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. Author manuscript; available in PMC: 2015 Aug 1.
Published in final edited form as: Hypertension. 2014 May 27;64(2):369–377. doi: 10.1161/HYPERTENSIONAHA.114.03654

The Prostaglandin EP4 Receptor Mediates Angiotensin II-Induced (Pro)Renin Receptor Expression in the Rat Renal Medulla

Fei Wang 1,2, Xiaohan Lu 1,2, Kexin Peng 1, Yaomin Du 3, Shu-Feng Zhou 4, Aihua Zhang 5, Tianxin Yang 1,2
PMCID: PMC4445967  NIHMSID: NIHMS587185  PMID: 24866147

Abstract

AngII stimulates (pro)renin receptor (PRR) expression in the renal collecting duct (CD), triggering the local renin response in the distal nephron. Our recent study provided evidence for involvement of COX-2-PGE2 pathway in AngII-dependent stimulation of PRR expression in the CD. Here we tested the role of EP subtypes acting downstream of COX-2 in this phenomenon. In primary rat inner medullary collecting duct (IMCD) cells, AngII treatment for 12 h induced a 1.8-fold increase in the full-length PRR protein expression. To assess the contribution of EP receptor, the cell were pre-treated with specific EP receptor antagonists: SC-51382 (for EP1), L-798106 (for EP3), and L-161982 (for EP4) and ONO-AE3-208 (ONO, a structurally distinct EP4 antagonist). The upregulation of PRR expression by Ang II was consistently abolished by L-161982 and ONO, and partially suppressed by SC-51382, but was unaffected by L-798106. The PRR expression was also significantly elevated by the EP4 agonist CAY10598 in the absence of AngII. Sprague-Daley rats were subsequently infused for 1 or 2 weeks with vehicle, AngII alone or in combination with ONO. AngII infusion induced parallel increases in renal medullary PRR protein, and renal medullary and urinary renin activity and total renin content, all of which were blunted by ONO. Both tail cuff plethysmography and telemetry demonstrated attenuation of AngII hypertension by ONO. Overall, these results have established a crucial role of the EP4 receptor in mediating the upregulation of renal medullary PRR expression and renin activity during AngII hypertension.

Keywords: (Pro)renin receptor, prostaglandin E2, EP4 receptors, renin activity, inner medullary collecting duct

Introduction

In recent years, there is rising interest about the local renin-angiotensin system (RAS) in a variety of tissues including the kidney1, 2. Within the kidney, angiotensinogen is expressed in the proximal tubule and renin in the connecting tubules3 and cortical and medullary collecting ducts (CDs)4, 5, forming the anatomic basis of intrarenal RAS. In response to angiotensin II (AngII), the intrarenal RAS is activated as reflected by increased renin mRNA and protein expression in the CD6 whereas the systemic RAS is suppressed, highlighting the difference in the two RAS system. Several lines of evidence demonstrate a critical role of intrarenal RAS in AngII-induced hypertension. Experiments in rats infused with Val5-Ang II, an isoform of AngII that can be separated from endogenous AngII (Ile5-Ang II) by high-performance liquid chromatography, demonstrated that the chronic Val5-Ang II (exogenous AngII) infusion induces renal Ile5-Ang II (endogenous Ang II) synthesis 7. In another study, when endogenous AngII production was reduced by ACE inhibition, AngII–infused mice became normotensive8, 9. The genetic absence of kidney ACE substantially blunts the hypertension induced by AngII infusion10. In experiments involving kidney cross-transplantation between global AT1 KO mice and wild-type controls, AngII is shown to cause hypertension through stimulation of AT1 receptors in the kidney 11. Lastly, overexpression of renin in the CD causes spontaneous hypertension12. However, evidence also exists to suggest that some components of the RAS may be of extrarenal origin. For example, renal angiotensinogen and AngII are shown to originate from liver13.

(Pro)renin receptor (PRR) is a newly discovered component of the RAS, being capable of binding renin and prorenin with almost equal affinity to increase their catalytic activity14. PRR is considered to play an important role in regulation of tissue renin activity thereby controlling the activity of local RAS. Within the kidney, PRR expression is predominantly expressed in the intercalated cells of the CD 15. Chronic infusion of AngII in rats increased renal PRR transcript levels and augmented the PRR activity in renal medullary tissues, which may contribute to increased renin activity in the CD during AngII hypertension16. The activation of renal medullary PRR may serve as an important mechanism triggering the local renin response that may participate in regulation of blood pressure and fluid metabolism during AngII hypertension 16.

The biologic action of PGE2 is mediated by G protein-coupled E-prostanoid receptors designated EP1, EP2, EP3 and EP4 17. These four subtypes of EP receptor couple to distinct signaling pathways. Among the four EP subtypes, the EP4 receptor plays a dominant role in regulation of renin release from the juxtaglomerular apparatus18, 19. We hypothesize that the EP4 receptor may participate in AngII-induced renin response in the renal medulla through an impact on PRR. To test this hypothesis, we employed pharmacological inhibitors and activators of the EP4 receptor to study their effect on renal medullary PRR expression and renin activity, and hypertension development following AngII treatment.

Methods

Animals

Male Sprague-Dawley rats (220-250 g, Charles River Laboratories, Wilmington, MA) were cage- housed and maintained in a temperature-controlled room with a 12:12-h light-dark cycle, with free access to tap water and standard rat chow for 14 days. The animal protocols were approved by the Animal Care and Use Committee at Sun Yat-sen University, China. Rats randomly received either sham operation, AngII infusion (Human AngII, Sigma, St. Louis, MO) via a subcutaneous osmotic minipump (Alzet model 2002, Alza, Palo Alto, CA) at a rate of 100 ng/min, or co-administered with ONO-AE3-208 (ONO) (MedChemexpress LLC, Princeton, NJ) 20 at 0.2 mg/kg/d for 14 days. Under isoflurane anesthesia, the minipump was subcutaneously implanted in the back of the neck area. At the end of the experiment systolic blood pressure (SBP) was monitored by tail cuff plethysmography; the rats were placed in metabolic cages for 24-h urine collections. At day 14, under isoflurane anesthesia, blood was withdrawn from vena cava and kidneys were harvested and cut into cortex and inner medulla. To validate the blood pressure results, telemetry was performed in a separate experiment to monitor daily mean arterial pressure (MAP) in rats infused with AngII alone or in combination with ONO for 7 days at the same doses.

Primary cultures of rat inner medullary collecting duct (IMCD) cells

Primary cultures enriched in IMCD cells were prepared from pathogen-free male Sprague-Dawley rats (40∼100 g body wt) as previously described21. After 24 hours of serum deprivation, The IMCD cells were pretreated for 1 h with structurally distinct EP4 antagonists, ONO at 1 μM or L-161982 at 10 μM (Cayman chemical, Ann Arbor, MI), an EP1 antagonist SC-51382 at 10 nM (Cayman chemical, Ann Arbor, MI), or an EP3 antagonist L-798106 at 10 μM, (Tocris Bioscience, United Kingdom), followed by AngII treatment at 100 nM or 1 μM for various time periods. To study the effect of EP4 agonism on PRR expression, the IMCD cells were exposed to an EP4 agonist CAY10598 0.1 μM 10 (Cayman chemical, Ann Arbor, MI) in the absence of AngII. After these treatments, the cells were harvested for gene expression analysis or renin assay.

Sample preparation for renin activity assay

The blood samples were collected into tubes with 5.0 mmol/l EDTA and PRA were assayed. Urine and cell culture medium were applied to MW 10,000 cut-off centrifugal tubes (Amicon Ultra) to concentrate proteins higher than ∼30 kDa. The renal inner medulla and cortex were homogenized in 2.6 mM EDTA, 3.4 mM hydroxyquinoline, 5 mM ammonium acetate, 200 μM PMSF, and 0.256 μM dimercaprol. The homogenates were centrifuged at 4,000 rpm at 4°C for 30 min and the supernatant was collected.

Assay of renin activity

The samples were spiked with 1 μM synthetic renin substrate tetradecapeptide (RST; Sigma) for plasma, urine, and kidney tissues and with a final concentration of 1 μM angiotensinogen for cell culture medium. Following incubation at 37 °C for 1 h, the AngI generation was assayed using an EIA kit according to the manufacture's instruction (S-1188 Angiotensin-I EIA kit from Bachem). To exclude the effect of peptidases, identical urine samples-RST with the specific renin inhibitor WFML peptide (AnaSpec, Fremont, CA) were used as controls. The values were expressed as nanograms per milliliter per hour of generated AngI. Trysinization activates prorenin to renin22. For trypsinization, samples incubated with trypsin from bovine pancreas (T1426 from sigma) in 37 °C for 18 h and the reaction was then terminated with Soybean Trypsin Inhibitor (100 g/L) for 10 min on ice. Renin activity was determined in the native condition, active renin content with excessive angiotensinogen, and total renin content with excessive angiotensinogen plus trypsinization.

Immunoblotting

Renal tissues were lysed and subsequently sonicated in PBS that contained 1% Triton x-100, 250 μM phenylmethanesulfonyl fluoride (PMSF), 2 mM EDTA, and 5 mM dithiothrietol (DTT) (pH 7.5). Protein concentrations were determined by the use of Coomassie reagent. 40 μg of protein for each sample was denatured in boiling water for 10 min, then separated by SDS-PAGE, and transferred onto nitrocellulose membranes. The blots were blocked overnight with 5% nonfat dry milk in Tris-buffered saline (TBS), followed by incubation for 1 h with rabbit anti-PRR antibody (Cat#:ab40790, Abcam). In Figs. 1A, 2A, and 5, the membranes were stripped and reprobed with mouse anti-β-actin antibody (Cat#:A1978, Sigma). In Figs. 1C, 1E, 2A, 6A, and 6D, independent anti-β-actin immunobotting was performed since the stripping protocol did not yield optical results. After washing with TBS, blots were incubated with goat anti-rabbit horseradish peroxidase (HRP)-conjugated secondary antibody and visualized using Enhanced Chemiluminescence (ECL). The blots were quantitated by using Imagepro-plus.

Fig. 1.

Fig. 1

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Effect of EP4 antagonists on AngII-induced PRR protein expression in primary IMCD cells. The cells were pretreated for 1 h with two structurally distinct EP4 antagonists, L-16982 (A) and ONO (B), and then treated for 12 h with 1 μM AngII. In a separate experiment, the cells were treated for 12 h with ONO in the absence of AngII (C). PRR protein expression was analyzed by immunoblotting. (A, C, and E) Representative PRR immunoblot from 2-3 independent experiments. The full-length PRR protein was detected as a 43-kDa band. (B, D, and F) Densitometric analysis of PRR protein and normalized by β-actin. N = 6 per group. Data are mean ± SE.

Fig. 2.

Fig. 2

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Effect of EP4 agonism on PPR expression in primary IMCD cells. The cells were treated with the EP4 agonist CAY10598 at 0.1 μM for 12 h. PRR protein expression was analyzed by immunoblotting. (A) Representative PRR immunoblot from 3 independent experiments. (B) Densitometric analysis of PRR protein and normalized by β-actin. N = 6-8 per group. Data are mean ± SE.

Fig. 5.

Fig. 5

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Validation of the effect of EP4 antagonism on PRR expression in response to a lower dose of AngII. The IMCD cells were exposed to 100 nM AngII for 3 h in the presence or absence of 1 μM ONO. (A) Representative PRR immunoblot from 2 independent experiments. (B) Densitometric analysis of PRR protein and normalized by β-actin. N = 6 per group. Data are mean ± SE.

Fig. 6.

Fig. 6

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The expression of PRR in the renal inner medulla of rats treated with vehicle, AngII, or AngII + ONO. (A) Representative PRR immunoblot from 3 independent experiments. (B) Densitometric analysis of PRR protein. The PRR protein expression was normalized by β-actin. N = 5 per group. (C) qRT-PCR analysis of PRR mRNA. The mRNA expression was normalized by GAPDH. (D) Effect of ONO on baseline PRR protein expression in the rat renal inner medulla. The expression was determined by using immunoblotting analysis. N = 5 per group. Data are mean ± SE.

qRT-PCR

Total RNA isolation and reverse transcription were performed as previously described 23. Oligonucleotides were designed using Primer3 software (available at http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi). Primers for PRR were 5′-tgggaagcgttatggagaag-3′ (sense) and 5′-ggttgtagggactttgggtgt-3′ (antisense); primers for GAPDH were: 5′-gtcttcactaccatggagaagg-3′ (sense) and 5′-tcatggatgaccttggccag-3′ (antisense).

Blood pressure measurement

SBP was measured by tail cuff plethysmography using a Visitech BP2000 Blood Pressure Analysis System (Apex, NC) 24. All animals were habituated to the Bp measurement device for 7 days. They all underwent two cycles of 20 measurements reordered per day for a minimum of three days. Telemetry measurement of MAP was performed as previously described 25. Daily MAP was recorded as mean values of 4-h recordings from 5:00PM to 9:00PM.

Statistical Analysis

Data are summarized as means ± SE. Statistical analysis was performed using ANOVA with the Bonferroni test for multiple comparisons or by unpaired student t-test for two comparisons. p < 0.05 was considered statistically significant.

Results

In vitro investigation of the role of the EP receptor in mediating AngII-induced PRR expression in primary rat IMCD cells

We attempted to examine the EP subtypes involved in regulation of PRR expression in primary rat IMCD cells following AngII treatment. IMCD cells were isolated from the inner medulla of SD rats and gown in 6-well plates. After reaching confluence, the cells were exposed to AngII in the presence of absence of an EP antagonist. In our previous study, we observed that 1 μM AngII induced a peak stimulation of PRR protein expression at 12 h. accordingly; this experimental condition was used in the subsequent experiments to investigate the involvement of EP receptors. By immunoblotting, the full-length PRR was detected as a 43-kDa band. Exposure to 1 μM AngII for 12 h elevated the total protein abundance of PRR (Fig. 1). The increase of PRR protein abundance was abolished by an EP4 antagonist L-161982 at 10 μM (Fig. 1A&B). The same result was obtained by using a structurally distinct EP4 antagonist ONO at 1 μM (Fig. 1C&D). In a separate experiment, we found that the basal PRR protein expression was significantly suppressed by EP4 antagonism (Fig. 1E&F). To further validate EP4 regulation of PRR expression, we examined the effect of the EP4 agonist CAY10598 on PRR expression in primary IMCD cells. Following 12 h exposure to 100 nM CAY10598, PRR protein expression was significantly increased (Fig. 2).

In separate experiments, we examined the involvement of EP1 and EP3 by using the respective EP antagonists. The EP1 antagonist SC-51382 at 10 nM effectively attenuated AngII-induced PRR protein expression but the EP3 antagonist L-798106 at 10 μM was without an effect. Fig. 3A showed the densitometry results from these Western blots. qRT-PCR was performed to examine PRR mRNA expression following the treatment with AngII alone or in combination with an EP antagonist (for EP1, EP3, or EP4). However, we found no change in PRR mRNA in any of the experimental group (Fig. 3B).

Fig. 3.

Fig. 3

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(A) Effects of EP1 and EP3 antagonists on AngII-induced PRR protein expression in primary IMCD cells. The cells were pretreated for 1 h with the EP1 antagonist SC-51382 and the EP3 antagonist L798106 and then treated for 12 h with 1 μM AngII. Shown is the densitometric result from PRR immunoblotting. (B) Effects of EP antagonists on PRR mRNA expression during AngII treatment. The cells were pretreated for 1 h with SC-51382, ONO, or L-798106 and then then treated for 12 h with 1 μM AngII. PRR mRNA was detected by qRT-PCR and normalized by GAPDH. N = 6 per group. Data are means ± SE.

In light of the potential role of PRR in regulation of renin activity, we performed assay for renin activity in the medium of primary IMCD cells exposed to 1 μM AngII alone or in combination with ONO. The assay was performed using angiotensin I ELISA kit. To validate specificity of this kit, we examined its cross-activity with AngII. The cross-activity was undetectable for 1 or 100 μM AngII and 3/100,000 for 1 mM AngII, consistent with the report from the manufacturer, Peninsula Laboratories, LLC. As shown in Fig. 4, 1 μM AngII treatment for 12 h significantly increased medium renin activity and this increase was almost completely abolished by ONO (Fig. 4A). Similar results were obtained for active renin content (Fig. 4B). By qRT-PCR, renin mRNA was altered in a similar fashion as renin activity and active renin content (Fig. 4C).

Fig. 4.

Fig. 4

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Effect of EP4 antagonism on AngII-induced renin activity and renin content in primary IMCD cells. The cells were exposed to 1 μM AngII for 12 h in the presence or absence of 1 μM ONO. (A) Medium renin activity. (B) Medium active renin content. (C) qRT-PCR detection of renin mRNA. N= 3 per group. Data are means ± SE.

Given the concern about the high concentration of AngII used in the above-described experiments, we validated some of the major results from these experiments by using 100 nM AngII. Time course studies demonstrated that in response to 100 nM AngII treatment, PRR protein expression was induced at 3 h and decreased rapidly thereafter. EP4 antagonism with ONO completely abolished the PRR induction by 100 nM AngII and also reduced the basal PRR expression, thus confirming the results with the higher dose of AngII (Fig. 5).

In vivo investigation of the role of the EP4 receptor in mediating AngII-induced PRR expression in rat renal inner medulla

SD rats were treated for 14 days with AngII in combination with or without ONO. The endpoints included renal medullary expression of PRR expression, plasma, urinary, and tissue renin activity and content, as well as blood pressure. 14-day AngII infusion significantly increased PRR protein expression in the inner medulla as assessed by immnoblotting (4.2 ± 0.4 vs. 1.0 ± 0.2, p<0.05) (Fig. 6A&B). EP4 antagonism with ONO completely abolished the upregulation of renal medullary PRR expression by AngII (0.6±0.2 in the AngII + ONO group) (Fig. 6A&B). In contrast, qRT-PCR detected no change in PRR mRNA expression in the inner medulla following AngII treatment (Fig. 6C). In a separate experiment, we examined the effect of ONO on the baseline PRR protein expression in the rat renal inner medulla. A 14-day ONO treatment induced a small but significant reduction of the baseline PRR expression in the inner medulla (0.84 ± 0.15 vs. 1.0 ± 0.17, n = 4, p<0.05) (Fig. 6D).

Plasma, the renal cortex, and the inner medulla were subjected to measurement of renin activity, active renin content, and total renin content. All renin parameters including renin activity, active and total renin content in plasma were significantly suppressed following AngII infusion, which was unaffected by ONO treatment (Fig. 7A-C). A similar pattern of changes in these renin parameters were observed in the renal cortex (Fig. 7D-F). In a sharp contrast, these parameters were all elevated in urine following AngII infusion, which was almost completely reversed by ONO treatment (Fig. 8A-C). These results were almost identical to those in the inner medulla (Fig. 8D-F). These results reflect the opposite responses of systemic and renal medullary renin system and also suggest that urinary renin is of renal medullary origin.

Fig. 7.

Fig. 7

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Renin levels in plasma and renal cortex of rats treated with vehicle, AngII, or AngII + ONO. (A) Plasma renin activity. (B) Plasma active renin content. (C) Plasma total renin content. (D) Renal cortical renin activity. (E) Renal cortical active renin content. (F) Renal cortical total renin content. N = 5 per group. Data are mean ± SE.

Fig. 8.

Fig. 8

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Renin levels in urine and renal inner medulla of rats treated with vehicle, AngII, or AngII + ONO. (A) Urine renin activity. (B) Urine active renin content. (C) Urine total renin content. (D) Cortical renin activity in the inner medulla. (E) Active renin content in the inner medulla. (F) Total renin content in the inner medulla. N = 5 per group. Data are mean ± SE.

Systolic blood pressure (SBP) was measured by using tail cuff plethysmography. SBP was significantly higher in the AngII group than in the control group (182.3± 14.5 in the AngII group vs. 116.2 ±6 mmHg in the Control group, p<0.05) and the increase in SBP was less in the AngII + ONO group (143.5± 7.0 mmHg) (Fig. 9A). To validate this result, in a separate experiment, we compared MAP between the AngII group and the AngII + ONO group using telemetry. Again, the MAP was lower in the AngII + ONO group than in the AngII group (Fig. 9B). In another separate experiment, we examined the effect of ONO on baseline MAP in rats. The baseline MAP was unaffected over a 7-day ONO treatment (106.7± 6.4 mmHg in the ONO group vs. 107.5± 5.6 mmHg in the control group, n = 5 per group, p>0.05). Together, these results suggest that the EP4 receptor mediates AngII-induced hypertension but may play no role in the control of baseline blood pressure.

Fig. 9.

Fig. 9

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Effect of ONO on AngII-induced hypertension in SD rats. (A) Measurement of systolic blood pressure (SBP) by tail cuff plethysmography. (B) Measurement of mean arterial pressure (MAP) by telemetry. *, p<0.05 and **, p<0.01 vs. AngII at the corresponding period. N = 5 per group. Data are mean ± SE.

Discussion

The present study examined the EP subtypes involved. In primary cultures of rat IMCD cells, EP4 antagonism with structurally distinct EP4 antagonists completely abolished AngII-induced PRR expression and EP4 agonism alone elevated the expression. In SD rats, EP4 antagonism effectively suppressed the increases in renal medullary PRR expression, renal medullary and urinary renin levels, as well as blood pressure in response to AngII infusion. Interestingly, in vitro data also suggested involvement of the EP1 but not the EP3 subtype in AngII-induced PRR expression.

PGE2 is a major prostanoid produced in the kidney, particularly in the CD. As an autocrine/paracrine factor, PGE2 exerts a diverse range of action at the site of its production, affecting renal medullary blood flow and tubular sodium and water transport, as well as cell survival 26, 27. The biologic action of PGE2 is mediated by four distinct EP4 receptors (EP1-4). We for the first time demonstrated a dominant role of the EP4 receptor in mediating AngII-induced PRR expression and renin activity in the renal medulla. The evidence for this conclusion is compelling. First, EP4 antagonists were highly efficient in inhibiting PRR expression in that they not only blocked AngII-induced PRR expression but also remarkably reduced the basal expression. The use of structurally distinct EP4 antagonists has resolved the specificity issue related to the pharmacological approach. Second, the involvement of EP4 has also been demonstrated by EP4 agonism. Lastly, the observation with EP4 antagonist was initially made in vitro and was subsequently confirmed in vivo.

The detailed signaling pathway downstream of the EP4 receptor in the CD is not known. This EP subtype is presumed to signal through the Gs protein, which elevates intracellular cAMP. It seems reasonable to speculate that cAMP pathway may be involved in EP4-depenent stimulation of PRR expression during AngII hypertension. Currently, the direct evidence supporting this notion is lacking. On the other hand, the cGMP-PKG signaling pathway is shown to mediate the upregulation of PRR expression in IMCD cells exposed to salt depletion28. Although the relationship between cAMP and cGMP is generally antagonistic to each other, the two mediators can couple together to simulate renin secretion in JG cells. The cooperation between the two signaling pathways in renin regulation is reflected by the fact that cGMP inhibits phosphodiesterases (PDE) 3 that degrades cAMP. Future studies need to assess the relative importance of the two signaling pathways in regulation of PRR in the CD during AngII hypertension.

Abundant in vitro evidence demonstrates that PRR binds renin and prorenin to increase their catalytic activity14, 29-32. Accordingly, PRR is considered as a potential regulator of tissue RAS. However, solid in vivo evidence to support this notion is still lacking. Overexpression of human PRR in rats resulted in proteinuria and nephropathy but did not elevate blood pressure or renal AngII levels 33, 34. The lack of viable PRR null mice, systemic or tissue-specific, has made it difficult to convincingly prove PRR as a key player in RAS 35. In the present study, we observed that the decreased renal medullary PRR expression by EP4 antagonism was in parallel with the reduction of renal medullary renin levels and blood pressure. This observation represents indirect evidence supporting PRR being involved in renin regulation in the renal medulla during AngII treatment.

The PGE2/EP4 pathway has an established role in the macula densa signal for release of renin. For example, infusion of PGE2 into the kidney stimulates renin secretion in various ex vivo and in vitro JG cell culture models 36, 37 and this effect depends on cAMP 38. Deletion of EP4 receptors reduces renin stimulation by 70% following furosemide administration whereas deletion of EP2 has no effect19, supporting the dominant role of this receptor in renin regulation. Therefore, we can't rule out the possibility that EP4 activation may directly regulate renin expression, activity, or release in the CD independently of PRR.

The EP1 receptor was originally described as a smooth muscle constrictor 17. This receptor generally signals through intracellular calcium and protein kinase C. In the present study, we found that EP1 antagonism effectively blocked AngII-induced PRR expression, suggesting involvement of the EP1 receptor in the upregulation of PRR. It seems possible that the corporation between EP1 and EP4 subtypes may be required for the full PRR response during AngII hypertension. Whether intracellular calcium and PKC mediate the effect of EP1 activation is not known. Of note, like the EP1 receptor, the EP3 receptor also signals through intracellular calcium and protein kinase C and induces vasoconstriction 39, 40. However, we found no effect of EP3 antagonism on PRR expression. This may be related to the complex signaling properties of the EP3 receptors via Gi (inhibition of cAMP formation), Gs (stimulation of cAMP formation), and Gq (stimulation of intracellular Ca2+ release), which may exert diverse influence on PRR expression 41. Of note, the present study is limited in that the functional role of the EP2 receptor in PRR regulation was not tested due to the lack of commercially available antagonist for this receptor subtype.

The PRR-mediated local renin response is expected to contribute to AngII hypertension. Indeed, COX-2 deficiency or inhibitors like refecoxib and nimesulide exhibit potent antihypertensive action in rodent models of AngII hypertension42-44. The present study has extended these observations by elucidating the EP4 receptor as the responsible receptor subtype acting downstream of COX-2 in mediating the hypertensive response to AngII. In support of this notion, EP4 antagonism effectively lowered blood pressure accompanied with suppressed renal medullary PRR expression and renin levels. The blood pressure lowering effect of EP4 antagonism was initially observed by using tail cuff plethysmography and subsequently confirmed by using telemetry. Paradoxically, the EP4 receptor mediates PGE2-elicited acute vasodilator response by coupling with eNOS 45, 46. In addition, the EP4 activation promotes salt and water excretion during the blockade of the Na-K-2Cl co-transporter NKCC2 18. Together, the vasodilator and natriuretic/diuretic actions of the EP4 receptor will predict an antihypertensive action, which apparently opposes its prohypertensive action due to the activation of the local RAS in the distal nephron. The net effect of the EP4 activation may rely on the balance of prohypertensive and antihypertensive actions.

Perspectives

PRR is a newly discovered component of the RAS and has received a great deal of attention due to its implication in the pathogenesis of hypertension and chronic kidney disease. Despite diverse signaling properties, PRR appears to function as an important regulator of prorenin/renin activity, thereby modulating tissue activity the RAS. The present study for the first time examined the contribution of PGE2 EP subtypes to AngII-induced PRR expression. We provide in vitro and in vivo evidence supporting the EP4 receptor as a major regulator of PRR expression in the renal medulla during AngII hypertension. These results provide new insight into the interaction between renal medullary PRR and PGE2 in AngII signaling. More importantly, the results suggest that EP antagonism may represent a novel intervention in management of hypertension and kidney disease by targeting PRR.

Novelty and Significance.

What Is New?

  • -

    First demonstration of the EP4 receptor as a major mediator of AngII-induced PRR expression and renin activity in the renal medulla.

What Is Relevant?

  • -

    Defining the EP4 receptor as a regulator of PRR, a key component of the local RAS in the kidney will help understand the mechanism of human hypertension and also provide a new target for development of antihypertensive therapy.

Summary

The present study for the first time provides evidence for the link between the EP4 receptor and PRR in the renal medulla, offering new insight into the role of the local renin response during AngII hypertension.

Acknowledgments

We thank Aihua Lu (Sun Yat-sen University) and Hong Wang (Sun Yat-sen University) for their technical and administrative assistance.

Sources of Funding: This work was supported by National Natural Science Foundation of China Grant No. 31330037, VA Merit Review, National Institutes of Health Grant DK094956, National Basic Research Program of China 973 Program 2012CB517600 (No.2012CB517602). T. Yang is an Established Investigator from American Heart Association and Research Career Scientist in Department of Veterans Affairs.

Footnotes

Conflict of Interest: None.

References

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