Prostaglandin E-Prostanoid4 Receptor Mediates Angiotensin II −Induced (Pro)Renin Receptor Expression in the Rat Renal Medulla Fei Wang, Xiaohan Lu, Kexin Peng, Yaomin Du, Shu-Feng Zhou, Aihua Zhang and Tianxin Yang Hypertension. published online May 27, 2014; Hypertension is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231 Copyright © 2014 American Heart Association, Inc. All rights reserved. Print ISSN: 0194-911X. Online ISSN: 1524-4563
The online version of this article, along with updated information and services, is located on the World Wide Web at: http://hyper.ahajournals.org/content/early/2014/05/27/HYPERTENSIONAHA.114.03654
Permissions: Requests for permissions to reproduce figures, tables, or portions of articles originally published in Hypertension can be obtained via RightsLink, a service of the Copyright Clearance Center, not the Editorial Office. Once the online version of the published article for which permission is being requested is located, click Request Permissions in the middle column of the Web page under Services. Further information about this process is available in the Permissions and Rights Question and Answer document. Reprints: Information about reprints can be found online at: http://www.lww.com/reprints Subscriptions: Information about subscribing to Hypertension is online at: http://hyper.ahajournals.org//subscriptions/
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Original Article Prostaglandin E-Prostanoid4 Receptor Mediates Angiotensin II–Induced (Pro)Renin Receptor Expression in the Rat Renal Medulla Fei Wang, Xiaohan Lu, Kexin Peng, Yaomin Du, Shu-Feng Zhou, Aihua Zhang, Tianxin Yang Abstract—Angiotensin II (Ang II) stimulates (pro)renin receptor (PRR) expression in the renal collecting duct, triggering the local renin response in the distal nephron. Our recent study provided evidence for involvement of cyclooxygenase2–prostaglandin E2 pathway in Ang II-dependent stimulation of PRR expression in the collecting duct. Here, we tested the role of E-prostanoid (EP) subtypes acting downstream of cyclooxygenase-2 in this phenomenon. In primary rat inner medullary collecting duct cells, Ang II treatment for 12 hours induced a 1.8-fold increase in the full-length PRR protein expression. To assess the contribution of EP receptor, the cell was pretreated with specific EP receptor antagonists: SC-51382 (for EP1), L-798106 (for EP3), 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 Ang II. Sprague-Dawley rats were subsequently infused for 1 or 2 weeks with vehicle, Ang II alone, or in combination with ONO. Ang II 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 Ang II 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 Ang II hypertension. (Hypertension. 2014;64:00-00.) Key Words: dinoprostone
I
n recent years, there has been rising interest about the local renin–angiotensin system (RAS) in a variety of tissues including the kidney.1,2 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 (Ang II), the intrarenal RAS is activated as reflected by increased renin mRNA and protein expression in the CD,6 whereas the systemic RAS is suppressed, highlighting the difference in the 2 RAS system. Several lines of evidence demonstrate a critical role of intrarenal RAS in Ang II–induced hypertension. Experiments in rats infused with Val5-Ang II, an isoform of Ang II that can be separated from endogenous Ang II (Ile5-Ang II) by high-performance liquid chromatography, demonstrated that the chronic Val5-Ang II (exogenous Ang II) infusion induces renal Ile5-Ang II (endogenous Ang II) synthesis.7 In another study, when endogenous Ang II production was reduced by angiotensin-converting enzyme inhibition, Ang II–infused mice became normotensive.8,9 The genetic absence of kidney angiotensin-converting
■
XXX
enzyme substantially blunts the hypertension induced by Ang II infusion.10 In experiments involving kidney cross-transplantation between global angiotensin II receptor-1 knockout mice and wild-type controls, Ang II is shown to cause hypertension through stimulation of angiotensin II receptor-1 receptors in the kidney.11 Last, overexpression of renin in the CD causes spontaneous hypertension.12 However, evidence also exists to suggest that some components of the RAS may be of extrarenal origin. For example, renal angiotensinogen and Ang II are shown to originate from liver.13 (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 activity.14 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 Ang II in rats increased renal PRR transcript levels and augmented the PRR activity in renal medullary tissues, which may contribute to increased renin
Received April 1, 2014; first decision April 10, 2014; revision accepted April 16, 2014. From the Institute of Hypertension, Sun Yat-sen University School of Medicine, Guangzhou, China (F.W., X.L., K.P., T.Y.); Department of Internal Medicine, University of Utah, Salt Lake City (F.W., X.L., T.Y.); Veterans Affairs Medical Center, Salt Lake City, UT (F.W., X.L., T.Y.); Guangdong Provincial People’s Hospital and Guangdong Academy of Medical Sciences, Guangzhou, China (Y.D.); Department of Pharmaceutical Sciences, College of Pharmacy, University of South Florida, Tampa (S.-F.Z.); and Department of Nephrology, Nanjing Children’s Hospital, Affiliated to Nanjing Medical University, Nanjing, China (A.Z.). Correspondence to Tianxin Yang, Division of Nephrology and Hypertension, University of Utah and Veterans Affairs Medical Center, 30N 1900E, RM 4C224, Salt Lake City, UT 84132. E-mail [email protected] © 2014 American Heart Association, Inc. Hypertension is available at http://hyper.ahajournals.org
DOI: 10.1161/HYPERTENSIONAHA.114.03654
Downloaded from http://hyper.ahajournals.org/ 1 at University of Arizona on June 13, 2014
2 Hypertension August 2014 activity in the CD during Ang II hypertension.16 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 Ang II hypertension.16 The biological action of prostaglandin E (PGE)2 is mediated by G protein–coupled E-prostanoid (EP) receptors designated EP1, EP2, EP3, and EP4.17 These 4 subtypes of EP receptor couple to distinct signaling pathways. Among the 4 EP subtypes, the EP4 receptor plays a dominant role in regulation of renin release from the juxtaglomerular apparatus.18,19 We hypothesize that the EP4 receptor may participate in Ang II–induced renin response in the renal medulla through an effect on PRR. To test this hypothesis, we used pharmacological inhibitors and activators of the EP4 receptor to study their effect on renal medullary PRR expression and renin activity and hypertension development after Ang II 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-hour 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 sham operation, Ang II infusion (Human Ang II, Sigma, St. Louis, MO) via a subcutaneous osmotic minipump (Alzet model 2002, Alza, Palo Alto, CA) at a rate of 100 ng/min, or coadministered with ONO-AE3-208 (ONO; MedChemexpress LLC, Princeton, NJ)20 at 0.2 mg/kg per day 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-hour 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 Ang II alone or in combination with ONO for 7 days at the same doses.
Primary Cultures of Rat Inner Medullary Collecting Duct Cells Primary cultures enriched in inner medullary collecting duct (IMCD) cells were prepared from pathogen-free male Sprague-Dawley rats (40–100 g body wt) as previously described.21 After 24 hours of serum deprivation, the IMCD cells were pretreated for 1 hour with
Vehicle
AngII
AngII + L-161982
PRR
43 kDa
β-Actin
43 kDa
PRR densitometry/β-actin
B
A
3.0 p
The online version of this article, along with updated information and services, is located on the World Wide Web at: http://hyper.ahajournals.org/content/early/2014/05/27/HYPERTENSIONAHA.114.03654
Permissions: Requests for permissions to reproduce figures, tables, or portions of articles originally published in Hypertension can be obtained via RightsLink, a service of the Copyright Clearance Center, not the Editorial Office. Once the online version of the published article for which permission is being requested is located, click Request Permissions in the middle column of the Web page under Services. Further information about this process is available in the Permissions and Rights Question and Answer document. Reprints: Information about reprints can be found online at: http://www.lww.com/reprints Subscriptions: Information about subscribing to Hypertension is online at: http://hyper.ahajournals.org//subscriptions/
Downloaded from http://hyper.ahajournals.org/ at University of Arizona on June 13, 2014
Original Article Prostaglandin E-Prostanoid4 Receptor Mediates Angiotensin II–Induced (Pro)Renin Receptor Expression in the Rat Renal Medulla Fei Wang, Xiaohan Lu, Kexin Peng, Yaomin Du, Shu-Feng Zhou, Aihua Zhang, Tianxin Yang Abstract—Angiotensin II (Ang II) stimulates (pro)renin receptor (PRR) expression in the renal collecting duct, triggering the local renin response in the distal nephron. Our recent study provided evidence for involvement of cyclooxygenase2–prostaglandin E2 pathway in Ang II-dependent stimulation of PRR expression in the collecting duct. Here, we tested the role of E-prostanoid (EP) subtypes acting downstream of cyclooxygenase-2 in this phenomenon. In primary rat inner medullary collecting duct cells, Ang II treatment for 12 hours induced a 1.8-fold increase in the full-length PRR protein expression. To assess the contribution of EP receptor, the cell was pretreated with specific EP receptor antagonists: SC-51382 (for EP1), L-798106 (for EP3), 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 Ang II. Sprague-Dawley rats were subsequently infused for 1 or 2 weeks with vehicle, Ang II alone, or in combination with ONO. Ang II 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 Ang II 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 Ang II hypertension. (Hypertension. 2014;64:00-00.) Key Words: dinoprostone
I
n recent years, there has been rising interest about the local renin–angiotensin system (RAS) in a variety of tissues including the kidney.1,2 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 (Ang II), the intrarenal RAS is activated as reflected by increased renin mRNA and protein expression in the CD,6 whereas the systemic RAS is suppressed, highlighting the difference in the 2 RAS system. Several lines of evidence demonstrate a critical role of intrarenal RAS in Ang II–induced hypertension. Experiments in rats infused with Val5-Ang II, an isoform of Ang II that can be separated from endogenous Ang II (Ile5-Ang II) by high-performance liquid chromatography, demonstrated that the chronic Val5-Ang II (exogenous Ang II) infusion induces renal Ile5-Ang II (endogenous Ang II) synthesis.7 In another study, when endogenous Ang II production was reduced by angiotensin-converting enzyme inhibition, Ang II–infused mice became normotensive.8,9 The genetic absence of kidney angiotensin-converting
■
XXX
enzyme substantially blunts the hypertension induced by Ang II infusion.10 In experiments involving kidney cross-transplantation between global angiotensin II receptor-1 knockout mice and wild-type controls, Ang II is shown to cause hypertension through stimulation of angiotensin II receptor-1 receptors in the kidney.11 Last, overexpression of renin in the CD causes spontaneous hypertension.12 However, evidence also exists to suggest that some components of the RAS may be of extrarenal origin. For example, renal angiotensinogen and Ang II are shown to originate from liver.13 (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 activity.14 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 Ang II in rats increased renal PRR transcript levels and augmented the PRR activity in renal medullary tissues, which may contribute to increased renin
Received April 1, 2014; first decision April 10, 2014; revision accepted April 16, 2014. From the Institute of Hypertension, Sun Yat-sen University School of Medicine, Guangzhou, China (F.W., X.L., K.P., T.Y.); Department of Internal Medicine, University of Utah, Salt Lake City (F.W., X.L., T.Y.); Veterans Affairs Medical Center, Salt Lake City, UT (F.W., X.L., T.Y.); Guangdong Provincial People’s Hospital and Guangdong Academy of Medical Sciences, Guangzhou, China (Y.D.); Department of Pharmaceutical Sciences, College of Pharmacy, University of South Florida, Tampa (S.-F.Z.); and Department of Nephrology, Nanjing Children’s Hospital, Affiliated to Nanjing Medical University, Nanjing, China (A.Z.). Correspondence to Tianxin Yang, Division of Nephrology and Hypertension, University of Utah and Veterans Affairs Medical Center, 30N 1900E, RM 4C224, Salt Lake City, UT 84132. E-mail [email protected] © 2014 American Heart Association, Inc. Hypertension is available at http://hyper.ahajournals.org
DOI: 10.1161/HYPERTENSIONAHA.114.03654
Downloaded from http://hyper.ahajournals.org/ 1 at University of Arizona on June 13, 2014
2 Hypertension August 2014 activity in the CD during Ang II hypertension.16 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 Ang II hypertension.16 The biological action of prostaglandin E (PGE)2 is mediated by G protein–coupled E-prostanoid (EP) receptors designated EP1, EP2, EP3, and EP4.17 These 4 subtypes of EP receptor couple to distinct signaling pathways. Among the 4 EP subtypes, the EP4 receptor plays a dominant role in regulation of renin release from the juxtaglomerular apparatus.18,19 We hypothesize that the EP4 receptor may participate in Ang II–induced renin response in the renal medulla through an effect on PRR. To test this hypothesis, we used pharmacological inhibitors and activators of the EP4 receptor to study their effect on renal medullary PRR expression and renin activity and hypertension development after Ang II 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-hour 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 sham operation, Ang II infusion (Human Ang II, Sigma, St. Louis, MO) via a subcutaneous osmotic minipump (Alzet model 2002, Alza, Palo Alto, CA) at a rate of 100 ng/min, or coadministered with ONO-AE3-208 (ONO; MedChemexpress LLC, Princeton, NJ)20 at 0.2 mg/kg per day 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-hour 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 Ang II alone or in combination with ONO for 7 days at the same doses.
Primary Cultures of Rat Inner Medullary Collecting Duct Cells Primary cultures enriched in inner medullary collecting duct (IMCD) cells were prepared from pathogen-free male Sprague-Dawley rats (40–100 g body wt) as previously described.21 After 24 hours of serum deprivation, the IMCD cells were pretreated for 1 hour with
Vehicle
AngII
AngII + L-161982
PRR
43 kDa
β-Actin
43 kDa
PRR densitometry/β-actin
B
A
3.0 p
