.

Similarly, a recent study described an association between missense mutations in the extracellular domain of the PRR with a ‘congenital glycosylation disorder’ manifested by liver disease, immunodeficiency and psychomotor impairment in humans ¹⁰⁰. The authors studied this in a mouse model with liver-specific knockdown of PRR and proposed that the clinical symptoms of the glycosylation disorder were likely due to impaired vacuolar H⁺-ATPase assembly leading to defects in autophagy ¹⁰⁰. Taken together, these studies highlight a novel role of the PRR in lipid metabolism and liver function.

Clinical studies

Limited clinical data exists on the role of the PRR in glucose and lipid metabolism. Mutations in the ATP6AP2 gene were associated with disorders of glycosylation and autophagy in a small group of patients ^100,101. Elevated plasma sPRR levels were described in several studies of gestational diabetes mellitus ^68,102,103 yet plasma sPRR levels in diabetic patients were similar to those in healthy controls ⁷⁵. Ongoing research on the tissue-specific role of the PRR in glucose and lipid metabolism will help clarify these conflicting results and determine if the PRR is a reasonable therapeutic target. Table 2 summarizes current evidence for the PRR in glucose and lipid metabolism.

Table 2.

Experimental and clinical models of the PRR in glucose and lipid metabolism

Experimental studies	Species	Model studied	Phenotypic characteristics	Organ specific effects	Reference
Handle region peptide (HRP) from 4 weeks to 28 weeks of age (0.1 mg/kg)	Rat	STZ induced Type 1 diabetes	No change in body weight or hyperglycemia	↓ proteinuria and development of diabetic nephropathy	5
HRP from 4 weeks to 16 weeks of age (1mg/kg/day)	Mice	db/db mice	No change in body weight or hyperglycemia	↓ proteinuria and development of diabetic nephropathy	81
HRP (0.1 mg/kg) or Imidapril (0.015% in water) from 8 weeks to 24 weeks of age	Mice	STZ induced Type 1 diabetes in AT1-receptor KO mice	No change in body weight or hyperglycemia but BP lower in ATKO mice and after Imidapril treatment	HRP prevented proteinuria and development of diabetic nephropathy while Imidapril ↓ proteinuria	82
HRP (0.2 mg/kg) or Valsartan (2 mg/kg/day) or both for 2 weeks	Rats	STZ induced Type 1 diabetes	No change in body weight, kidney weight or hyperglycemia	Both treatments ↓ proteinuria & inflammatory cytokines	83
HRP (0.1 mg/kg) or Imidapril (0.015% in water) from 17 weeks to 29 weeks of age	Rats	STZ induced Type 1 diabetes	No change in body weight or hyperglycemia but BP lower after Imidapril treatment	HRP reverses diabetic glomerulosclerosis while Imidapril ↓ renal further damage	86
HRP (0.1 mg/kg/day) for 10 weeks	Mice	High fat diet	HRP ↓ weight gain, plasma glucose, insulin and triglyceride levels	↓ plasma and adipocyte leptin levels and adipocyte inflammatory markers	64
HRP (1mg/kg/day) or Aliskiren (10mg/kg/day) or combined treatment for 3 weeks	Rats	STZ induced Type 1 diabetes in heterozygousRen2 rats	Aliskiren alone decreased BP	Aliskiren alone ↓ proteinuria and glomerulosclerosis while HRP counteracts the effects of Aliskiren	23
HRP (1mg/kg/day) or Aliskiren (10mg/kg/day) or combined treatment for 3 weeks	Rats	STZ induced Type 1 diabetes in heterozygousRen2 rats	No change in blood glucose but BP lower in HRP and Aliskiren treatments	Aliskiren alone improved vascular dysfunction in diabetic rats and addition of HRP diminished the effects	87
HRP (0.1 mg/kg/day) or Imidapril (0.015% or 0.060% in water) or combined treatment for 4 weeks	Rats	STZ induced Type 1 diabetes stroke prone SHR	HRP had no effect on BP while Imidapril & combined treatment lowered BP, no change in blood glucose	Combined treatment ↓ cardiac weight, proteinuria & glomerulosclerosis than HRP or Imidapril alone	85
HRP (0.1 mg/kg/day) or Quinapril (50 mg/kg/day) or combined treatment for 8 weeks	Rats	STZ induced Type 1 diabetes	No change in blood glucose or body weight, blood pressure not measured	Combined treatment and Quinapril ↓ proteinuria and tubular injury but not HRP;HRP & Quinapril ↓ glomerular but no additive effects	84
Ceacam1 KO mice (model of insulin resistance)	Mice	Normal fat diet	↑ blood pressure	↑ expression of medullary PRR and activation of tubular RAS components	93
Ceacam1 KO mice (model of insulin resistance)	Mice	High fat diet for 8 weeks	Visceral obesity and postprandial hyperglycemia	↑ expression of medullary PRR and activation of tubular RAS components	94
Renal tubule PRR KO	Mice	High fat diet for 10 weeks	↓ obesity induced hypertension	↓ α ENaC abundance in the kidney	95
Adipocyte PRR KO (Adiponectin-Cre)	Mice	High fat diet for 16 weeks	Prevented development of obesity	↑ plasma insulin levels and improved insulin sensitivity	21
Adipocyte PRR KO (Adipocyte protein 2-Cre)	Mice	High fat diet for 9 weeks	↓ weight gain, ↑ basal metabolic rate	↑ plasma insulin and adiponectin levels and improved insulin sensitivity	22
Antisense oligonucleotide to block hepatic PRR	Mice	Normal or high fat diet	↓ diet induced obesity and hepatosteatosis	↑ plasma LDL levels, ↑ triglyceride levels	99
Adeno-Cre injection to reduce liver PRR	Mice	Floxed PRR mice	↑ plasma cholesterol	↑ liver injury and autophagy defects	100
Clinical studies	Species	Mutations	Predominant characteristics	Other features
Missense mutations in PRR (N=3)	Human	c.293T>C c.212G>A	Liver cirrhosis and immunodeficiency	Intellectual disability, ataxia, and cutis laxa	100,101

STZ - streptozotocin; ENaC – epithelial Na channel; RAS – renin angiotensin system blocker; AT1 – angiotensin-II Type 1 receptor; LDL- low density lipoprotein

Role of the (pro)renin receptor in fibrosis

As described earlier, the PRR is involved in the Wnt/β-catenin signaling pathway and plays an important role in kidney development and cell differentiation. New research suggests that PRR activation of Wnt/β-catenin signaling could contribute to renal fibrosis ¹⁰⁴. Renal PRR expression is up-regulated in mice with chronic kidney disease following unilateral ureteral obstruction, adriamycin-induced renal injury or following chronic Ang-II infusion ¹⁰⁴. In these conditions, the renal PRR, via activation of the Wnt/β-catenin pathway, enhances expression of downstream profibrotic markers such as fibronectin, plasminogen activator inhibitor 1 and α– smooth muscle actin (α-SMA). Consistent with this, PRR immunostaining was markedly enhanced in human kidney biopsy specimens with diabetic nephropathy, membranous nephropathy or lupus nephritis compared to control normal kidney biopsies ¹⁰⁴. While PRR activation of the Wnt/β-catenin signaling was independent of prorenin in the above study ¹⁰⁴, others report that prorenin treatment in cultured renal tubular epithelial cells, albeit at higher doses than seen in vivo, enhances expression of the PRR, vacuolar H⁺-ATPase and profibrotic markers ^105,106. Further, inhibition of the PRR or vacuolar H⁺-ATPase activity partially reduced prorenin-induced fibronectin and α-SMA expression, suggesting that prorenin, via the PRR, promotes renal fibrosis ¹⁰⁵. However, mice with constitutive global overexpression of PRR did not manifest cardiac or renal fibrosis despite marked increases in renal and cardiac PRR expression ⁴⁴. Interpretation of these findings is difficult; however, the bulk of evidence suggests that the PRR, at least under some conditions, may play a pro-fibrotic role.

Perspectives and future studies

The PRR exerts a multitude of effects involving numerous tissues; these have physiological and pathophysiological consequences. The PRR may be of pathological significance in hypertension, metabolic syndrome, diabetes and other disorders. While clearly additional studies are necessary, it is instructive to speculate on what our current knowledge of the PRR might imply with regard to clinical relevance and, in particular, its utility as a clinical marker and/or therapeutic target. Several studies raise the interesting possibility of using sPRR as a clinical marker; however, it remains to be seen whether circulating and/or urinary sPRR, possibly together with prorenin, will have prognostic or diagnostic utility. With regard to therapeutic potential, it is first important to distinguish targeting PRR from effects elicited by other RAS antagonists: angiotensin receptor blockers, angiotensin converting enzyme inhibitors or direct renin inhibitors. None of these agents interfere with prorenin or renin binding to, or activating of, the PRR, i.e., the Ang-II independent effects of the PRR would not be targeted. Since Ang-II independent effects of PRR activation include stimulation of profibrogenic pathways and, as described herein, targeting the PRR reduces end organ pathology independent of targeting Ang-II, it is appropriate to further investigate the potential for using PRR antagonists in disease. However, such agents would have to block prorenin/renin-stimulated PRR signaling without interfering with PRR modulation of the vacuolar H⁺-ATPase. Since PRR regulation of the vacuolar H⁺-ATPase is independent of prorenin/renin, such agents may be possible; clearly, much additional work is needed in this area.