RILLKKMPSV Influences the Vasculature, Neurons and Glia, and (Pro)Renin Receptor Expression in the Retina
The (pro)renin receptor [(P)RR] is implicated in organ pathology. We examined the cellular location of the (P)RR and whether a putative (P)RR antagonist, RILLKKMPSV, corresponding to the handle region of the prorenin prosegment (handle region peptide [HRP]) influences angiogenesis, inflammation, and neuronal and glial function in rat retina. The (P)RR was localized to retinal vessels, endothelial cells, and pericytes, but most immunolabeling was in ganglion cells and glia. HRP (1 mg/kg per day by IP injection) reduced physiological angiogenesis in developing retina. Moreover, HRP (0.1 mg/kg per day by subcutaneous minipump) reduced pathological retinal angiogenesis, inflammation, and vascular endothelial growth factor and intercellular adhesion molecule-1 mRNA in rats with oxygen-induced retinopathy (OIR) to an extent similar to valsartan (10 mg/kg per day, IP). In contrast to its effects on vasculature, HRP compromised the electroretinogram in shams and OIR and increased phosphorylated extracellular-signal–related protein kinase 1/2 immunolabeling in shams but not in OIR, whereas valsartan did not affect the electroretinogram and reduced extracellular-signal–related protein kinase 1/2 immunolabeling in OIR. Retinal (P)RR mRNA levels were increased in OIR; HRP, but not valsartan, increased (P)RR mRNA levels in shams, whereas both HRP and valsartan reduced (P)RR mRNA levels in OIR. A control peptide (VSPMKKLLIR, 0.1 mg/kg per day) did not influence retinal vasculopathy or function. Circulating HRP levels in rats administered 1 mg/kg per day HRP were undetectable (<3 pmol/L). We conclude that HRP had protective effects on the retinal vasculature similar to those of valsartan; however, unlike valsartan, HRP injured neuro-glia, which may involve the (P)RR, although the undetectable circulating HRP level makes a direct effect of HRP on retinal (P)RR function unlikely.
The retina contains a local renin-angiotensin-aldosterone system, and angiotensin-converting enzyme and angiotensin type 1 receptor blockade (ARB) are known to improve pathology in ocular diseases such as retinopathy of prematurity and diabetic retinopathy.1 A pathogenic role for prorenin in retina is suggested by its elevation in the plasma and vitreous of patients with diabetic retinopathy,2 and plasma of patients with retinopathy of prematurity.3 This idea has gained renewed interest since the cloning of the (pro)renin receptor [(P)RR].4 The current view is that the (P)RR acts in 2 different ways.5,6 First, the binding of prorenin to the (P)RR activates prorenin, resulting in the generation of angiotensin II. Second, prorenin binding to the (P)RR initiates cellular signaling mechanisms, such as mitogen-activated protein kinase, which, via phosphorylation of extracellular-signal–related protein kinases (p-ERK) 1/2, results in cellular pathology that is independent of angiotensin II. To date, the role of the (P)RR in retinal disease is not understood. However, the finding of high (P)RR expression in the central nervous system, with associations with ocular development7 and X-linked mental retardation and epilepsy,8 suggests that it may influence retinal function. Also to be considered when interpreting the actions of (P)RR is that it may also function as an accessory protein of vacuolar-ATPase (v-ATPase).9
A decoy-peptide epitope corresponding to amino acids 10 to 19 of the prorenin prosegment has been developed and termed the handle region peptide (HRP).10,11 It has been suggested that HRP prevents binding of renin and prorenin to the (P)RR inhibitor,10,11 and it has potent beneficial effects in diabetic glomerulosclerosis,12 cardiac fibrosis,13 and ocular pathology.10,11,14 Despite these findings, there is ongoing debate about these effects of HRP and whether the (P)RR is involved.5,6,15 Our goal was to evaluate the distribution of (P)RR in retina to gain insight into its cellular function and make a comprehensive evaluation of HRP’s effects by studying angiogenesis, inflammation, and neuro-glial function in retinal development and a rat model of retinopathy of prematurity known as oxygen-induced retinopathy (OIR). Comparisons were made with the ARB valsartan.
Materials and Methods
Animals and Treatments
Procedures using Sprague-Dawley rats complied with the National Health and Medical Research Council of Australia’s Guidelines for the care of animals in scientific research. HRP (sequence RILLKKMPSV)12 and a control HRP peptide with the reverse sequence (VSPMKKLLIR, designated HRP-R) were syntheiszed by Invitrogen. Valsartan was obtained from Novartis Pharma.
In rats, the retina develops postnatally, with most physiological angiogenesis occurring between birth and postnatal day (P) 7. Rat pups were administered either vehicle (0.9% NaCl) or 1 mg/kg HRP in 0.9% NaCl by daily IP injection from birth until P7. This dose and route of administration were based on a recent report in which plasma levels of HRP were measured.10 Administration by osmotic minipump was not possible because of the small size of rat pups.
OIR represents a model of marked retinal pathology in response to changes in inspired oxygen that occurs between birth and P18. On P0, pups and their mothers were randomly assigned to the following groups: (1) sham, exposed to room air from birth until P18; (2) sham+HRP; (3) sham+valsartan; (4) OIR control; (5) OIR+HRP; (6) OIR+valsartan; and (7) OIR+HRP-R. OIR was induced using a published method.16 Pups were exposed from P0 until P11 to 80% oxygen for 22 hours/day and to room air for 2 hours/day, a process that suppresses normal retinal physiological angiogenesis. On P12, pups were placed in room air until P18, which causes the upregulation of angiogenic and inflammatory factors and pathological angiogenesis. OIR is also associated with deficits in neuronal and glial function. In OIR, treatments were given during the period of retinal pathology (P12 to P18). In shams, treatments were also given between P12 and P18. For comparability with most previous studies, HRP was delivered at 0.1 mg/kg per day via osmotic minipump (1007D, Alzet; in 0.9% saline at 0.5 μL/hour in a 100-μL volume), which was inserted into the flank. HRP-R was administered at 0.1 mg/kg per day by osmotic minipump (0.9% saline). Valsartan was given by IP injection (10 mg/kg per day in 0.1 mol/L Tris buffer). For detailed Materials and Methods, please see the Supplemental Data, available online at http://hyper.ahajournals.org.
HRP, HRP-R, and valsartan had no effect on body weight (Supplemental Table I).
In retina, most (P)RR immunolabeling was apparent in ganglion cells and Müller cells in the inner nuclear layer (Figure 1 and Supplemental Figure I). A glial site for (P)RR was confirmed in cultured rat glia (Figure 1). In retina, (P)RR immunolabeling was also identified in some retinal blood vessels, which was confirmed in cultured retinal endothelial cells and pericytes (Figure 1).
HRP Reduces Angiogenesis in Developing Retina and OIR
HRP reduced vascular growth from the optic disk to peripheral retina compared with untreated controls (Figure 2).
In OIR controls, blood vessel profiles were increased throughout the inner retina compared with sham controls, and they protruded into the vitreous. HRP and valsartan had no effect on the number of blood vessel profiles in the inner retina in shams, but they reduced blood vessel profiles to sham control levels in OIR, whereas HRP-R had no effect on retinal blood vessel profiles (Figure 2 and Supplemental Figure II).
HRP Reduces Inflammation in OIR
In shams, few leukocytes were detected in retinal vessels, and neither HRP nor valsartan influenced leukostasis (Figure 3). Leukostasis was increased in OIR controls compared with sham groups, and it was reduced by both HRP and valsartan to sham levels, with no difference between treated groups. HRP-R had no effect on leukostasis (Figure 3 and Supplemental Figure II).
Retinal Vascular Endothelial Growth Factor, Intercellular Adhesion Molecule-1, and (P)RR mRNA in Shams and OIR
Vascular Endothelial Growth Factor and Intercellular Adhesion Molecule-1
In shams, neither HRP nor valsartan affected retinal vascular endothelial growth factor (VEGF) or intercellular adhesion molecule-1 (ICAM-1) mRNA levels (Figure 4). In OIR, both VEGF and ICAM-1 mRNA levels were increased compared with sham groups, and HRP and valsartan reduced these to sham control levels, whereas HRP-R had no effect on retinal VEGF or ICAM-1 mRNA levels in OIR.
In shams, HRP increased (P)RR mRNA, whereas valsartan had no effect. In OIR, (P)RR mRNA was increased compared with sham control, and both HRP and valsartan reduced (P)RR mRNA levels to those of the sham control, whereas HRP-R had no effect (Figure 4).
HRP Reduces Ganglion and Glial Cell Viability and Retinal Function
In sham controls, p-ERK1/2 immunolabeling was barely detected, and HRP caused a marked increase in p-ERK1/2 immunolabeling in ganglion cells and glia in the inner nuclear layer (Figure 5). These findings with HRP treatment in shams were consistent with a decline in the electroretinogram (ERG). In particular, the postphotoreceptor responses of the b wave and the amacrine cell-derived oscillatory potentials were significantly reduced (Figure 5 and Supplemental Figure III). There was also a reduction in the photoreceptor response (a wave), indicating photoreceptor dysfunction in shams treated with HRP (Supplemental Figure III). In contrast, valsartan had no effect on p-ERK1/2 or the ERG in shams (Figure 5). In OIR controls, as expected, p-ERK1/2 was increased, and there was a decline in the ERG photoreceptor response (a wave) and postphotoreceptor responses (b wave, oscillatory potential amplitude), compared with sham controls (Figure 5 and Supplemental Figure III). In OIR, HRP had no significant effect on p-ERK1/2 immunolabeling and slightly reduced the ERG responses, whereas valsartan reduced p-ERK1/2 immunolabeling without effect on the ERG. HRP-R had no effect on either p-ERK1/2 immunolabeling or the ERG in OIR (Figure 5 and Supplemental Figures II and III).
HRP Concentrations in Plasma and Blood
Plasma HRP concentrations were undetectable (<3 pmol/L) for rats infused with either 0.1 or 1 mg/kg HRP per day (Supplemental Figure IV). We examined the metabolism of HRP in EDTA-plasma by high-performance liquid chromatography with monitoring of absorbance at 214 nm and found HRP to be degraded with a half-life of ≈5 minutes at 37°C, whereas it remained stable for at least 30 minutes at 0°C (data not shown). To exclude the possibility that plasma HRP levels were undetectable because of ex vivo metabolism, we measured HRP in blood collected directly into 4 mol/L guanidine thiocyanate to prevent HRP metabolism ex vivo. Consistent with the plasma HRP measurements, blood HRP concentrations were undetectable (<25 pmol/L) for rats infused with either 0.1 or 1 mg/kg HRP per day (Supplemental Figure IV).
Here we make the first report that the (P)RR is expressed on the retinal vasculature, neurons, and glia. Our evaluation of HRP in retina revealed distinct antiangiogenic and antiinflammatory effects that in OIR were comparable to ARB. Of particular interest was that in retinal neurons and glia where (P)RR was most abundantly expressed, HRP induced injury with increased p-ERK1/2 immunolabeling and a worsened ERG. HRP’s ability to modulate (P)RR expression in retina suggests that its actions involve the (P)RR through mechanisms yet to be defined.
Our previous demonstration of prorenin on retinal blood vessels suggested a vascular role for prorenin.17 However, the major location of prorenin was macroglial Müller cells, the site of renin expression.18 Given that Müller cells contribute to retinal vasculopathy, it is possible that prorenin located in Müller cells has a paracrine effect on the vasculature. Here we report the localization of the (P)RR to retinal endothelial cells and pericytes, and also to glia in the inner nuclear layer, the site of Müller cell nuclei. Most notable was (P)RR immunolabeling in ganglion cells, which is consistent with reports of high levels of (P)RR expression in brain neurons.19,20 In contrast to our findings, a previous study reported both activated prorenin and (P)RR immunolabeling on retinal blood vessels, with no evidence of a neuro-glial localization.14
We confirmed the previously reported antiinflammatory actions of HRP in OIR14 and found HRP’s actions to be similar to those of valsartan in OIR. In addition, we found HRP attenuated physiological angiogenesis in the developing retina to an extent similar to that previously reported for angiotensin-converting enzyme inhibition.17 Our finding that neither HRP nor ARB had an effect on angiogenesis and inflammation in shams, yet reduced angiogenesis in developing retina at P7, is not surprising given that the retinal vasculature in shams at P12 is largely established and less vulnerable to antiangiogenic agents.21 The finding that HRP-R did not alter vasculopathy in OIR further highlights the bioactive properties of HRP and suggests that the actions of HRP on the retina were sequence specific.
Retinal neurons and glia are damaged in OIR, which is reflected by a substantial reduction in retinal function as assessed by the ERG.22 In retinal diseases such as diabetes,23 ERK1/2 is activated in neurons and glia and acts as a stress sensor in cellular proliferation, differentiation, and repair.24 One of our most interesting findings was in shams, where HRP markedly reduced the a and b waves and oscillatory potentials of the ERG and increased p-ERK1/2 expression in neurons and glia. This may suggest that in shams, although the vasculature was not influenced by HRP, neurons and glia were particularly sensitive to HRP. HRP also had a slight detrimental effect on the ERG in OIR despite the already heavily compromised ERG. p-ERK1/2 expression was near-maximal in OIR, and therefore it is not surprising that there was no further increase with HRP. Given these findings and that retinal neuro-glia are immunolabeled for (P)RR, it is tempting to speculate that the detrimental effects of HRP were linked to the (P)RR. Our finding that HRP modulated retinal (P)RR mRNA levels in shams and OIR was suggestive of this possibility.
Our previous studies indicated that ARB prevents glial (astrocyte) apoptosis in OIR25 and restores deficits in the ERG of diabetic animals.26 Given these findings, it was not surprising that valsartan had no effect on the ERG and p-ERK1/2 in shams and improved p-ERK1/2 in OIR. It might be expected that valsartan would restore the ERG in OIR; however, this was unlikely given that although improvements in individual neuronal and glial cell populations can occur in OIR, recovery of overall retinal function can take much longer than the P18 time frame of the present study.22 Overall, our findings highlight the injurious effects of HRP on retinal neurons and glia compared with ARB. Of interest was the reduction in retinal (P)RR mRNA by valsartan in OIR, indicating a possible interaction between ARB and (P)RR expression in retinal disease.
Our finding that the concentration of HRP in both plasma and blood was undetectable for rats infused with either 0.1 or 1 mg/kg HRP per day suggested rapid metabolism of the peptide in vivo, and this interpretation was supported by our finding that HRP was metabolized with a half-life of 5 minutes in EDTA-plasma at 37°C. Satofuka et al10 reported transient plasma HRP concentrations of ≈100 nmol/L in mice administered 1 mg/kg HRP by IP injection. There are several possible reasons why we measured lower circulating HRP levels than reported by Satofuka et al10 In contrast to the single IP injection of HRP by Satofuka et al,10 we administered HRP by continuous subcutaneous infusion. HRP may have been absorbed into blood more rapidly and with less metabolism from the peritoneal cavity in their studies than from the subcutaneous space in our studies. It is also of note that the HRP assay used by Satofuka et al10 measured immunoreactive HRP and may have detected HRP metabolites, whereas the high-performance liquid chromatography–based radioimmunoassay used in our study measured only intact HRP. We added plasma and blood to 4 mol/L guanidine thiocyanate before extraction to prevent any metabolism of HRP during sample processing. If HRP were in complex with the soluble (P)RR,27 guanidine thiocyante would have dissociated the complex, thereby allowing HRP to be measured by our assay.
Several studies suggest that the effects of HRP are mediated by concentrations less than those required to influence prorenin binding to the (P)RR. Satofuka et al10 found that 10 μmol/L HRP produced only 50% inhibition of prorenin-stimulated ERK1/2 phosphorylation in brain-derived capillary endothelial cells. Although these authors reported peak plasma concentrations of ≈100 nmol/L in mice injected with 1 mg/kg IP, similar effects on retinopathy were seen with 0.1 mg/kg, and in another study, 0.01 mg/kg suppressed ocular inflammation.11 Moreover, HRP administration at 1.8 to 3.6 μg/kg per day by subcutaneous minipump prevented and reversed diabetic nephropathy in rats.12,28 The very low circulating concentration of HRP in our studies raises the question of its site of action in mediating its effects on the retina. Emerging evidence indicates that interleukins, complement, and circulating angiogenic cytokines are associated with retinopathy of prematurity.29 One possibility is that HRP has actions in the subcutaneous tissue at the site of infusion, possibly modulating cytokine release or cells of the immune system. Overall, the present study raises important questions about the mechanisms by which HRP produces its effects, which may be particularly relevant to the retina.
In summary, we identified that HRP clearly exhibited antiangiogenic and antiinflammatory effects but also induced neuronal and glial injury in retina. Our findings that the (P)RR was expressed in retinal neurons and glia and that HRP modulated retinal (P)RR expression suggest that changes in (P)RR expression may mediate in part the effects of HRP on retinal function. This would be consistent with an emerging role for (P)RR in the central nervous system.
Of possible relevance to the actions of the (P)RR is its role as an accessory protein of v-ATPase.6 v-ATPase is implicated in the stimulation of angiogenesis, inflammation, and neurotransmitter uptake.9 Indeed, inhibition of v-ATPase with bafilomycin reduces these effects by inducing cellular autophagy.9 Our results are consistent with HRP having similar actions to a v-ATPase inhibitor in retina. These properties of HRP and particularly its detrimental effects on neuro-glia may be confined to retinal development and OIR, and additional studies are required to determine whether HRP has a role in the treatment of ocular pathologies.
The authors thank Kylie McMaster, Lainie Sutton, and Frosa Katsis for technical assistance.
Sources of Funding
This work was supported by National Health and Medical Research Council grant 491058. J.L.W.-B. and D.J.C. are National Health and Medical Research Council Senior Research Fellows. A.G.M. is a Juvenile Diabetes Research Foundation Post-Doctoral Scholar. M.E.C. is an Australian Research Fellow and a Juvenile Diabetes Research Foundation Scholar.
- Received November 30, 2009.
- Revision received December 19, 2009.
- Accepted March 15, 2010.
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