ALDOSTERONE AND ENAC IN HEALTH AND DISEASE: THE KIDNEY AND BEYOND PROGRAM BOOK
ESTES PARK, COLO. OCTOBER 2–6, 2019
#PhysiologyConf | the-aps.org
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MembershipBenefits_Conferences_MayJune_8.5x11.indd 1 5/30/2019 2:48:17 PM 2019 APS Aldosterone and ENaC in Health and Disease: The Kidney and Beyond October 2 – 6, 2019 The Stanley Hotel Estes Park, Colorado
Conference Organizing Committee: Iris Jaffe Daniela Rotin Peter Snyder Co-chair Co-chair Co-chair
Thomas Kleyman David Pearce Olivier Staub Alicia McDonough
Acknowledgements: The conference organizers and the American Physiological Society and gratefully recognize the generous financial support from the following:
Sponsors:
American Heart Association, National Institute of Diabetes Council on the Kidney in and Digestive and Kidney Cardiovascular Disease* Diseases (NIDDK)**
* This program is supported in part by the American Heart Association’s Council on the Kidney in Cardiovascular Disease.
** Research reported in this publication was supported by the National Institute Of Diabetes And Digestive And Kidney Diseases of the National Institutes of Health under Award Number R13DK122821. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
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2019 APS ALDOSTERONE AND ENAC IN HEALTH AND DISEASE: THE KIDNEY AND BEYOND OCTOBER 2–6, 2019 THE STANLEY HOTEL ESTES PARK, CO TABLE OF CONTENTS
Organizing Committee and Acknowledgments ...... Page 1 General Information...... Page 6 Week-at-a-Glance ...... Page 8 Venue Map ...... Page 10 Daily Schedule ...... Page 11 Abstracts of Invited and Volunteered Presentations ...... Page 23 1.0 Keynote Lecture: Human kidney organoids ...... Page 25 2.0 Symposium 1A: Aldosterone and mineralocorticoid receptors in the kidney and hypertension ...... Page 26 3.0 Symposium 1B: Metabolic and sex differences in aldosterone responses .. Page 30 4.0 Symposium 2A: Structure function of ENaC and related transporters ...... Page 33 5.0 Symposium 2B: ENaC function and regulation in tissues ...... Page 36 6.0 Poster Session 1 ...... Page 38 7.0 Symposium 3A: signaling and regulation of ENaC and other epithelial channels/transporters I ...... Page 48 8.0 Symposium 3B: signaling and regulation of ENaC and other epithelial channels/transporters II ...... Page 51 9.0 Symposium 4A: ENaC biogenesis and trafficking...... Page 54 11.0 Poster Session 2 ...... Page 56 12.0 Symposium 5A: MR in the vasculature ...... Page 67 13.0 Symposium 5B: MR structure and role in the heart and lungs ...... Page 70 14.0 Clinical Plenary Lecture: The role of non-steroidal MR antagonists and new potassium binders for the treatment of cardiovascular disease ...... Page 73 15.0 Symposium 6A: Normal and pathogenic regulation of aldosterone biosynthesis ...... Page 74 16.0 Symposium 6B: Integrated regulation of renal ion transport ...... Page 76 Author Index ...... Page 81
5 General Information
LOCATION: INCLUDED IN YOUR REGISTRATION: The 2019 APS Aldosterone and ENaC in Your registration to this meeting includes Health and Disease: The Kidney and entry into all oral and poster scientific Beyond is held at The Stanley Hotel, 333 E sessions; all meals, welcome reception, Wonderview Ave, Estes Park, CO 80517. morning and afternoon breaks, awards Phone: (970) 577-4000 banquet during the conference; and a program book which serves at the conference proceedings. MEETING REGISTRATION HOURS: Wed., October 2...... 3:00 p.m.–8:30 p.m. Thurs., October 3...... 7:00 a.m.–9:00 p.m. Registration is nontransferable. You must Fri., October 4...... 7:00 a.m.–9:00 p.m. pay the entire fee regardless of the number Sat., October 5...... 7:00 a.m.–6:00 p.m. of sessions/events you attend. Guests of Sun., October 6...... 7:00 a.m.–10:30 a.m. attendees are not permitted in the oral scientific sessions, poster sessions, opening reception, meals, conference breaks or STUDENT REGISTRATION: social events. Any student member or regularly matriculated student working toward a degree in one of the biomedical sciences is PRESS REGISTRATION: eligible to register at the student fee. Press badges will be issued at the Nonmember postdoctoral fellows, hospital Conference Registration Desk to working residents and interns, and laboratory members of the press and freelance writers technicians do not qualify as students. Non- bearing a letter of assignment from an member students who register on-site must editor. Representatives of allied fields provide a valid university student ID card. (public relations, public affairs, etc.) must APS student members should present their register as nonmembers. current APS membership card indicating their student category status. PHOTOGRAPH/VIDEO RECORDING: Photo or video capture of any scientific POSTDOCTORAL REGISTRATION: presentation, whether an oral or poster Any person who has received a PhD degree presentation in whole or in part, is expressly in physiology or related field within five prohibited. Recording or taking photography years of the conference start date, as of another person without their explicit attested to by their department head, is permission is prohibited. eligible to register at the postdoctoral fee. A statement signed by the department head Individuals observed photographing or must accompany the registration form and videotaping any presentation, in whole or in remittance when registering. part, will be asked to leave the conference immediately, forfeiting the registration fee.
6 SOCIAL MEDIA POLICY: Additional information on APS’ Conference APS encourages the use of social media Policies can be found on our website: during our conferences and conferences as https://www.the-aps.org/conferences- a way of connecting with other attendees awards/conferences/conference- and expanding the reach of science being events/control-of-renal-function/Code-of- presented. If you plan on using social media Conduct?SSO=Y to discuss the conference, please use the official APS conference hashtag, #PhysiologyConf. PROGRAM OBJECTIVE: The goal of this conference is to bring Sharing of specific research presentations, together clinical and basic researchers from poster or slides is prohibited without the around the world with an interest in ENaC express permission of the presenter. (and related transporters) and aldosterone (and the MR), to move forward the understanding of the basic biology of these CODE OF CONDUCT: molecules, their role in renal function and APS is committed to providing a safe, hypertension, and the emerging role in productive and welcoming environment for other tissues and diseases. This conference all conference participants and staff. All will bring together aldosterone and ENaC participants including, but not limited to, experts in basic, clinical, and translational attendees, speakers, volunteers, APS staff, research to: hotel staff, service providers and others are Present and discuss the latest research expected to abide by the APS Conference in the field and outline the future Code of Conduct which maintains that all direction of that work. individuals should: Facilitate participation and career Be treated with respect and development for trainees, early career consideration, valuing a diversity of investigators, and underrepresented views and opinions; groups. This will allow for the Be considerate, respectful and enhanced opportunities to present collaborative; their new work, and also benefit from Communicate openly and with respect, exposure to the research and thinking critiquing ideas rather than individuals; of leaders in the field. avoid personal attacks; Create an environment in which clinical Be mindful of your surroundings and and basic scientists in the field can fellow participants; and, exchange ideas with diverse Be respectful of the rules and the perspectives; novel ideas and conference venue. approaches to these fields which can initiate collaboration in the Contact the APS staff at the Conference investigation of aldosterone/MR, ENaC, Registration Desk if you notice a dangerous and related areas. situation or someone in distress or in violation of this Code of Conduct.
7 2019 APS ALDOSTERONE AND ENAC IN HEALTH AND DISEASE: THE KIDNEY AND BEYOND OCTOBER 2 – 6, 2019 THE STANLEY HOTEL ESTES PARK, COLORADO
Wednesday Thursday Friday Saturday Sunday October 2 October 3 October 4 October 5 October 6 6:30 a.m. - Registration Registration Registration 9:30 p.m. 6:30 a.m. - Registration 10:00 a.m. 7:00 a.m. - Breakfast Breakfast Breakfast Breakfast 8:00 a.m. Symposium 1A: Symposium 3A: Symposium 5A: Symposium 6A: Aldosterone and Signaling and MR in the Normal and mineralocorticoid regulation of vasculature pathogenic 8:00 a.m. - receptors in the ENaC and other regulation of 10:00 a.m. kidney and epithelial aldosterone hypertension channels/trans- biosynthesis porters I 10:00 a.m. - Break 10:15 a.m. 10:00 a.m. - Break Break Break 10:30 a.m. Symposium 6B: Integrated 10:15 a.m. - regulation of 12:00 p.m. renal ion transport Symposium 1B: Symposium 3B: Symposium 5B: Metabolic and Signaling and MR structure sex differences regulation of and role in the 10:30 a.m. - in aldosterone ENaC and other heart and lungs 12:00 p.m. responses epithelial channels/trans- porters II
12:00 p.m. - Box lunches & Lunch Lunch Lunch 1:00 p.m. departure Symposium 2A: Symposium 4A: Structure ENaC biogenesis 1:00 p.m. - function of ENaC and trafficking 3:00 p.m. and related transporters
8
Wednesday Thursday Friday Saturday Sunday October 2 October 3 October 4 October 5 October 6 Clinical Plenary Lecture: The role of non-steroidal MR antagonists 1:30 p.m. - and new 2:30 p.m. potassium binders for the treatment of cardiovascular disease 3:00 p.m. - Break 3:30 p.m. APS Career 3:00 p.m. – Development 5:00 p.m. Workshop/Hike 3:00 p.m. - Registration 10:00 p.m. Symposium 2B: ENaC function 3:30 p.m. - and regulation in 5:00 p.m. tissues
6:00 p.m. - Dinner Dinner Dinner 7:00 p.m. APS Banquet 6:00 p.m. - and Awards 9:00 PM Presentation 7:00 p.m. - Poster Session 1 Poster Session 2 9:30 p.m. Keynote 7:30 p.m. - Lecture:
8:30 p.m. Kidney organoids
8:30 p.m. - Welcome 10:00 p.m. Reception
9
DAILY SCHEDULE WEDNESDAY, OCTOBER 2, 2019 6:00 p.m. – Dinner 7:00 p.m. Pavilion, 1st Floor
7:30 p.m. – 1.0 Keynote Lecture 8:30 p.m. Pavilion Theater, 1st Floor Chair: Daniela Rotin, Hospital for Sick Children and Univ. of Toronto
1.1 Human kidney organoids Joseph Bonventre, Brigham and Women’s Hospital and Harvard Medical School
8:30 p.m. – Welcome Reception 10:00 p.m. Pavilion, 1st Floor
THURSDAY, OCTOBER 3, 2019 7:00 a.m. – Breakfast 8:00 a.m. Pavilion, 1st floor
8:00 a.m. – 2.0 Symposium 1A: Aldosterone and mineralocorticoid receptors in the kidney and 10:00 a.m. hypertension Pavilion Theater, 1st Floor Chairs: David Calhoun, Univ. of Alabama Birmingham Celso Gomez Sanchez, Univ. of Mississippi
8:00 a.m. – 2.1 Aldosterone-independent activation of mineralocorticoid receptor in salt- 8:30 a.m. sensitive hypertension and glomerular diseases Toshiro Fujita, The Univ. of Tokyo
8:30 a.m. – 2.2 The expanding spectrum of primary aldosteronism 9:00 a.m. Anand Vaidya, Brigham and Women's Hospital and Harvard Medical School
9:00 a.m. – 2.3 The intercalated cell mineralocorticoid receptor regulates pendrin diretly and 9:15 a.m. regulates principal cell ENaC indirectly over a wide range in serum K+ Susan Wall, Emory Univ.
9:15 a.m. – 2.4 Kir5.1-mediated changes in renin-angiotensin-aldosterone system balance in salt 9:30 a.m. sensitive hypertension Anna Manis, Medical College of Wisconsin
9:30 a.m. – 2.5 A proof-of-principle study of sodium loading in prehypertension 9:45 a.m. J. Brian Byrd, Univ. of Michigan Medical School
9:45 a.m. – Coffee Break 10:30 a.m. Pavilion, 1st Floor
11 10:30 a.m. – 3.0 Symposium 1B: Metabolic and sex differences in aldosterone responses 12:00 p.m. Pavilion Theater, 1st Floor Chairs: Massi Caprio, IRCCS James Luther, Vanderbilt Univ.
10:30 a.m. – 3.1 Biological sex-specific differences in the aldosterone responses to angiotensin II 11:00 a.m. in humans and rodents Jose Romero, Brigham and Women’s Hospital and Harvard Medical School
11:00 a.m. – 3.2 The leptin-aldosterone-mineralocorticoid receptor axis: a major contributor to 11:30 a.m. cardiovascular disease in obese females Eric J. Belin de Chantemele, Medical College of Georgia at Augusta Univ.
11:30 a.m. – 3.3 Hypersensitivity of renal ENaC to aldosterone is a sex-specific determinant of 11:45 a.m. blood pressure control in females Mykola Mamenko, Augusta Univ.
11:45 a.m. – 3.4 Female mice exhibit higher increases in aldosterone synthase expression and 12:00 p.m. aldosterone production than males in response to low salt diet Jessica Faulkner, Medical College of Georgia at Augusta Univ.
12:00 p.m. – Lunch 1:00 p.m. Pavilion, 1st Floor
1:00 p.m. – 4.0 Symposium 2A: Structure function of ENaC and related transporters 3:00 p.m. Pavilion Theater, 1st Floor Chairs: Peter Snyder, Univ. of Iowa Bernard Rossier, Univ. of Lausanne
1:00 p.m. – 4.1 ENaC structure by cryo-electron microscopy 1:30 p.m. Isabelle Baconguis, Oregon Health and Science Univ.
1:30 p.m. – 4.2 ENaC gating regulation by biliary factors 2:00 p.m. Ossama Kashlan, Univ. of Pittsburgh
2:00 p.m. – 4.3 ENaC subunit N-glycans have different roles for the ability of the channel to 2:15 p.m. respond to shear force Martin Fronius, Univ. of Otago
2:15 p.m. – 4.4 N-methyl-D-aspartate (NMDA) receptor interacts with ENaC to induce renal 2:30 p.m. vasodilation in the connecting tubule. Cesar Romero, Emory Univ. School of Medicine
2:30 p.m. – 4.5 Proton block of the epithelial sodium channel 2:45 p.m. Daniel Collier, Univ. of Tennessee Health Science Center
12 DAILY SCHEDULE 2:45 p.m. – Coffee Break 3:30 p.m. Pavilion, 1st Floor
3:30 p.m. – 5.0 Symposium 2B: ENaC function and regulation in tissues 5:10 p.m. Pavilion Theater, 1st Floor Chairs: Larry Palmer, Cornell Univ. Edith Hummler, Univ. of Lausanne
3:30 p.m. – 5.1 Regulation of ENaC by extracellular Na 4:00 p.m. Thomas Kleyman, Univ. of Pittsburgh
4:00 p.m. – 5.2 ENaC regulation by aldosterone and proteases 4:30 p.m. Christoph Korbmacher, Friedrich-Alexander Univ. Erlangen-Nürnberg (FAU)
4:30 p.m. – 5.3 Potassium sensing in the distal nephron 4:50 p.m. David Penton Ribas, Univ. of Zurich
4:50 p.m. – 5.4 The hypertension pandemic: an evolutionary perspective 5:10 p.m. Bernard Rossier, Univ. of Lausanne
6:00 p.m. – Dinner 7:00 p.m. Pavilion, 1st Floor
7:00 p.m. – 6.0 Poster Session 1 9:30 p.m. Pavilion, 2nd Floor
Board # 1 6.1 Ubiquitination of ENaC subunits in vivo Lawrence Palmer, Gustavo Frindt, Lei Yang, Weill-Cornell Medical College; Weill- Cornell Medical College; Weill-Cornell Medical College
3 6.2 Modulation of ENaC mediated sodium transport by the basolateral Kir4.1/Kir5.1 channels Oleg Palygin, Elena Isaeva, Alexey Shalygin, Ruslan Bohovyk, Mykhailo Fedoriuk, Christine Klemens, Ashraf El-Meanawy, Jerod Denton, Alexander Staruschenko, Medical College of Wisconsin; Medical College of Wisconsin; Medical College of Wisconsin; Medical College of Wisconsin; Medical College of Wisconsin; Medical College of Wisconsin; Medical College of Wisconsin; Vanderbilt Univ. Medical Center; Medical College of Wisconsin
5 6.3 Regulation of ENaC by paraoxonase 3 Shujie Shi, Nicolas Montalbetti, Xueqi Wang, Stephanie Mutchler, Allison Marciszyn, Marcelo Carattino, Thomas Kleyman, Univ. of Pittsburgh; Univ. of Pittsburgh; Univ. of Pittsburgh; Univ. of Pittsburgh; Univ. of Pittsburgh; Univ. of Pittsburgh; Univ. of Pittsburgh
13 Board # 7 6.4 Is the membrane-bound serine protease Tmprss3 implicated in ENaC-mediated sodium absorption? Deepika Vijayan, Thibaud Peyrollaz, Simona Frateschi, Edith Hummler, Univ. of Lausanne; Univ. of Lausanne; Univ. of Lausanne; Univ. of Lausanne
9 6.5 Regulation of endothelial sodium channel (EnNaC) by mTOR and SGK-1 signaling. Michael Hill, Yan Yang, Guanghong Jia, Annayya Aroor, James Sowers, Univ. of Missouri; Univ. of Missouri; Univ. of Missouri; Univ. of Missouri; Univ. of Missouri
11 6.6 What is new about ENaC regulation by the serine protease CAP1/Prss8 in kidney? Asma Mechakra, Yannick Jaeger, Chloe Sergi, Thibaud Peyrollaz, Anne-Marie Merillat, Ivan Gautschi, Stephan Kellenberger, Simona Frateschi, Edith Hummler, Univ. of Lausanne; Univ. of Lausanne; Univ. of Lausanne; Univ. of Lausanne; Univ. of Lausanne; Univ. of Lausanne; Univ. of Lausanne; Univ. of Lausanne; Univ. of Lausanne
13 6.7 Renal denervation improves sodium excretion in rats with chronic heart failure: effects on ENaC and angiotensin II-aldosterone actions Hong Zheng, Xuefei Liu, Kenichi Katsurada, Kaushik Patel, Univ. of South Dakota; Univ. of South Dakota; Univ. of Nebraska Medical Center; Univ. of Nebraska Medical Center
15 6.8 Withdrawn
17 6.9 Sprinonolactone treatment reverses arterial ENaC expression and improves arterial responsiveness in hypertensive Cyp1a1-Ren2 rats Martin Fronius, Sama Mugloo, Zoe Ashley, Ivan Sammut, Catherine Leader, Rob Walker, Fiona McDonald, Univ. of Otago; Univ. of Otago; Univ. of Otago; Univ. of Otago; Univ. of Otago; Univ. of Otago; Univ. of Otago
19 6.10 High salt diet and aldosterone: focus on CaSR-RAS on plasma and urinary outcomes in salt-sensitive rats. Charles Okechukwu, Emmanuel Awumey, North Carolina Central Univ.; North Carolina Central Univ.
21 6.11 Aldosterone-independent regulation of ENaC and Na and K excretion Lei Yang, Gustavo Frindt , Lawrence Palmer, Weill Cornell Medical College; Weill Cornell Medical College; Weill Cornell Medical College
23 6.12 Mineralocorticoid receptor antagonism in acute hyperkalemia: is aldosterone a must-have? Frederico Fazan, Eduardo Colombari, Federal Univ. of São Paulo; UNESP/FOAr
14 DAILY SCHEDULE Board # 25 6.13 Probiotics effectively restore the function and structure of damaged kidney in gout Rostyslav Bubnov, Mykola Spivak, Zabolotny Institute of Microbiology and Virology, National Academy of Sciences of Ukraine; Zabolotny Institute of Microbiology and Virology, National Academy of Sciences of Ukraine
27 6.14 Sex-dependent protection from high fat diet-induced metabolic disease in mice lacking normal levels of TrpC6 and/or ASIC2 and βENaC proteins Heather Drummond, Zachary Mitchell, Emily Hildebrandt, David Stec, Univ. of Mississippi Medical Center; Univ. of Mississippi Medical Center; Univ. of Mississippi Medical Center; Univ. of Mississippi Medical Center
29 6.15 Effects of dietary potassium restriction and K+ loading on blood pressure and renal tubular Na+ transport Cary Boyd-Shiwarski, Rebecca Beacham, Claire Weaver, Daniel Shiwarski, Kelly Connolly, Allison Marciszyn, Arohan Subramanya, Univ. of Pittsburgh; Univ. of Pittsburgh; Univ. of Pittsburgh; Carnegie Mellon Univ.; Univ. of Pittsburgh; Univ. of Pittsburgh; Univ. of Pittsburgh
31 6.16 PIP2 regulates ENaC through multiple electrostatic interactions Crystal Archer, Chase Carver, Benjamin Enslow, Yinghua Chen, James Stockand, Univ. of Texas Health San Antonio; Univ. of Texas Health San Antonio; Univ. of Texas Health San Antonio; Case Western Reserve Univ.; Univ. of Texas Health San Antonio
33 6.17 Effects of dietary habits on hypertension among medical students Suyog Mhamankar, Spartan Health Science Univ.
35 6.18 A mechanism to reduce cysts in autosomal dominant polycystic kidney disease by inducing fluid absorption via CFTR, NHE3 and ENaC Liudmila Cebotaru, Muraly Yanda, Johns Hopkins Univ.; Johns Hopkins Univ.
FRIDAY, OCTOBER 4, 2019 7:00 a.m. – Breakfast 8:00 a.m. Pavilion, 1st floor
8:00 a.m. – 7.0 Symposium 3A: Signaling and regulation of ENaC and other epithelial 10:00 a.m. channels/transporters I Pavilion Theater, 1st Floor Chair: Doug Eaton, Emory Univ.
8:00 a.m. – 7.1 ENaC regulation by the mTORC2-SGK1 signaling module 8:30 a.m. David Pearce, Univ Calif San Francisco
8:30 a.m. – 7.2 Role of microRNAs in aldosterone signaling and ENaC regulation 9:00 a.m. Michael Butterworth, Univ. of Pittsburgh
15 9:00 a.m. – 7.3 AMPK regulates ENaC via a tripartite inhibitory complex 9:30 a.m. Kenneth R. Hallows, Univ. of Southern California
9:30 a.m. – 7.4 Elevated sodium activates the NLRP3 inflammasome in antigen presenting cells 9:45 a.m. through an ENaC-dependent mechanism Ashley Pitzer, Vanderbilt Univ. Medical Center
9:45 a.m. – 7.5 Postprandial effects on ENaC-mediated sodium absorption 10:00 a.m. Christine A. Klemens, Medical College of Wisconsin
10:00 a.m. – Coffee Break 10:30 a.m. Pavilion, 1st Floor
10:30 a.m. – 8.0 Symposium 3B: Signaling and regulation of ENaC and other epithelial 12:00 p.m. channels/transporters II Pavilion Theater, 1st Floor Chairs: Alexander Staruschenko, Medical College of Wisconsin Jim Stockand, Univ. of Texas, San Antonio
10:30 a.m. – 8.1 WNK kinases and cell volume regulation 11:00 a.m. Arohan Subramanya, Univ. of Pittsburgh
11:00 a.m. – 8.2 From SPLUNC1 to SPX-101, novel peptidomimetics to treat sodium 11:30 a.m. hyperabsorption in the CF lung Robert Tarran, Univ. of North Carolina
11:30 a.m. – 8.3 Direct effect of potassium on ENaC regulation and potassium secretion in 11:45 p.m. collecting duct cells: role of mTORC2/SGK1 signaling Bidisha Saha, Univ. of California, San Francisco
11:45 a.m. – 8.4 High mobility group box-1 protein regulates lung epithelial sodium channel 12:00 p.m. activity via the receptor for advanced glycation end products Garett J. Grant, Univ. of Utah
12:00 p.m. – Lunch 1:00 p.m. Pavilion, 1st Floor
1:00 p.m. – 9.0 Symposium 4A: ENaC biogenesis and trafficking 3:00 p.m. Pavilion Theater, 1st Floor Chair: Daniela Rotin, Hospital for Sick Children and Univ. of Toronto
1:00 p.m. – 9.1 Novel mechanisms of diuretic resistance revealed by single cell analysis 1:30 p.m. Vivek Bhalla, Stanford Univ. School of Medicine
1:30 p.m. – 9.2 Regulation of renal ion transport by ubiquitylation and phosphorylation networks 2:00 p.m. Olivier Staub, Univ. of Lausanne
16 DAILY SCHEDULE 2:00 p.m. – 9.3 Regulation of ENaC by the ER lumenal, molecular chaperone, GRP170 2:30 p.m. Teresa Buck, Univ. of Pittsburgh
2:30 p.m. – 9.4 A conserved region in the N-terminus of α-ENaC regulates proteolytic processing 2:45 p.m. during anterograde transport Pradeep Kota, Univ. of North Carolina at Chapel Hill
3:00 p.m. – 10.0 Career development workshop/hike 5:00 p.m. The purpose of the workshop/hike is to provide career advice from senior investigators to junior faculty, postdoctoral fellows and students on areas including grant writing, academic promotion, manuscript writing, mentorship and other faculty activities.
6:00 p.m. – Dinner 7:00 p.m. Pavilion, 1st Floor
7:00 p.m. – 11.0 Poster Session 2 9:30 p.m. Pavilion, 2nd Floor
Board # 2 11.1 Myeloid mineralocorticoid receptor contributes to lung inflammation and vascular remodeling in experimental pulmonary hypertension Guanming Qi, Rod R. Warburton, Krishna C. Penumatsa, Mary E. Moss, Joshua Man, Qing Lu, Seung Kyum Kim, Nicholas S. Hill, Iris Z. Jaffe, Ioana R. Preston, Tufts Medical Center; Tufts Medical Center; Tufts Medical Center; Tufts Medical Center; Tufts Medical Center; Tufts Medical Center; Tufts Medical Center; Tufts Medical Center; Tufts Medical Center; Tufts Medical Center
4 11.2 Endothelial mineralocorticoid receptor deletion protects female mice from obesity-induced endothelial dysfunction and modulates vascular TRPV4 expression and function Lauren Biwer, Brigett Carvajal, Qing Lu, Iris Jaffe, Tufts Medical Center; Tufts Medical Center; Tufts Medical Center; Tufts Medical Center
6 11.3 Long extracellular beta strand in ENaC gating Shaohu Sheng, Jingxin Chen, Xueqi Wang, Thomas Kleyman, Univ. of Pittsburgh; Univ. of Pittsburgh; Univ. of Pittsburgh; Univ. of Pittsburgh
8 11.4 ENaC-α agonism can induce endothelial dysfunction in control female mice only independently of endothelial mineralocorticoid receptors Jessica Faulkner, Simone Kennard, Iris Jaffe, Rudolf Lucas, Eric Belin de Chantemèle, Medical College of Georgia at Augusta Univ.; Medical College of Georgia at Augusta Univ.; Tufts Medical Center; Medical College of Georgia at Augusta Univ.; Medical College of Georgia at Augusta Univ.
10 11.5 Effect of amiloride on epithelial sodium channel among adult Nigerian subjects Simiat Elias, Olusoga Sofola, Smith Jaja, Lagos State Univ. College of Medicine; College of Medicine, Univ. of Lagos; College of Medicine, Univ. of Lagos
17 Board # 12 11.6 Epithelial sodium channel modulation by acid and anions in salt taste Alan Ryan, Daniel Collier, Peter Snyder, Univ. of Iowa; Univ. of Tennessee Health Science Center; Univ. of Iowa
14 11.7 The stimulation of the basolateral Kir4.1/Kir5.1 in distal convoluted tubule is involved in augmenting NCC activity of Nedd4-2-deficient mice Peng Wu, Xiaotong Su, Zhong-Xiuzi Gao, Olivier Staub, Wenhui Wang, Daohong Lin, New York Medical College; Oregon Health & Science Univ.; New York Medical College; Univ. of Lausanne; New York Medical College; New York Medical College
16 11.8 MLP regulates ENaC activity in renal distal convoluted tubule cells Chang Song, Janet Le, Qiang Yue, Monisha Mistry, Otor Al-Khalili, Brandi Wynne, Douglas Eaton, Emory Univ. School of Medicine; Univ. of Texas; Emory Univ. School of Medicine; Emory Univ. School of Medicine; Emory Univ. School of Medicine; Emory Univ. School of Medicine; Emory Univ. School of Medicine
18 11.9 The differential effects of aldosterone and high salt on kidney injury Stephanie Mutchler, Mahpara Hasan, Roderick Tan, Thomas Kleyman, Univ. of Pittsburgh; Carnegie Mellon Univ.; Univ. of Pittsburgh; Univ. of Pittsburgh
20 11.10 Characterization of mice with a hypomorphic ENaC γ subunit Evan C. Ray, Jenna Barbour, Austin Lieber, Aaliyah Winfrey, Stephanie Mutchler, Allison Marciszyn, Yaakov Barak, Harry Blair, Thomas Kleyman, Univ. of Pittsburgh; Univ. of Pittsburgh; Univ. of Pittsburgh; Univ. of Pittsburgh; Univ. of Pittsburgh; Univ. of Pittsburgh; Magee-Women’s Research Institute; Univ. of Pittsburgh; Univ. of Pittsburgh
22 11.11 Epithelial sodium channel (ENaC) in endothelium modulates vascular reactivity with a high salt diet Stephanie Mutchler, Thomas Kleyman, Univ. of Pittsburgh; Univ. of Pittsburgh
24 11.12 Epithelial Na+ channel activation by cleavage coevolved with the terrestrial migration of vertebrates Xueping Wang, Deidra M. Balchak, Clayton Gentilcore, Nathan L. Clark, Ossama B. Kashlan, Univ. of Pittsburgh; Children's Hospital of Pittsburgh; Univ. of Pittsburgh; Univ. of Pittsburgh; Univ. of Pittsburgh
26 11.13 Wnt4(+) cell mapping and changes in ENaC regulatory genes in furosemide- treated mice George Kunkel, Steven Schaffert, Aram Krauson, Yanli Qiao, Purvesh Khatri, Vivek Bhalla, Stanford Univ. School of Medicine; Stanford Univ. School of Medicine; Stanford Univ. School of Medicine; Stanford Univ. School of Medicine; Stanford Univ. School of Medicine; Stanford Univ. School of Medicine
18 DAILY SCHEDULE Board # 28 11.14 Angiotensin and aldosterone receptor antagonism attenuates angiotensin ii- induced hypertension in Sprague Dawley rats Stacy Tletlepantzi, Univ. of California, Merced
30 11.15 Serum and glucocorticoid-regulated kinase 1 (SGK1) up-regulates ENaC in the uterus for pro-inflammatory shift in term and preterm labor Ye Chun Ruan, The Hong Kong Polytechnic Univ.
32 11.16 Primary aldosteronism decreases insulin secretion and increases insulin clearance in humans James Luther, Gillian Murray, Adina Turcu, Nian Hui, Chang Yu, Dungeng Peng, Gail Adler, Vanderbilt Univ. Medical Center; Brigham and Women’s Hospital; Univ. of Michigan; Vanderbilt Univ.; Vanderbilt Univ.; Vanderbilt Univ. Medical Center; Brigham and Women’s Hospital
34 11.17 Differential effects of acute vs. chronic dietary potassium intake on plasma potassium concentration and NCC phosphorylation and expression Jonathan Nizar, Tim Davie, Vivek Bhalla, Univ. of Iowa; Univ. of Iowa; Stanford Univ.
36 11.18 Age and sex-specific reference ranges are needed for the aldosterone/renin ratio Jun Yang, Pravik Solanki, Stella May Gwini, Peter J Fuller, Jimmy Shen, James CG Doery, Kay Weng Choy, Dept. of Endocrinology, Monash Health, Clayton; Dept. of Medicine, Monash University, Clayton; University Hospital Geelong, Barwon Health, Geelong; Hudson Institute of Medical Research, Clayton, Victoria, Australia
SATURDAY, OCTOBER 5, 2019 7:00 a.m. – Breakfast 8:00 a.m. Pavilion, 1st floor
8:00 a.m. – 12.0 Symposium 5A: MR in the vasculature 10:00 a.m. Pavilion Theater, 1st Floor Chairs: Frederic Jaisser, INSERM Iris Jaffe, Tufts Medical Center
8:00 a.m. – 12.1 Sex-specific mechanisms of resistance vessel endothelial dysfunction in obesity 8:30 a.m. Ana Davel, Univ. of Campinas
8:30 a.m. – 12.2 Sex differences in the role of the smooth muscle cell mineralocorticoid receptor 9:00 a.m. in cardiovascular aging Jennifer DuPont, Tufts Medical Center
9:00 a.m. - 12.3 Role of the myeloid mineralocorticoid receptor in vascular inflammation in 9:15 a.m. atherosclerosis Joshua Man, Tufts Medical Center
19 9:15 a.m. – 12.4 Aldosterone and angiotensin II increase aortic stiffness and endothelial 9:30 a.m. dysfunction via an action of oxidative stress on the endothelial sodium channel James Sowers, Univ. of Missouri
9:30 a.m. – 12.5 The novel non-steroidal MR antagonist finerenone improves metabolic 9:45 a.m. parameters via ATGL-mediated lipolysis of brown adipose tissue in high-fat diet fed mice Vincenzo Marzolla, IRCCS San Raffaele Pisana
9:45 a.m. – Coffee Break 10:30 a.m. Pavilion, 1st Floor
10:30 a.m. – 13.0 Symposium 5B: MR structure and role in the heart and lungs 12:00 p.m. Pavilion Theater, 1st Floor Chairs: Gail Adler, Brigham and Women’s Hospital Shawn Bender, Univ. of Missouri
10:30 a.m. – 13.1 Enhanced endothelium epithelial sodium channel signaling prompts left 11:00 a.m. ventricular diastolic dysfunction in obese female mice Guanghong Jia, Univ. of Missouri School of Medicine
11:00 a.m. – 13.2 Structural determinants of activation of the mineralocorticoid receptor: an 11:30 a.m. evolutionary perspective Peter Fuller, Hudson Institute of Medical Research
11:30 a.m. – 13.3 The quaternary structure of the mineralocorticoid receptor depends on ligand 11:45 a.m. and DNA binding Diego Alvarez de la Rosa, Universidad de La Laguna
11:45 a.m. – 13.4 Vascular cell-specific roles of mineralocorticoid receptors in pulmonary 12:00 p.m. hypertension Ioana Preston, Tufts Medical Center
12:00 p.m. – Lunch 1:00 p.m. Pavilion, 1st Floor
1:30 p.m. – 14.0 Clinical Plenary Lecture 2:30 p.m. Pavilion Theater, 1st Floor Chair: Maria-Christina Zennaro, Paris Cardiovascular Research Center
1:30 p.m. – 14.1 The role of non-steroidal MR antagonists and new potassium binders for the 2:30 p.m. treatment of cardiovascular disease Bertram Pitt, Univ. of Michigan
2:30 p.m. – Free Time 6:00 p.m.
20 DAILY SCHEDULE 6:00 p.m. – Banquet and Awards Presentation 9:00 p.m. Pavilion, 1st Floor
SUNDAY, OCTOBER 6, 2019 7:00 a.m. – Breakfast 8:00 a.m. Pavilion, 1st floor
8:00 a.m. – 15.0 Symposium 6A: Normal and pathogenic regulation of aldosterone biosynthesis 10:00 a.m. Pavilion Theater, 1st Floor Chairs: Eleanor Davies, Univ. of Glasgow Jun Yang, Monash Univ.
8:00 a.m. – 15.1 Development of an inducible mouse model of aldosteronism 8:30 a.m. William Rainey, Univ. of Michigan
8:30 a.m. – 15.2 A gain of function mutation in CLCN2 chloride channel gene causes primary 9:00 a.m. aldosteronism Fabio Fernandes-Rosa, INSERM U970
9:00 a.m. – 15.3 Circulating microRNAs as diagnostic biomarkers of primary aldosteronism 9:15 a.m. Eleanor Davies, Univ. of Glasgow
9:15 a.m. – 15.4 The retinoic acid receptor a contributes to the development of primary 9:30 a.m. aldosteronism by regulating adrenal cortex structure through interactions with Wnt and Vegfa signaling Sheerazed Boulkroun, INSERM U970
9:30 a.m. – Coffee Break 10:15 a.m. Pavilion, 1st Floor
10:15 a.m. – 16.0 Symposium 6B: Integrated regulation of renal ion transport 12:00 p.m. Pavilion Theater, 1st Floor Chairs: David Ellison, Oregon Health Science Univ. Johannes Loffing, Univ. of Zurich
10:15 a.m. – 16.1 The role of ENaC in hyperoxia-induced preterm lung injury 10:45 a.m. My Helms, Univ. of Utah
10:45 a.m. – 16.2 Regulation of renal ion transport and blood pressure by the CRL3-WNK-SPAK 11:15 a.m. pathway James McCormick, Oregon Health and Science Univ.
11:15 a.m. – 16.3 WNK regulation of ion transport in the malpighian tubule 11:45 a.m. Aylin Rodan, Univ. of Utah
21 11:45 a.m. – 16.4 Interleukin 6 activation of the epithelial sodium channel in the distal convoluted 12:00 p.m. tubule and cortical collecting duct via Rac1 Oishi Paul, Emory Univ.
12:00 p.m. Boxed lunches available upon departure Pavilion, 1st floor
22 2019 APS ALDOSTERONE AND ENAC IN HEALTH AND DISEASE: THE KIDNEY AND BEYOND OCTOBER 2–6, 2019 THE STANLEY HOTEL ESTES PARK, CO TABLE OF CONTENTS
Abstracts of Invited and Volunteered Presentations ...... Page 23 1.0 Keynote Lecture: Human kidney organoids ...... Page 25 2.0 Symposium 1A: Aldosterone and mineralocorticoid receptors in the kidney and hypertension ...... Page 26 3.0 Symposium 1B: Metabolic and sex differences in aldosterone responses .. Page 30 4.0 Symposium 2A: Structure function of ENaC and related transporters ...... Page 33 5.0 Symposium 2B: ENaC function and regulation in tissues ...... Page 36 6.0 Poster Session 1 ...... Page 38 7.0 Symposium 3A: signaling and regulation of ENaC and other epithelial channels/transporters I ...... Page 48 8.0 Symposium 3B: signaling and regulation of ENaC and other epithelial channels/transporters II ...... Page 51 9.0 Symposium 4A: ENaC biogenesis and trafficking...... Page 54 11.0 Poster Session 2 ...... Page 56 12.0 Symposium 5A: MR in the vasculature ...... Page 67 13.0 Symposium 5B: MR structure and role in the heart and lungs ...... Page 70 14.0 Clinical Plenary Lecture: The role of non-steroidal MR antagonists and new potassium binders for the treatment of cardiovascular disease ...... Page 73 15.0 Symposium 6A: Normal and pathogenic regulation of aldosterone biosynthesis ...... Page 74 16.0 Symposium 6B: Integrated regulation of renal ion transport ...... Page 76 Author Index ...... Page 81
23 Special thanks to our conference sponsors.
Aldosterone and ENaC in Health and Disease: The Kidney and Beyond Join our sponsors in their effort Conference to support physiology. Your donation will make a lasting impact on the next generation 2019 SPONSORS of physiologists. By giving, AMERICAN HEART ASSOCIATION you ensure that the best and This program is supported in part by the American Heart Association’s Council on the brightest are empowered to Kidney in Cardiovascular Disease. make the research discoveries needed to help us understand NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES life and improve health. Research reported in this publication was supported by the National Institute of Diabetes and Digestive and Kidney Diseases of the National Institutes of Health (NIH) under Award Number R13DK122821. The content is solely the responsibility of the authors and does not necessarily represent the official views of NIH. the-aps.org/giving
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ENaC Conference Thank You to Sponsors.indd 1 8/26/2019 12:56:51 PM 2019 APS ALDOSTERONE AND ENAC IN HEALTH AND DISEASE: THE KIDNEY AND BEYOND ABSTRACTS OF INVITED AND VOLUNTEERED PRESENTATIONS
Organoids can be used to study kidney This program is the official conference
proceedings of the 2019 APS Aldosterone and development, mechanisms of disease processes
ENaC in Health and Disease: The Kidney and nephrotoxicity of drugs. Epithelial toxins and Beyond conference. Find the program at: pro-inflammatory agents, such as IL-1 and www.the-aps.org/conferenceproceedings. TGF can cause increased numbers of stromal cells with characteristics of myofibroblasts and To cite your abstract, use the format listed deposition of collagen. Kidney organoids are below: effective tools to study other genetic and non- Bonventre J. Human kidney organoids genetic disorders of the kidney. As an example, (Abstract). APS Conference: Aldosterone organoids derived from iPSCs generated from and ENaC in Health and Disease: The individuals with autosomal dominant or Kidney and Beyond, Estes Park, CO, 2-6, October September 2019, p. A1.1 recessive polycystic kidney disease can be induced to form cysts while organoids derived from subjects without cystic disease do not. 1: KEYNOTE LECTURE: The convergence of “personalized” kidney organoids with genome editing (e.g. CRISPR-Cas 1.1 9 approaches) and single cell sequencing Human kidney organoids technology holds great promise to result in Joseph Bonventre1 better insight to disease, better human cell 1Renal Division, Brigham and Women’s Hospital disease models, more predictive toxicology, and and Dept. of Medicine, Harvard Medical School potentially “clinical trials in a dish”. There is a This human kidney differentiation system has great deal of interest in functional assays but spawned a rapidly moving field of kidney most success at this point is limited to biology with potential for transformative impact demonstration of secretory ability of the on models of disease and drug development. proximal tubule and improvement in these We have developed an efficient, chemically assays are ongoing. Another area of research is defined protocol for differentiating human enrichment of nephron structures, such as embryonic stem (ES) cells and induced podocytes, proximal or distal tubule cells which pluripotent stem cells (iPSCs) into multipotent is of particular interest for further studies of nephron progenitor cells (NPCs) that can form these segments or retrieval of particular cell nephron-like structures. By recapitulating types from the organoids. Placing organoids metanephric kidney development in vitro we under the kidney capsule has been found to generate SIX2+SALL1+WT1+PAX2+ NPCs with enhance differentiation and vascularization and 75-90% efficiency within 8-9 days of initiation of connections with the host circulation. differentiation. The NPCs form PAX8+LHX1+ As the organoid field develops there is renal vesicles that self-organize into nephron increasing opportunity at the boundary of structures. NPCs form kidney organoids biology and engineering to control and modify containing epithelial nephron-like structures differentiation and maturation and also analysis expressing markers of podocytes, proximal of function. Construction of cellular tubules, loops of Henle and distal nephrons in microfabricated hybrid devices are guided by an organized, continuous arrangement that anatomical and functional microenvironmental resembles the nephron in vivo. The organoids relationships that characterize the kidney. For express genes of many transporters seen in example printed channels can be lined with adult metanephric-derived kidney. Stromal cells epithelial cells or endothelial cells to mimic the are also generated with the presence of tubule and it’s adjacent vasculature. Challenges PDGFR+ (pericyte), CD31+endomucin+ are many in this “organ on chip” approach. One (endothelial cell), or -SMA+ (myofibroblast) key feature which must be recapitulated by the interstitial cells. The entire procedure is cell-matrix and microfluidic environmental performed with completely defined conditions conditions in the maintenance of viable without the need for embryonic spinal cord.
25 2019 APS ALDOSTERONE AND ENAC IN HEALTH AND DISEASE: THE KIDNEY AND BEYOND ABSTRACTS OF INVITED AND VOLUNTEERED PRESENTATIONS functional differentiated cells in vitro. This has hypertension through increased NaCl been challenging as isolated cells ex vivo have reabsorption (Hypertension 2017). 11β -HSD2 is a strong tendency to undergo de-differentiation also present in placenta, and placental 11β - and a metabolic shift from oxidative to glycolytic HSD2 block the penetration of high cortisol from metabolism. pregnant mother into fetus. But, malnutrition In conclusion, human kidney organoids during pregnancy downregulates placental 11β - represent an important addition to our HSD2 activity, leading to cortisol excess in fetus, armamentarium to better understand kidney and in turn, excessive cortisol during the fetal physiological and pathophysiological processes period causes prenatal programmed salt- and to bring new therapeutic agents to growing sensitive hypertension in adult life. We also number of individuals with kidney disease. clarified epigenetic mechanism of prenatal programmed hypertension (JCI-Insight 2018). 2) 2: SYMPOSIUM 1A: ALDOSTERONE AND Previously, we found that Rac1, a member of MINERALOCORTICOID RECEPTORS IN RhoGTPases, activate MR, aldosterone- THE KIDNEY AND HYPERTENSION independently (Nat Med 2008). In salt-loaded Dahl S hypertensive rats, treatments of Rac1 2.1 inhibitor, as well as eplerenone, decreased BP Aldosterone-independent activation of and albuminuria, associated with mineralocorticoid receptor in salt-sensitive downregulation of Sgk1, a downstream molecule hypertension and glomerular diseases of MR signaling, indicating that salt-induced Toshiro Fujita1 activation of Rac1-MR pathway contribute to salt- 1Research Center for Academic Sciences & sensitive hypertension and glomerular injury (JCI Technology, The Univ. of Tokyo 2011). Moreover, podocyte-specific RhoGDI-KO Obesity is associated with increased salt- mice showed albuminuria and podocte damage sensitivity of blood pressure (BP). Obesity- by Rac1 activation, but both podocyte injury and induced salt-sensitive hypertension is caused by albuminuria were ameliorated by eplerenone. hyperaldosteronism that is due to adipocyte- Consistently, Hildebrandt et al. found steroid- derived aldosterone releasing factors. Moreover, resistant congenital nephrotic syndrome in high salt diet aggravates BP elevation and patients with RhoGDI mutation, associated with cardio-renal injury with the inadequate activation of Rac1-MR pathway (JCI 2013). We suppression of plasma aldosterone, but recently established the diabetic nephropathy- treatment of the mineralocorticoid receptor (MR) model mice showing massive albuminuria with antagonist eplerenone improved these intense staining of active Rac1 in podocytes and abnormalities. Indeed, MR is activated by upregulation of Sgk1. But, the Rac1 inhibitor and aldosterone excess and high salt, but MR antagonist decreased albuminuria. aldosterone-independent MR activation is Moreover, we tried to have the RCT to evaluate involved in salt-sensitive hypertension and the effect of eplerenone on urinary albumin glomerular diseases, despite normal, even low, excretion in hypertensive CKD patients circulating aldosterone concentration. 1) Cortisol receiving RAS blockades. Eplerenone and aldosterone, in vitro, equally bind to MR, but decreased urinary albumin excretion, in an 11β -HSD2 in the kidney converts cortisol to aldosterone-independent manner (Lancet cortisone, an inactive metabolite; aldosterone, Endocrinol & Diabetes 2017). Both BP and but not cortisol, serves as a sole ligand of MR in albuminuria increased during 1-year treatment in the kidney. Recently, we found that deletion of the placebo group with higher salt intake, 11β -HSD2 in aldosterone-sensitive distal despite receiving RAS blockades, but not in the nephron activate ENaC through MR activation lowest salt group, suggesting salt-induced induced by corticosterone excess. Resultantly, resistance to RAS blockades. But, salt-induced hypokalemia and alkalosis activate NCC and increase in BP and albuminuria disappeared in pendrin, respectively, resulting in salt-sensitive eplerenone-treated patients. This result
26 2019 APS ALDOSTERONE AND ENAC IN HEALTH AND DISEASE: THE KIDNEY AND BEYOND ABSTRACTS OF INVITED AND VOLUNTEERED PRESENTATIONS supports our hypothesis that progression of 2.3 albuminuria and BP elevation on the high salt The intercalated cell mineralocorticoid receptor diet may be mediated by salt-induced activation regulates pendrin diretly and regulates principal of Rac1-MR pathway, aldosterone- cell ENaC indirectly over a wide range in serum independently. K+ Susan Wall1, Truyen Pham1, Yanhua Wang1, 2.2 Cesar Romero1, Qiang Yue1, Chao Chen2, The expanding spectrum of primary Monika Thumova1, Douglas Eaton1, Yoskaly aldosteronism Lazo-Fernandez1, Jill Verlander2 Anand Vaidya1 1Medicine, Emory Univ.; 2Medicine, Univ. of 1Endocrinology, Brigham and Women's Hospital, Florida Harvard Medical School Background: Aldosterone activates the Primary aldosteronism is characterized by intercalated cell (IC) mineralocorticoid receptor autonomous aldosterone production that is (MR), which is enhanced during hypokalemia. independent of renin and angiotensin II and Whether the intercalated cell MR directly sodium status. For decades, the traditional regulates the intercalated cell Cl-/HCO3- approaches to defining and diagnosing primary exchanger, pendrin, K+’s role in this response, aldosteronism have been relatively constant and and the receptor’s impact on IC and principal generally focused on detecting severe and cell function in the cortical collecting duct (CCD) classical presentations of the disease. However, are unresolved. Methods: We measured CCD Cl- accruing evidence indicates that the prevalence absorption, transepithelial voltage (VT), ENaC of primary aldosteronism is much greater than channel activity, plus pendrin abundance and previously recognized, and that milder and non- subcellular distribution in wild type and IC- classical forms or primary aldosteronism that specific MR null mice. Results: In CCDs from impart heightened cardiovascular risk may be aldosterone-treated mice, IC MR ablation common and unrecognized. This presentation reduced Cl- absorption as well as ENaC channel will review the physiologic approach to activity and ENaC open probability despite considering primary aldosteronism and will principal cell MR expression in the KO mice. summarize evidence from human studies that Since Cl- absorption in CCDs of aldosterone- have characterized a broad severity spectrum of treated mice occurs in large part through primary aldosteronism, ranging from mild to pendrin-mediated, Cl- absorption, we asked if overt, that correlates with cardiovascular risk. pendrin protein abundance and/or pendrin's The expanding spectrum of primary relative abundance in the region of the apical aldosteronism may have important implications membrane is lower in kidneys from aldosterone- for understanding the pathogenesis and treated IC MR KO relative to wild type mice. We treatment of hypertension and cardiovascular observed that pendrin total protein abundance disease. and pendrin's relative abundance in the region of the apical plasma membrane are lower in kidneys from IC MR null relative to wild type mice following treatment models associated with high circulating aldosterone concentration. To determine if the IC MR directly regulates pendrin and the impact of serum K+ on this response, we made use of the fact that occasional intercalated cells from the IC MR null mice express the MR. Thus, we measured pendrin label intensity and subcellular distribution in MR (+) and (-) intercalated cells taken from the same IC MR KO mice. Under
27 2019 APS ALDOSTERONE AND ENAC IN HEALTH AND DISEASE: THE KIDNEY AND BEYOND ABSTRACTS OF INVITED AND VOLUNTEERED PRESENTATIONS conditions associated with high serum based method to quantify plasma aldosterone aldosterone, pendrin’s relative abundance in the and angiotensin metabolites in SSKcnj16-/- rats apical membrane region and pendrin and SSWT control rats. We found that SSKcnj16- abundance/cell were lower in the MR (-) than the /- rats had substantial increases in all RAAS MR (+) intercalated cells, whether serum K+ was hormones compared to SSWT rats. A high salt high or low. Moreover, application of diet magnified the observed increases in aldosterone in vitro increased pendrin label angiotensin and its metabolites but caused a intensity in cultured CCDs. Conclusions: In significant attenuation in aldosterone preceding treatment models associated with high deaths. Furthermore, blocking aldosterone circulating aldosterone, IC MR ablation reduces action in SSKcnj16-/- rats with spironolactone did CCD Cl- absorption and indirectly reduces not prevent these salt-induced deaths, but itself principal cell ENaC abundance and function. caused 100% mortality within 48 hours of Aldosterone and the IC MR directly regulate administration, even during low salt treatment. pendrin. IC MR gene ablation reduces pendrin Supplementation of the diet with high K+ protein abundance and pendrin’s relative showed strong attenuation in the RAAS abundance in the apical membrane region over response to high salt and was protective against a wide range in serum K+. mortality resulting from aldosterone-mediated mechanisms. Administration of angiotensin 2.4 receptor blocker, Losartan, and ACE inhibitor, Kir5.1-mediated changes in renin-angiotensin- Captopril, did not impact survival. These studies aldosterone system balance in salt sensitive reveal that the deletion of Kir5.1 resulted in hypertension marked RAAS modulation and the resulting Anna Manis1, Oleg Palygin1, Sherif Khedr1, amplification in aldosterone (but not angiotensin) Vladislav Levchenko1, Matthew Hodges1, signaling is required for survival without this Alexander Staruschenko1 channel, revealing disparate effects of 1Physiology, Medical College of Wisconsin angiotensin and aldosterone pathways on Kir5.1 (encoded by Kcnj16 gene) is an inwardly Kir4.1/Kir5.1-mediated control of K+ balance. rectifying K+ channel (Kir) subunit which is highly expressed in the aldosterone-sensitive distal 2.5 nephron, where it forms a functional A proof-of-principle study of sodium loading in heterotetramer with Kir4.1. Our previous studies prehypertension revealed that a knockout of Kcnj16 in the Dahl J. Brian Byrd1,2, Brian Bazzell1, William Rainey3, salt-sensitive rat (SSKcnj16-/-) resulted in Richard Auchus1, Davide Zocco4, Marco Bruttini5, hypotension, salt wasting tubulopathy, and Scott Hummel1,6 hypokalemia, which was exacerbated and led to 1Internal Medicine, Univ. of Michigan Medical mortality within a few days when SSKcnj16-/- rats School; 2Internal Medicine, Univ. of Michigan were fed a high salt diet. Moreover, we found Comprehensive Hypertension Center; that accompanying the 4% NaCl challenge with 3Molecular and Integrative Physiology, Univ. of increased dietary potassium (1.41% K+) or Michigan Medical School; 4Research and administering the ENaC blocker benzamil (but Development, Exosomics Siena; 5Life Sciences, not a furosemide or HCTZ) prevented all Univ. Degli Studi di Siena; 6Section of occurrences of salt-induced mortality. However, Cardiology, Ann Arbor Veterans Affairs Medical specific mechanisms pertaining to cardiorenal Center abnormalities in SSKcnj16-/- rats and the Background: mRNA shielded by extracellular interaction of Kir4.1/Kir5.1-mediated potassium vesicles and perhaps other carriers such as transport with the renin-angiotensin-aldosterone proteins is found in urine supernatant. This system (RAAS) remain unclear. The objective of mRNA might encode information about renal this study was to evaluate the role of Kir5.1 in and cardiorenal pathophysiology, including RAAS regulation. We used a mass spectrometry- hypertension. However, whether the mRNA
28 2019 APS ALDOSTERONE AND ENAC IN HEALTH AND DISEASE: THE KIDNEY AND BEYOND ABSTRACTS OF INVITED AND VOLUNTEERED PRESENTATIONS transcriptome found in urine supernatant assays for MR-regulated targets (SCNN1A, reflects that of renal tissues and whether SCNN1G, TSC22D3) changed after sodium changes in renal physiology are detectable loading, and MR-regulated targets (SCNN1A, using US-mRNA has not been known. Like other SCNN1G, SGK1, and TSC22D3) gene expression nuclear hormone receptors, the correlated significantly with serum aldosterone mineralocorticoid receptor (MR) is a ligand- and inversely with urinary sodium excretion. activated transcription factor. Objective: The first Conclusions: RNA-sequencing of urinary objective was to use RNA-Seq data to compare extracellular vesicles shows concordance with human urinary extracellular vesicles mRNA human kidney. Perturbation in human endocrine content to human renal cortex. The second signaling (MR activation) was accompanied by objective was to characterize the effect of low- changes in mRNA in urine supernatant. Our sodium diet and subsequent sodium-loading on findings could be useful for individualizing transcripts regulated by mineralocorticoid pharmacological therapy in patients with receptor activation assayed in the supernatant disorders of mineralocorticoid signaling, such as of human urine. Methods: Using publicly resistant hypertension. More generally, these available data, we compared the transcriptomes insights could inform discovery of novel non- of a healthy male volunteer's human urinary invasive biomarkers of disordered renal and extracellular vesicles and deceased males' cardiorenal physiology. Sources of Funding Dr human renal cortex. To avoid similarities Byrd was supported by National Institutes of attributable to ubiquitously expressed genes, we Health grant K23HL128909 and the Michigan separately analyzed ubiquitously expressed and MTRAC Kickstart Award. Disclosures Drs Byrd, highly kidney-enriched genes. To determine Rainey, and Auchus are inventors of a whether mRNA in human urine supernatant provisional patent related to detecting reflects changes in renal gene expression, we mineralocorticoid receptor activation. Dr Zocco assayed cell-depleted urine for transcription is an employee of a company, Exosomics Siena factor activity of MR using per-amplified probe- SpA, that develops sample preps in the field of based quantitative polymerase chain reaction. liquid biopsy. He is an inventor of patents The urine was collected from prehypertensive owned by this company. This work was individuals (n=18) after 4 days on low-sodium published in Circulation: Genomic and Precision diet to stimulate MR activity and again after Medicine in September 2018 and is submitted to suppression of MR activity via sodium infusion. this meeting because the presenter thinks it will All work was conducted in accordance with the interest the audience and they may not have guidelines for experimental procedures set forth seen the work yet. The full manuscript is in the Declaration of Helsinki, and all study available on an open access basic at: participants gave informed consent. Results: In https://www.ahajournals.org/doi/full/10.1161/CIRC comparing mRNA in human urine supernatant GEN.118.002213 and human kidney cortex, expression of 55 highly kidney-enriched genes correlated strongly (rs=0.82) while 8,457 ubiquitously expressed genes correlated moderately (rs=0.63). Standard renin-angiotensin- aldosterone system phenotyping confirmed the expected response to sodium loading. Assays were designed for genes expected to be regulated by MR activation and genes expected not to be regulated in this way, with more than one assay per gene in view of the possibility that the mRNA would be fragmented. Gene expression inferred from cycle threshold for
29 2019 APS ALDOSTERONE AND ENAC IN HEALTH AND DISEASE: THE KIDNEY AND BEYOND ABSTRACTS OF INVITED AND VOLUNTEERED PRESENTATIONS
3: SYMPOSIUM 1B: METABOLIC AND SEX aldosterone production in female than male DIFFERENCES IN ALDOSTERONE zona glomerulosa cells basally and in response RESPONSES to both AngII and potassium (P<0.0001). We then studied a rat model of aldosterone- 3.1 mediated cardiovascular disease induced by Biological sex-specific differences in the increased AngII and low NO. In this model, aldosterone responses to angiotensin II in circulating aldosterone levels (P<0.01), humans and rodents myocardial damage (P<0.001), and proteinuria Jose Romero1, Gail Adler1, Gordon Williams1, (P<0.05) were greater in female than male rats Luminita Pojoga1 despite having similar BP responses. Thus, 1Medicine, Brigham and Women’s Hospital and increased aldosterone production likely Harvard Medical School contributes to sex differences in cardiovascular There is abundant evidence showing that in disease, suggesting that women may be more humans and rodents, chronic activation of the responsive to mineralocorticoid receptor mineralocorticoid receptor (MR) and disordered blockade than men. (This work was supported, levels of angiotensin II (AngII) and aldosterone in part, by the National Institute of Health, lead to increased blood pressure (BP) and National Heart, Lung and Blood Institute Grants cardiovascular and renal damage. However, the R01HL096518 and R01HL114765) Hypertension. impact of biological sex on the renin– 2018; 71:1083-1090. DOI: angiotensin–aldosterone system (RAAS) is 10.1161/HYPERTENSIONAHA.117.11087. unclear. We hypothesized that sex would modify the interaction between aldosterone responses 3.2 to salt intake and AngII. To test this hypothesis, The leptin-aldosterone-mineralocorticoid in over 1500 subjects from the well-controlled receptor axis: a major contributor to Hypertensive Pathotype cohort, we compared cardiovascular disease in obese females the responses of women and men to chronic (BP Eric J. Belin de Chantemele1 response to dietary salt) and acute (aldosterone 1Vascular Biology Center and Dept. of Medicine responses to AngII infusion) manipulations. (Cardiology), Medical College of Georgia at Women had a 30% higher salt sensitivity of BP Augusta Univ. than men (P<0.0005) regardless of age and a While female sex steroids protect 15% greater aldosterone response to AngII on premenopausal women from cardiovascular both restricted and liberal salt diets (P<0.005). disease, recent clinical evidence indicates that The means of aldosterone responses to AngII obesity, which is more prevalent and severe in on a liberal salt diets were grouped according to women compared to men, ablates the protective quartiles of age. The aldosterone responses effects of female sex hormones and predisposes decrease with age in both sexes (linear young women to cardiovascular disease. regression analyses in women P<0.001 and men However, the mechanisms whereby obesity P<0.005) and was higher in women than men promotes vascular disease and hypertension in from the lowest quartile of age and reached females remain ill-defined. Excess production in similar levels in women and men at the highest the adipocyte-derived hormone leptin quartile. Furthermore, we observed that contributes to hypertension in males via aldosterone responses to AngII on a restricted overactivation of the sympathetic nervous salt diet correlated with estradiol levels in system. Despite higher leptin levels than males, women <51 years old (P=0.033, obese premenopausal women do not exhibit coefficient=0.154). We assessed potential sympatho-activation but show inappropriately mechanisms for this sex effect by comparing high aldosterone levels which positively and contrasting aldosterone responses to AngII correlate with the level of adiposity and blood and potassium from ex vivo rat zona pressure. However, the origin of these glomerulosa cells. We observed greater inappropriately high aldosterone levels as well
30 2019 APS ALDOSTERONE AND ENAC IN HEALTH AND DISEASE: THE KIDNEY AND BEYOND ABSTRACTS OF INVITED AND VOLUNTEERED PRESENTATIONS as the potential contribution of aldosterone to 3.3 obesity-related cardiovascular disease remained Hypersensitivity of renal ENaC to aldosterone is to be determined. My group tacked these a sex-specific determinant of blood pressure questions and reported that leptin as well as control in females leptin receptor deficiency or leptin receptor Mykola Mamenko1, Alena Cherezova1, Katherine blockade ablated obesity-mediated increases in Hatcher1, Michael Brands1, Vadym Buncha1 adrenal aldosterone synthase (CYP11B2) 1Dept. of Physiology, Augusta Univ. expression and aldosterone production. Excessive renal Na+ retention is a common Concomitantly, we showed that increases in pathogenic determinant of hypertension. Final leptin signaling in female mice or adrenocortical adjustments of sodium transport in the kidney cells in culture elevated adrenal CYP11B2 occur in the aldosterone sensitive distal nephron expression and aldosterone production. With through regulation of the Epithelial Na+ Channel these data, we demonstrated for the first time (ENaC). Both males and females employ that leptin is a direct regulator of aldosterone aldosterone to stimulate ENaC-dependent Na+ production. In parallel, we showed that both reabsorption and maintain their hydromineral reduction in leptin signaling and inhibition in homeostasis. Recent studies suggest the aldosterone action via mineralocorticoid existence of profound sex disparities pertaining receptor (MR) blockade abolished obesity- to aldosterone-induced regulation of ENaC associated endothelial dysfunction, function during hypertension. Specifically, hypertension and cardiac fibrosis. The effects of aldosterone antagonism with mineralocorticoid aldosterone blockade were specific to females, receptor (MR) inhibitors is insufficient to reduce which led to the proposal that obesity triggers excessive ENaC activity in the kidney and fails to cardiovascular disease via sex-specific effectively decrease blood pressure (BP) in mechanisms. We extended our findings by hypertensive males. At the same time, inability reporting that endothelial cell MR contribute to to suppress circulating aldosterone levels has predispose females to obesity-induced been associated with elevated BP in females cardiovascular disease. We showed that female that is successfully treated with MR antagonists. mice and human express more endothelial MR The contribution of aldosterone-driven ENaC- than males and that selective deletion of mediated renal sodium reabsorption to the endothelial MR protects female mice from leptin- increased BP observed in the hypertensive induced endothelial dysfunction. Finally, we females is unclear. The objective of this study reported that progesterone positively regulates was to test if enhanced sensitivity of renal ENaC endothelial MR expression providing an to aldosterone is a sex-specific determinant of explanation for the sex-difference in endothelial excessive Na+ retention and elevated BP in MR expression. All together our findings suggest females. We employed a rat model of Ang II that progesterone-dependent up-regulation of dependent hypertension. Sprague Dawley rats endothelial MR receptor and leptin-mediated of both sexes were infused with a slow-pressor aldosterone production are two parallel dose of Ang II (400 ng·kg−1·min−1) via mechanisms whereby obesity ablates the subcutaneously implanted mini-osmotic pumps cardiovascular protective effects of female sex for 14 days. This resulted in a graduate increase hormones. of BP reaching a steady state after 6-9 days of infusion. To assess the contribution of aldosterone-dependent mechanisms to the observed increase in BP, a group of Ang II infused males and females received aldosterone antagonist spironolactone (30 mg·kg−1·day−1) in the drinking water. Where necessary, non- hypertensive rats were used as controls. Ang II infusion markedly elevated circulating
31 2019 APS ALDOSTERONE AND ENAC IN HEALTH AND DISEASE: THE KIDNEY AND BEYOND ABSTRACTS OF INVITED AND VOLUNTEERED PRESENTATIONS aldosterone levels in rats of both sexes. 3.4 However, tail cuff plethysmography Female mice exhibit higher increases in demonstrated that treatment with aldosterone synthase expression and spironolactone significantly decreased BP only aldosterone production than males in response in hypertensive females, from 205± 4 mmHg to to low-salt diet 178± 3 mmHg (BP in spironolactone untreated Jessica Faulkner1, Daisy Harwood1, Galina and treated males was 217± 2 and 215± 5 mmHg Antonova1, Jennifer Sullivan2, Wendy Bollag2, respectively). Metabolic cage studies showed Sebastien Faure3, Nicolas Clere3, Eric Belin de that spironolactone markedly improved renal Chantemèle1 capacity to excrete sodium at lower BP in Ang II 1Vascular Biology Center, Medical College of infused females. Patch-clamp recordings of Georgia at Augusta Univ.; 2Physiology Dept., single-channel ENaC currents from freshly Medical College of Georgia at Augusta Univ.; isolated split-opened collecting ducts showed 3Micro & Nanomédecines Translationnelles- that spironolactone was markedly more effective MINT, Univ. Angers at inhibiting ENaC activity in Ang II infused It is well established that the renin-angiotensin- females. The activity of the channel returned to aldosterone system responds to changes in basal levels, observed in non-hypertensive dietary salt consumption to promote either salt female controls. MR inhibition primarily retention or excretion. However, the response of diminished the abundance of functional ENaCs aldosterone production to changes in dietary on the membrane and only moderately salt may be sex specific. Our previous published decreased the channel’s open probability. In work indicates that aldosterone levels and contrast, aldosterone antagonism was only adrenal aldosterone synthase (CYP11B2) partially effective in Ang II infused males. After expression are not suppressed in female mice in treatment with spironolactone ENaC activity response to a high-salt diet, in contrast to males, remained at least two times higher than in consistent with similar findings in humans. This normotensive male rats. RT-qPCR analysis failure of high-salt diet to suppress aldosterone revealed that MR expression is significantly in female mice in our previous study was higher in the kidneys of Ang II infused females, associated with an increase in blood pressure when compared to males. Semi-quantitative and development of endothelial dysfunction, western blotting demonstrated that which was ablated by mineralocorticoid receptor spironolactone significantly increased the antagonism. We therefore hypothesized that activity of Nedd4-2 in the kidneys extracted from high aldosterone levels induced by a low-salt hypertensive females, pointing to the facilitated diet will result in endothelial dysfunction in ENaC internalization. Treatment with female mice. Wild-type male and female mice spironolactone did not affect the abundance of were fed either a normal-salt (0.4%NaCl) or a other distal nephron sodium transporters, NCC low-salt (0.05% NaCl) diet for either 14 or 28 and NKCC2, and did not result in elevation of days. Plasma aldosterone levels were increased plasma K+ levels in hypertensives of either sex. in female mice to a greater extent than males Thus, in contrast to males, aldosterone is a following both 14 days (∆ increase from same- primary regulator of ENaC activity in sex normal-salt 125± 63 pg/ml male low-salt vs hypertensive females. MR inhibition effectively 236± 68 pg/ml female low-salt, *P<0.05) and 28 reduces high BP in females through days (∆ increase from same-sex normal-salt of improvement of pressure-natriuresis. This makes 96± 42 pg/ml male low-salt vs 246± 78 pg/ml MR antagonism a promising therapeutic female low-salt, P=0.05) of low-salt diet. In approach to combat hypertension in women. correlation, adrenal CYP11B2 protein expression was unchanged following 14-day feeding in male mice on low-salt diet (1.10± 0.15 normal-salt vs 1.21± 0.14 low-salt ratio to β -actin) but modestly increased in females on low-salt (1.10± 0.11
32 2019 APS ALDOSTERONE AND ENAC IN HEALTH AND DISEASE: THE KIDNEY AND BEYOND ABSTRACTS OF INVITED AND VOLUNTEERED PRESENTATIONS normal-salt vs 1.68± 0.28 low-salt ratio to β - 4: SYMPOSIUM 2A: STRUCTURE actin, P=0.06). CYP11B2 protein expression was FUNCTION OF ENAC AND RELATED similarly unchanged with low-salt diet in males TRANSPORTERS following 28 days of feeding (0.62± 0.23 normal- salt vs 0.53± 0.17 low-salt ratio to β -actin) 4.1 whereas low-salt diet significantly increased ENaC structure by cryo-electron microscopy CYP11B2 expression in females (0.91± 0.07 Isabelle Baconguis1, Sigrid Noreng2, Arpita normal salt vs 1.26± 0.16 ratio to β -actin, Bharadwaj1, Richard Posert2, Craig Yoshioka3 *P<0.05). No difference in adrenal renin, 1Vollum Institute, Oregon Health and Science angiotensin II type 1 receptor or Univ.; 2Biochemistry and Molecular Biology, angiotensinogen mRNA expression was Oregon Health and Science Univ.; 3Biomedical observed between male or female mice on Engineering, Oregon Health and Science Univ. normal- or low-salt diet. In addition, no The epithelial sodium channel (ENaC), a member differences were observed between male or of the ENaC/DEG superfamily, regulates Na+ female mice on low-salt diet in acetylcholine and water homeostasis. Structures of a relative (endothelium-dependent) or sodium of ENaC, acid-sensing ion channels (ASICs), nitroprusside (endothelium-independent) aorta have shown that the members of the ENaC/DEG relaxation responses nor phenylephrine- or KCl- superfamily are trimers with large extracellular induced contraction. However, female mice on domains and narrow transmembrane domains. high-salt diet displayed an increased While ASICs can form physiologically relevant constriction response to serotonin in response homomeric and heteromeric ion channels, to low-salt diet, whereas no such alteration was ENaCs assemble as heterotrimeric channels that observed in male mice on low-salt diet (2-way harbor protease-sensitive domains critical for ANOVA with repeated measures, *P<0.05). gating the channel. The structure of human Collectively, these data indicate that dietary ENaC in the uncleaved state determined by sodium intake is a more potent regulator of single-particle cryo-electron microscopy reveals aldosterone production in female mice than in that ENaC assembles with a 1:1:1 stoichiometry of male mice which may promote serotonin- α:β :γ subunits arranged in a counter-clockwise mediated constriction in females. Given that manner. Like ASIC, the shape of each subunit is studies are currently emerging that indicate that reminiscent of a hand holding a ball. females are more sensitive to the blood Surrounding the ball-domain are the palm, pressure lowering effects of mineralocorticoid finger, and thumb domains. The finger domain of receptor antagonists than males, the each subunit forges interaction with knuckle contribution of dietary salt to aldosterone levels domain of the adjacent subunit. Wedged in females may play a role in cardiovascular between the finger and thumb domains is the health in women both on high- and low-salt elusive protease-sensitive inhibitory domain, not diets. found in ASICs, and is poised to regulate conformational changes of the ‘finger’ and ‘thumb,’ domains that play critical roles in gating the ion channel.
4.2 ENaC gating regulation by biliary factors Ossama Kashlan1 1Medicine, Univ. of Pittsburgh The epithelial sodium channel (ENaC) mediates Na+ transport in several epithelia, including the aldosterone-sensitive distal nephron, distal colon, and biliary epithelium. Numerous factors
33 2019 APS ALDOSTERONE AND ENAC IN HEALTH AND DISEASE: THE KIDNEY AND BEYOND ABSTRACTS OF INVITED AND VOLUNTEERED PRESENTATIONS regulate the activity of the channel, including 4.3 several extracellular ligands and membrane- ENaC subunit N-glycans have different roles for resident lipids. Bile acids are abundant in the the ability of the channel to respond to shear biliary tree and intestinal tract, and can be force elevated in the urine of patients with advanced Martin Fronius1, Jan-Peter Baldin1, Daniel Barth1, liver disease. Using Xenopus oocytes and a Fenja Knöpp2 cultured mouse cortical collecting duct cell line, 1Physiology, Univ. of Otago; 2Excellence Cluster we found that bile acids regulate ENaC gating. Cardio-Pulmonary Institute, Univ. Giessen Regulation was dependent on specific bile acid Background: Post-translational modification of functional groups, rather than on their ENaC in form of N-glycans was identified to be biophysical properties. Enhanced regulation by important for channel maturation and trafficking. taurine-conjugated bile acids, which are The role of individual N-glycans for the ability of membrane impermeant, suggest that bile acids the channel to sense shear force is unknown. regulate the channel by binding sites accessible We hypothesized that ENaC N-glycans are from the extracellular side of the channel. important for shear force sensing by providing a Activating bile acids had greater effects on connection to the extracellular matrix. Material ENaC mutants with a very low open probability, and Methods: Human αβ γ ENaC subunits were and no effect on fully active trypsin-treated expressed in Xenopus oocytes and channels, consistent with regulation of ENaC transmembrane currents were recorded by two- gating. Modeling of the voltage-dependence of electrode voltage clamp under no flow regulation suggest at least two binding sites for conditions and after application of a fluid stream taurocholic acid, one of which is voltage- generating ~0.2 dynes/cm2 of shear force (SF). sensitive. To test whether bile acid regulation of ENaC–mediated current was determined with ENaC in the kidney could contribute to the amiloride (10 μM). To assess the role of N- pathophysiology of advanced liver disease, we glycans asparagines within glycosylation tested the ability of urines collected from consensus motifs of each subunit were advanced liver disease patients to regulate individually replaced against alanines by site- ENaC expressed in oocytes. Urine pH and Na+ directed mutagenesis. The normalized SF concentrations were adjusted to match control responses of channels lacking asparagines was buffers, and buffers were supplemented with compared with wild type (wt) ENaC. Results: In α mannitol to control for osmolarity. We found that ENaC two asparagines were identified that – if urines from some advanced liver disease replaced individually – reduced the normalized patients activated amiloride-sensitive currents in SF response by about 40% in comparison to wt ENaC expressing oocytes. Activation was ENaC. Combined replacement of both attenuated when urines were treated with asparagines reduced the SF response by ~80 % cholestyramine, a bile acid sequestering resin. indicating additive effects. In β ENaC two Our data suggest that bile acids regulate ENaC asparagines were identified that, if replaced in the biliary tract under physiological individually, resulted in channels that provided a conditions, and may regulate ENaC in the stronger response to SF compared with the wt nephron under pathophysiological conditions. channel. Replacement of N-glycans in γ ENaC This work was supported by NIDDK grant did not influenced the normalized current DK098204 and a grant from the Pittsburgh Liver response to SF. Conclusion: Results indicate that Research Center. N-glycans of α and β ENaC have different/antagonistic roles for the ability of the channel to respond to SF. N-glycans of α ENaC may facilitate a connection to the extracellular matrix since their removal impairs the ability to respond to SF. N-glycans of β ENaC seem to have a different role. It may be hypothesized,
34 2019 APS ALDOSTERONE AND ENAC IN HEALTH AND DISEASE: THE KIDNEY AND BEYOND ABSTRACTS OF INVITED AND VOLUNTEERED PRESENTATIONS that N-glycans of ENaC are tethers that facilitate amiloride-sensitive current in mpkCCD specific interactions with either the extracellular monolayers. In split open native mouse CCDs, matrix or adjacent channel subunits. NMDA activation increased channel activity, NPo (p=0.04). In pre-constricted AfA, in vitro glycine 4.4 plus glutamate application to the CNT lumen N-methyl-D-aspartate (NMDA) receptor interacts increased AfA dilation (p<0.001). This amino- with ENaC to induce renal vasodilation in the acid-induced dilation was blocked with ENaC connecting tubule inhibitors (benzamil) and blunted with the NMDA Cesar Romero1, Oishi Paul1, Hong Wang2, Yilin receptor blocker MK-801. Conclusions: NMDA Ren2, Brandy Wynne1, Qiang Yue1, Susan Wall1, receptor activation increased both amiloride- Douglas Eaton1 sensitive current in principal cell monolayers, as 1Renal Division, Emory Univ. School of Medicine; well as ENaC channel activity in native, mouse 2Hyperension and Vascular Research Division, split open CCDs. NMDA receptor activation Henry Ford Hospital increased amino acid-induced renal vasodilation Introduction: N-methyl-D-aspartate (NMDA) by increasing ENaC-dependent CNTGF receptors are expressed throughout the responses in the CNT. These data explain the nephron, including distal nephron principal cells physiological basis of renal functional reserve (PC), and induce renal vasodilation through an test and provide a potential signaling pathway, unknown mechanism. These receptors are which could be targeted to prevent the renal activated by glutamate and glycine, and mediate damage associated with a high protein diet. the vasodilation that follows a high protein diet, which is an indicator of renal functional reserve 4.5 commonly used in clinical practice. Epithelial Proton block of the epithelial sodium channel Na+ channel (ENaC), the primary mechanism of Daniel Collier1, Alan Ryan2, Zerubbable sodium absorption in the connecting tubule Peterson2, Noah Williford2, Peter Snyder2 (CNT) and cortical collecting duct (CCD) 1Pharmaceutical Sciences, Univ. of Tennessee mediates CNT-glomerular feedback (CNTGF), a Health Science Center; 2Internal Medicine, Univ. vasodilator feedback mechanism producing of Iowa afferent arteriole (AfA) vasodilatation following The Epithelial Sodium Channel (ENaC) is high distal delivery of sodium. We hypothesized exposed to wide variations in extracellular pH. In that NMDA receptors activation stimulate ENaC, the kidney, pH can range from 9 to 5. In the which increases CNTGF when amino acids tongue, many of the foods and beverages we reach the distal nephron. Methods: To ingest can be highly acidic (pH < 3). In previous determine the role of NMDA receptors in ENaC work, we found that H+ in these ranges channel activity and ENaC-mediated CNTGF we stimulate ENaC by altering its gating. Here we examined the effect of NMDA agonists and observed that H+ can also have an inhibitory antagonists on ENaC-mediated current in effect on ENaC that is independent of channel mpkCCD monolayers and ENaC channel activity gating. In Xenopus oocytes, once ENaC was in principal cell apical membrane patches from fully activated (e.g. DEG mutation, trypsin, pH split-open mouse CCDs. To determine the effect 2.25), addition of H+ to the extracellular solution of NMDA receptor on CNTGF-mediated AfA reduced ENaC current. Inhibition occurred over dilation, we measured glycine plus glutamate- a pH range of 6-2 (IC50~ 3.5). H+ inhibition was induced CNTGF-mediated AfA dilation in the voltage-dependent, suggesting that H+ might presence or absence of an NMDA receptor block the ENaC pore. To test whether H+ could antagonist (MK801), using a double-perfusion permeate ENaC, we replaced Na+ in the method of the AfA and CNT ex vivo in rabbits extracellular bathing solution with H+. Under and stop-flow pressure (SFP) technique in vivo in these conditions, we observed an inward H+ rats. Results: NMDA receptor activation current that was blocked by amiloride. Mutation increased, whereas NMDA blockade reduced, of pore residues altered H+ permeability relative
35 2019 APS ALDOSTERONE AND ENAC IN HEALTH AND DISEASE: THE KIDNEY AND BEYOND ABSTRACTS OF INVITED AND VOLUNTEERED PRESENTATIONS to Na+ and Li+; γS540C enhanced H+ 5.2 permeation, whereas γS542L reduced H+ ENaC regulation by aldosterone and proteases permeation. An additional residue within the Christoph Korbmacher1 inner vestibule also contributed to H+ 1Institute of Cellular and Molecular Physiology, permeation (β D546). Together, the data support Friedrich-Alexander Univ. Erlangen-Nürnberg a model in which H+ blocks ENaC Na+ current (FAU) by competition for binding sites within the The epithelial sodium channel (ENaC) channel pore. We speculate that this may play a constitutes the rate-limiting step for sodium role in modulating sour and salt taste sensation. absorption in the aldosterone-sensitive distal nephron (ASDN), which comprises the late distal 5: SYMPOSIUM 2B: ENAC FUNCTION AND convoluted tubule (DCT2), the connecting tubule REGULATION IN TISSUES (CNT) and the entire collecting duct. It is well known that ENaC activity in the cortical 5.1 collecting duct (CCD) is tightly regulated by Regulation of ENaC by extracellular Na aldosterone which stimulates channel activity by Thomas Kleyman1 activating the mineralocorticoid receptor (MR). In 1Dept. of Medicine, Univ. of Pittsburgh contrast, ENaC activity in the early part of the ENaC activity is regulated by number of ASDN appears to be independent of extracellular factors, including specific ions, aldosterone [1] but is likely to contribute to blood proteases and shear stress. Intracellular factors, pressure control [2]. To test the hypothesis that including acidic phospholipids and channel ENaC function in the early part of the ASDN is modifications by ubiquitination, palmitoylation aldosterone-independent but dependent on the and phosphorylation also affect ENaC activity. presence of MR, we used doxycycline-inducible Many of these factors likely work in a nephron-specific MR-deficient (MR KO) mice [3]. coordinated manner to regulate ENaC open Using whole-cell recordings, ENaC function was probability. Among the factors that affect assessed in nephron fragments from the channel open probability is extracellular Na, transition zone of DCT2/CNT or CNT/CCD. As which binds to specific extracellular site(s) and expected, ENaC activity was detectable in transitions the channel to a low open probability CNT/CCD cells of control mice but absent or state. Our group and others have identified a barely detectable in the majority of CNT/CCD growing number of rare human ENaC variants cells from MR KO mice. Importantly, ENaC (nsSNVs) that alter ENaC expression in currents in DCT2/CNT were greatly reduced in heterologous expression systems. Many of MR KO mice compared to ENaC currents these variants are in the extracellular regions of measured in DCT2/CNT of control mice. A ENaC subunits and affect the inhibitory characteristic feature of ENaC is its regulation by response of extracellular Na. The contributions proteases [4]. To explore a possible link of these gain- or loss-of-function variants to between the stimulatory effect of aldosterone human physiologic parameters, including blood and proteolytic ENaC activation, we used the pressure, largely remains to be determined. We mCCDcl1 cell line derived from microdissected are using mouse models to explore the function mouse CCD. ENaC-mediated sodium transport roles of ENaC-specific variants that modify the was assessed by recording the amiloride- inhibitory response of extracellular Na. sensitive equivalent short circuit current (ISC- Ami) in Ussing chambers [5]. Exposure of the mCCDcl1 cells to 3 nM aldosterone for 2.5 h stimulated ISC-Ami about 2.5-fold. Apical application of chymotrypsin had no stimulatory effect on ISC-Ami with or without aldosterone pretreatment. This suggests that in mCCDcl1 cells ENaC is fully activated by endogenous
36 2019 APS ALDOSTERONE AND ENAC IN HEALTH AND DISEASE: THE KIDNEY AND BEYOND ABSTRACTS OF INVITED AND VOLUNTEERED PRESENTATIONS proteases in the presence and absence of studied in detail. To address this question, we aldosterone. To inhibit these endogenous generated the mouse model proteases, we used an intracellularly acting AQP2cre:Kcnj10flox/flox with a deletion of convertase inhibitor (furin inhibitor-1) and an Kcnj10 specifically in the CS (CS-Kcnj10-KO). CS- extracellularly acting serine protease inhibitor Kcnj10-KO mice responded normally to standard (nafamostat). Pretreatment of mCCDcl1 cells with and high K+ diet. However, CS-Kcnj10-KO furin inhibitor-1 in combination with nafamostat exhibited a higher kaliuresis and lower plasma essentially prevented ENaC stimulation by K+ than control mice when treated with thiazide aldosterone. Subsequent application of diuretics. Likewise, CS-Kcnj10-KO displayed an chymotrypsin rapidly and completely rescued inadequately high kaliuresis and renal Na+ the stimulatory effect of aldosterone in mCCDcl1 retention upon dietary K+ restriction. In this cells treated with protease inhibitors. We condition, CS-Kcnj10-KO mice became conclude that in CCD cells stimulation of ENaC hypokalemic due to an insufficient activity by aldosterone requires proteolytic downregulation of the epithelial Na+ channel channel activation by endogenous proteases. (ENaC) and the renal outer medullary K+ channel Interestingly, ENaC activity in DCT2/CNT is (ROMK) in the CS. Consistently, the phenotype aldosterone-independent, but largely reduced in of CS-Kcnj10-KO was ameliorated by either the absence of MR. Further studies are needed pharmacological inhibition of ENaC or by to elucidate the complex regulatory interplay of genetic inactivation of ROMK in the CS. In aldosterone and proteases to modify renal conclusion, KCNJ10 in the CS contributes to the ENaC activity in a site-specific manner. [1] renal control of K+ homeostasis by regulating Nesterov et al. 2012, Am J Physiol 303:F1289– ENaC and ROMK. Impaired KCNJ10 function in F1299 [2] Nesterov et al. 2016, Hypertension the CS predisposes for thiazide- and low K+ diet- 67:1256-62 [3] Canonica et al. 2016, Pflügers induced hypokalemia and likely contributes to Arch 468:895–908 [4] Loffing and Korbmacher. the pathophysiology of renal K+ loss in 2009, Pflügers Arch 458:111-35 [5] Mansley et al. EAST/SeSAME syndrome. 2018, Pflügers Arch 470: 295-304 Funded by the Deutsche Forschungsgemeinschaft (DFG, 5.4 German Research Foundation) with project The hypertension pandemic: an evolutionary numbers 387509280 – SFB 1350 and KO perspective 1057/10-1 Bernard Rossier1 1Pharmacology and Toxicology, Univ. of 5.3 Lausanne Potassium sensing in the distal nephron The hypertension pandemic has become the David Penton Ribas1, Twinkle Vohra1, Eszter most critical and expensive public health Banki1, Agnieszka Wengi1, Maria Weigert2, Anna- problem. How and why a pandemic? Recent Lena Forst2, Sascha Bandulik2, Richard Warth2, progresses in genetics and population genetics, Johannes Loffing1 genomics, comparative genomics helped by 1Institute of Anatomy, Univ. of Zurich; 2Medical new sequencing methods and bioinformatics Cell Biology, Univ. of Regensburg tools pave the way to a better understanding of The basolateral K+ channel KCNJ10, is hypertension using a evolutionary based expressed in the renal distal convoluted tubule approach. I will discuss the importance of the (DCT) and controls the activity of the thiazide- renin-angiotensin-aldosterone system (RAAS) for sensitive NaCl cotransporter (NCC). Loss-of- the control of blood pressure, focusing on the function mutations of KCNJ10 cause evolution of the system and its critical EAST/SeSAME syndrome with salt wasting and importance for adaptation of vertebrates to a hypokalemia. KCNJ10 is also expressed in the terrestrial and dry environment. The evolution of principal cells of the collecting system (CS); blood pressure control during the evolution of however, its role in this segment has not been primates, hominins, and humans will be
37 2019 APS ALDOSTERONE AND ENAC IN HEALTH AND DISEASE: THE KIDNEY AND BEYOND ABSTRACTS OF INVITED AND VOLUNTEERED PRESENTATIONS discussed, together with the role of common of the subunits, contributing to retrieval and/or genetic factors and the possible causes of the disposal of channels expressed at the cell current hypertension pandemic in the light of surface. The enhanced ubiquitination may evolutionary medicine. reflect increased amounts of fully processed ENaC under these conditions resulting from 6: POSTER SESSION 1 stimulation of forward ENaC trafficking by elevated aldosterone. It does not support the 6.1 idea that diminished ubiquitination is a major Ubiquitination of ENaC subunits in vivo factor in hormonal regulation of the channel. Lawrence Palmer1, Gustavo Frindt1, Lei Yang1 Supported by NIH grant DK-5RO1-DK111380 1Physiology and Biophysics, Weill-Cornell Medical College 6.2 Ubiquitination is thought to control ENaC Modulation of ENaC mediated sodium transport trafficking in renal epithelial cells and may by the basolateral Kir4.1/Kir5.1 channels mediate hormonal regulation of the channels. Oleg Palygin1,2, Elena Isaeva1, Alexey Shalygin 1, We assessed ENaC subunit ubiquitination (ub- Ruslan Bohovyk1, Mykhailo Fedoriuk1, Christine ENaC) in rat kidney using affinity beads Klemens1,2, Ashraf El-Meanawy2,3, Jerod Denton4, (Cytoskeleton Inc) to bind ubiquitinated proteins. Alexander Staruschenko1,2 Kidneys were homogenized in 0.5M urea and 1Physiology, Medical College of Wisconsin; 0.5% SDS to solubilize membrane proteins. 2Cardiovascular Center, Medical College of Ubiquitinated proteins were captured and Wisconsin; 3Medicine, Medical College of subsequently eluted from the affinity beads, Wisconsin; 4Anesthesiology and Pharmacology, separated using SDS-PAGE and assessed with Vanderbilt Univ. Medical Center anti-ENaC antibodies. Ub-αENaC was observed Despite highly relevant clinical and translational as a series of proteins of apparent MW 40-70 evidence supporting benefits of high-potassium kDa, consistent with addition of variable diet, there is a substantial lag in our numbers of ubiquitin molecules to the N- understanding of underlying molecular terminal cleaved fragment (30 kDa) of the mechanisms. Furthermore, there is a critical subunit. No ubiquinated form of the full-length need in the identification of pharmacological protein was observed. Ub-γENaC subunit was tools for manipulating potassium homeostasis. also detected. This species ran at the same Inwardly rectifying (Kir) channels, specifically apparent MW as the fully cleaved C-terminal Kir4.1 and Kir5.1 (encoded by Kcnj10 and Kcnj16 fragment of the subunit (~60 kDa). This suggests genes, respectively) play a dominant role in that the N-terminus of γENaC is ubiquitinated determining blood potassium level and serve as and remains attached to the C-terminal moiety potassium sensors in the distal nephron. In until treatment with reducing agent. No addition to Na+-Cl− cotransporter (NCC) in the ubiquitinated form of the full-length protein was distal convoluted tubules (DCT), Kir4.1/Kir5.1 observed. No significant Ub-β ENaC was channels might control Na+ transport through detected. No protein was detected with control the epithelial Na+ channel (ENaC) in the cortical beads coated with probes lacking the affinity collecting ducts (CCD) and potentially serve as a site. These results are consistent with in vitro new compelling pharmacological target for the data indicating ubiquitination of the α and γ but blood pressure management. Here we have not β subunits (Staub et al EMBO J 16:6325, tested several potential Kir inhibitors on the 1997). To see how ubiquitination changed with activity of Kir4.1/Kir5.1 and ENaC channels. hormone status we fed rats either control or low- Single channel analysis of either Kir4.1 or Na diet for 7 days prior to kidney harvest. Na Kir4.1/Kir5.1 overexpressed in CHO cells depletion increased the amount of ub- αENaC revealed that Kir4.1 channel shows a high and ub-γENaC (by 3-5 fold). This suggests that sensitivity to fluoxetine (100 μM) with no or ubiquitination occurs after complete processing moderate block by amitriptyline (100 μM). In
38 2019 APS ALDOSTERONE AND ENAC IN HEALTH AND DISEASE: THE KIDNEY AND BEYOND ABSTRACTS OF INVITED AND VOLUNTEERED PRESENTATIONS contrast, amitriptyline inhibits Kir4.1/Kir5.1 6.3 heteromeric channel activity with no response to Regulation of ENaC by paraoxonase 3 fluoxetine, as we and others previously have Shujie Shi1, Nicolas Montalbetti1, Xueqi Wang1, shown. Further, to test native properties of Stephanie Mutchler1, Allison Marciszyn1, Marcelo basolateral channels in the kidney, we isolated Carattino1,2, Thomas Kleyman1,2,3 rat (N=5) and human (N=4) CCDs for the 1Medicine, Univ. of Pittsburgh; 2Dept. of Cell electrophysiological assessment of the Biology, Univ. of Pittsburgh; 3Dept. of basolateral potassium activity and Pharmacology and Chemical Biology, Univ. of pharmacological sensitivity. Our experiments Pittsburgh revealed the presence of Kir4.1/Kir5.1 channels The epithelial sodium channel (ENaC) mediates (48 and 54 pS for rat and human, respectively) the rate-limiting step of Na+ uptake across the and pharmacological blockade of single apical membrane of specific epithelia. ENaC- channels activity by amitriptyline. Moreover, dependent Na+ absorption in the kidney has using recently described inhibitor VU0134992 important roles in regulating extracellular fluid (Kharade et al., Mol Pharmacol, 2018; IC50 are volume, extracellular [K+] and blood pressure. 0.97 and 9 μM for Kir4.1 or Kir4.1/Kir5.1, ENaC functional expression is tightly regulated respectively), we demonstrated that potassium by multiple intracellular and exogenous factors, conductance in the basolateral membrane of including molecular chaperones that are CCD is predominantly mediated by the implicated in key steps during ENaC biogenesis. activation of Kir4.1/Kir5.1 (blocked by 30 μM We’ve recently shown that ENaC was regulated VU0134992). Furthermore, using short-circuit by paraoxonase 2 (PON2), a mammalian current measurements in polarized epithelial orthologue of the C. elegans MEC-6. MEC-6 is mCCDcl1 cells, we showed that application of an ER-resident chaperone that has an essential amitriptyline (100 μM), but not fluoxetine, role in the proper assembly and surface resulted in the inhibition of ENaC-mediated expression of the touch-sensing MEC-4/MEC-10 sodium current. Control experiments in CHO channel. There are significant sequence and cells overexpressed with ENaC subunits showed structural conservation between MEC-6 and that application of amitriptyline did not cause mammalian PONs. However, whether the any direct modulation on sodium conductance, chaperone function is also conserved within this confirming that the effects of amitriptyline on family remain undetermined. Like PON2, PON3 ENaC are mediated through inhibition of is expressed in principal cells of the distal Kir4.1/Kir5.1 channel. In conclusion, Kir4.1/Kir5.1 is nephron, where ENaC resides. We hypothesize the predominant channel in human, and rat CCD that mammalian PONs function as molecular and its inhibition by amitriptyline or VU0134992 chaperones and PON3 regulates ENaC strongly modulate electrolyte transport and functional expression. We first examined the indirectly block sodium conductance through effect of PON3 KD on endogenous ENaC in apical ENaC channel. Funding: NHLBI R35 mCCD cells, a mouse cortical collecting duct cell HL135749, T32 HL134643 CVC A.O. Smith line. ENaC-mediated Na+ transport was Fellowship and MCW CVC Michael H. Keelan determined with Ussing chambers recording. Research Foundation Grant We observed that PON3 KD led to an enhances Na+ transport in mCCD cells, which was associated with increased ENaC subunits processing. We furtherly examined the mechanism by which PON3 regulates ENaC expression in two heterologous expression systems, fisher rat thyroid (FRT) cells and Xenopus oocytes. When co-expressed in FRT cells, PON3 co-immunoprecipitated with all three ENaC subunits, and the presence of PON3
39 2019 APS ALDOSTERONE AND ENAC IN HEALTH AND DISEASE: THE KIDNEY AND BEYOND ABSTRACTS OF INVITED AND VOLUNTEERED PRESENTATIONS reduced both the whole-cell and surface an ENaC channel activating protease in the abundance of ENaC. In oocytes co-expressing kidney, as the animals do not show significant PON3, ENaC-mediated inward Na+ currents weight loss, indicative for disturbed ENaC were reduced, at least in part due to a reduced regulation. Further experiments are required ENaC surface expression. In addition, and ongoing to determine its role in ENaC chymotrypsin-mediated ENaC activation was not regulation and kidney function. References: 1. altered by PON3 in oocytes, suggesting that Guipponi, M. et al. The transmembrane serine PON3 did not affect the channel open protease (TMPRSS3) mutated in deafness probability. Together, our data suggest that DFNB8/10 activates the epithelial sodium PON3 regulates ENaC expression via affecting channel (ENaC) in vitro. Human Molecular its biogenesis and trafficking. Future studies will Genetics (2002). 2. Andreasen D et al. Activation investigate the physiological roles of PON3 in of Epithelial Sodium Channels by Mouse regulating ENaC activity, blood pressure and Channel Activating Proteases (mCAP) Expressed extracellular [K+] using the Pon3 KO mice. in Xenopus Oocytes Requires Catalytic Activity of mCAP3 and mCAP2 but not mCAP. JASN 6.4 (2006). 3. Tang T et al. A mouse knockout library Is the membrane-bound serine protease for secreted and transmembrane proteins. Nat Tmprss3 implicated in ENaC-mediated sodium Biotechnol (2010). 4. Fasquelle et al. Tmprss3, a absorption? transmembrane serine protease deficient in Deepika Vijayan1, Thibaud Peyrollaz2, Simona human DFNB8/10 deafness, is critical for Frateschi2, Edith Hummler2 cochlear hair cell survival at the onset of 1Dept. of Pharmacology and Toxicology, Univ. of hearing. J Biol Chem (2011). Lausanne; 2Dept. of Pharmacology and Toxicology, Univ. of Lausanne 6.5 Epithelial sodium channel (ENaC) plays a critical Regulation of endothelial sodium channel role in the maintenance of sodium homeostasis (EnNaC) by mTOR and SGK-1 signaling and regulating blood pressure. Previously, ENaC Michael Hill1,2, Yan Yang1, Guanghong Jia3, was shown to be activated and regulated by Annayya Aroor3, James Sowers1,3 channel activating proteases (CAPs) like e.g., 1Dalton Cardiovascular Research Center, Univ. Tmprss3 in Xenopus laevis oocytes (Andreasen of Missouri; 2Medical Pharmacology and D. et al, 2006, Guipponi M. et al, 2002). In Physiology, Univ. of Missouri; 3Diabetes and human, autosomal recessive missense Cardiovascular Center, Univ. of Missouri mutations in the TMPRSS3 gene cause non- Published studies provide strong support for a syndromic deafness characterized by hearing significant role for EnNaC in the increased loss. These variants failed to undergo proteolytic vascular and cardiac stiffness that is associated cleavage and activate ENaC in-vitro (Guipponi with endothelial cell mineralocorticoid receptor M. et al, 2002, Fasquelle et al., 2011). In mice, (ECMR) activation in pathophysiological states Tmprss3 mRNA transcript expression was such as diet induced-obesity and aldosterone detected in the whole kidney lysate using qPCR excess (Sowers et al., 2019). Currently, our (Guipponi M. et al, 2002) and localized in the mechanistic understanding of the molecular and endoplasmic reticulum membrane compartment cellular pathways regulating ENaC function is (Fasquelle et al., 2011). However, nothing is largely derived from studies of epithelial cells, known about its implication in the ENaC- leaving us to assume that similar mechanisms mediated sodium absorption in the kidney. are present in vascular ECs. Recent studies from Therefore, in-vivo experiments were performed our group show that ECMR activation by a using B6;129S5-Tmprss3tm1Lex/Mmucd KO western diet or aldosterone, stimulates mTOR2 mice model (Tang T et al, 2010). Preliminary data and serum glucocorticoid regulated kinase 1 from KO mice subjected to sodium-deficient diet (SGK-1), pathways known to contribute to do not support the hypothesis that Tmprss3 is membrane localization of ENaC (Gleason et al.,
40 2019 APS ALDOSTERONE AND ENAC IN HEALTH AND DISEASE: THE KIDNEY AND BEYOND ABSTRACTS OF INVITED AND VOLUNTEERED PRESENTATIONS
2015). Thus, using a pharmacological approach 6.6 the present studies examined the role of What is new about ENaC regulation by the mTOR2/SGK-1 signaling in the acute regulation serine protease CAP1/Prss8 in kidney? of lung vascular EnNaC activity, using a whole Asma Mechakra1, Yannick Jaeger2, Chloe Sergi2, cell patch clamp protocol. Cells were freshly Thibaud Peyrollaz2, Anne-Marie Mérillat2, Ivan isolated from mouse lung using a sequential Gautschi2, Stephan Kellenberger2, Simona two-step antibody (anti CD45 and CD31)-bound Frateschi2, Edith Hummler2 magnetic bead technique and cultured for 5-7 1Univ. of Lausanne; 2Pharmacology & days at 37oC, 5%CO2. The presence of Toxicology, Univ. of Lausanne functional EnNaC was verified by a reduction in The membrane-bound serine protease current in the presence of the Na+ channel CAP1/Prss8 (also called prostasin) has been inhibitor, amiloride (1 uM). SGK-1 inhibition with identified as a regulator of ENaC, also called GSK-650394 (20 uM) caused a substantial “channel-activating protease (CAP), thereby decrease (approximately 40% inhibition at -80 revealing a novel autocrine regulation of this mV) in whole cell Na+ current in lung ECs. sodium channel (Vallet et al., Nature, 1997). Further, 30 mins exposure to the mTORC2 Using gene targeting experiments, we inhibitor, PP242 (1 uM), caused a reduction previously showed that CAP1/Prss8 is implicated (approximately 53% inhibition at -80 mV) in Na+ in vivo in ENaC-mediated sodium absorption in current similar to that reported for renal lung (Planes et al., EMM, 2010) and colon epithelial cells (Gleason et al., 2015). In (Malsure et al., JASN, 2014 ). Nothing is known additional studies, aldosterone was found to yet about its function in kidney. We generated increase mTORC2 expression in association renal tubular-specific CAP1/Prss8 knockout mice with decreased levels of miR99a. Further, using the Pax8-rtTA-LC1-Cre system as treatment with a miR99a mimic (20 nM) described by (Traykova-Brauch et al., Nat Med, attenuated the effect of aldosterone on 2008). Ko (CAP1/Prss8PaxLC1) and control mTORC2 expression. Collectively, the data are (CAP1/Prss8Pax8, CAP1/Prss8LC1) mice on consistent with vascular EnNaC conductance sodium restriction were analyzed with respect to being, in part, regulated by mTORC/SGK-1 their physiological and metabolic parameters. signaling. Future studies will be aimed at Surprisingly, preliminary studies show that ko determining the relative contributions of cell mice do not show any salt-wasting phenotype, surface expression and channel activity. indicative for a disturbed ENaC activation by (Support provided by NHLBI and VA) References CAP1/Prss8. We are currently studying whether Jia, G. et al. Epithelial sodium channel in compensatory mechanisms take place, like e.g., aldosterone-induced endothelium stiffness and through upregulation of other serine proteases aortic dysfunction. Hypertension 72:731-738, (CAPs), or through other sodium transporting 2018. Sowers, J.R. et al. Epithelial sodium systems). channels in endothelial cells mediate diet- induced endothelium stiffness and impaired 6.7 vascular relaxation in obese female mice. Renal denervation improves sodium excretion in Metabolism 2019. Gleason, C.E. et al., mTORC2 rats with chronic heart failure: Effects on ENaC regulates renal tubule sodium uptake by and angiotensin II-aldosterone actions promoting ENaC activity. J. Clin. Invest. 125:117- Hong Zheng1, Xuefei Liu1, Kenichi Katsuada2, 128, 2015. Kaushik Patel2 1Basic Biomedical Sciences, Univ. of South Dakota; 2Cellular and Integrative Physiology, Univ. of Nebraska Medical Center Previously we have shown that increased expression of renal epithelial sodium channels (ENaC) may contribute to the renal sodium and
41 2019 APS ALDOSTERONE AND ENAC IN HEALTH AND DISEASE: THE KIDNEY AND BEYOND ABSTRACTS OF INVITED AND VOLUNTEERED PRESENTATIONS water retention observed during chronic heart 6.9 failure (CHF). The goal of this study was to Sprinonolactone treatment reverses arterial examine whether renal denervation (RDN) ENaC expression and improves arterial reduced Na+ retention through modulation of responsiveness in hypertensive Cyp1a1-Ren2 angiotensin II (ANG II)-aldosterone (ALDO) rats actions on renal ENaC in rats with CHF. CHF Martin Fronius1, Sama Mugloo1, Zoe Ashley1, Ivan was produced by left coronary artery ligation in Sammut2, Catherine Leader3, Rob Walker3, Fiona rats. Four weeks after ligation surgery, surgical McDonald1 bilateral RDN were performed. Seven days after 1Physiology, Univ. of Otago; 2Pharmacology, RDN, renal norepinephrine (NE), ANG II and Univ. of Otago; 3Medicine, Univ. of Otago ALDO were measured by ELISA using renal Background: Vascular ENaC has been tissue from four groups of rats (Sham, implicated in mediating endothelium-dependent Sham+RDN, CHF, CHF+RDN, n=6/group). Renal vasodilation and myogenic vasoconstriction. The ANG II AT1 receptors and ALDO receptors from role of vascular ENaC in hypertension and renal cortex and medulla were also measured whether it is a suitable target for by immunoblotting (n=4/group). We found that; 1) antihypertensive medication remains elusive. RDN reduced the renal NE concentration to We hypothesized that elevated vascular ENaC undetectable in both Sham and CHF rats, expression is associated with hypertension and confirming RDN; 2) RDN significantly decreased a potential target for mineralocorticoid receptor ANG II levels but did not change ALDO levels in antagonism. Material and Methods: The study the renal cortex and medulla of rats with CHF; 3) was approved by the Univ.’s animal ethics RDN reduced renal ANG II AT1 receptor committee. Male Cyp1a1-Ren2 rats were either expressions but not ALDO receptor expressions kept on normal diet (normotensive) or an indole- in CHF rats. We also examine renal 3-carbinol substituted diet (hypertensive). After adrenoceptors (alpha1, alpha2, beta1, beta3) two weeks on I3C diet or normal diet, animals protein expressions in renal cortex and medulla were also treated orally with 4.41 mg/day in the same four groups of rats. The results spironolactone (4 weeks). Conduit and showed that RDN reduced renal adrenoceptor resistance arteries were collected from these protein levels in CHF rats, particular for alpha1 animals. Arteries were used for analysing ENaC and beta1 adrenoceptors. As a functional test for subunit expression (mRNA and protein) and ENaC activation, diuretic and natriuretic pressure myography (mesenteric). Results: The responses to ENaC inhibitor Benzamil were mRNA and protein level of α ENaC was monitored in the same four groups of rats. There upregulated in hypertensive aorta, carotid, were significant increases in diuretic and femoral and mesenteric arteries. Amiloride natriuretic responses to ENaC inhibitor Benzamil increased acetylcholine (ACh)-induced in rats with CHF. RDN significantly reduced the vasodilation in normotensive carotid arteries but diuretic and natriuretic responses to Benzamil in did not change the ACh response in CHF rats. These results suggest that adrenergic hypertensive carotid arteries indicating a loss of and ANG II/ALDO systems are activated during ENaC function in hypertension. In contrast, in CHF and that RDN appears to reduce Na+ hypertensive mesenteric arteries amiloride retention through modulation of ANG II/ALDO elevated the ACh-response in hypertension actions on ENaC in the renal tubules of rats with indicating an elevated ENaC activity. CHF. (This work was supported by National Surprisingly, spironolactone did not change Institutes of Health grants 1R01DK114663.) systolic arterial blood pressure in hypertensive animals. However, spironolactone normalized 6.8 ENaC expression in all four types of arteries Withdrawn from hypertensive animals and improved ACh- mediated vasodilation in mesenteric arteries. Conclusion: The study identifies increased ENaC
42 2019 APS ALDOSTERONE AND ENAC IN HEALTH AND DISEASE: THE KIDNEY AND BEYOND ABSTRACTS OF INVITED AND VOLUNTEERED PRESENTATIONS expression as a contributing factor for impaired females compared to males. Thus, CaSR vaso-reactivity in this rat model of hypertension. signaling is linked to RAAS to control In addition, spironolactone treatment of hypertension. hypertensive rats normalized ENaC expression and ACh-mediated responses were similar to 6.11 the responses observed in arteries from Aldosterone-independent regulation of ENaC normotensive animals. This indicates that and Na and K excretion vascular ENaC is as a promising target for the Lei Yang1, Gustavo Frindt 1, Lawrence Palmer1 treatment of hypertension. 1Physiology and Biophysics, Weill-Cornell Medical College 6.10 We investigated the regulation of ENaC under High-salt diet and aldosterone: focus on CaSR- conditions of dietary Na depletion and K loading RAS on plasma and urinary outcomes in salt- in mice lacking the gene for aldosterone sensitive rats synthase (AS) and the ability to produce Charles Okechukwu1, Emmanuel Awumey1 aldosterone. We used amiloride-sensitive whole- 1Biological and Biomedical Sciences, North cell currents (INa) in principal cells of the cortical Carolina Central Univ. collecting duct and proteolytic cleavage of the The CaSR regulates Ca2+-mediated release of γENaC subunit in whole kidneys to assess the parathyroid hormone (PTH) from parathyroid activation of ENaC. Under control conditions no cells by sensing small changes in serum INa could be detected and a minimal amount of Ca2+concentration. The receptor is also cleaved γENaC was observed in both wild-type involved in the regulation of cellular Ca2+ levels (AS+/+) and knockout (AS-/-) animals. After 24 independent of PTH secretion. The renin- hours on a low-Na diet, the amount of cleaved angiotensin-aldosterone system (RAAS) plays γENaC increased in AS+/+ (1.7 ± 0.2 fold) but not vital roles in the development of hypertension, AS-/- animals (1.0 ± 0.2 fold). Under these which is associated with vascular and renal conditions urinary Na excretion was higher in dysfunction, and treatment is difficult because AS-/- (62 ± 12 nmoles/min) compared with AS+/+ the mechanisms are not fully understood. (11 ± 2 nmoles/min) mice. After 7 days of Na Studies show that adequate Ca2+ intake (1.0 to depletion INa was significantly higher in AS+/+ 1.5 g/day) is critical for optimal BP regulation, (270 ± 70 pA) than in AS-/- (54 ± 18 pA) animals, and randomized controlled trials have revealed and the amount of cleaved γENaC was also significant reductions in hypertension risk and significantly higher only in WT mice (2.3 ± 0.5 BP levels in humans. In the kidney, the CaSR is fold). Both genotypes had similar reductions in expressed along the nephron and plays an Na excretion at this time. However in the AS-/- important role, along with the Na+ /Ca2+ mice this was accompanied by a large reduction exchanger (NCX), in the regulation of tubular in GFR (AS+/+: 177 ± 5 ml/min; AS-/-: 51 ± 5 Ca2+, Na+, and water reabsorption. In the ml/min). These animals were able to completely present study, we analyzed the effects of a high conserve Na but at the expense of reduced salt diet on plasma aldosterone, sex hormones, glomerular filtration. Animals fed a high-K diet expression of the CaSR and its downstream (3% K) for 1 week lost weight and had only a signaling complements as well as RAAS proteins moderately increased rate of K excretion. We in male and female Dahl salt-sensitive (SS) rats. therefore assessed responses to an acute Our data show statistically significant decreases dietary K load. Animals were habituated to a the in both plasma and urinary aldosterone, renin, high-K diet for 48 hours, fasted overnight and and estradiol in SS females than males fed high then re-fed with either a high-K or a low-K diet salt diets. Furthermore, the high salt diet for 5 hours. AS-/- and AS+/+ mice ate increased the expression of NCX1 and CaSR and comparable amounts of high-K food and decreased its downstream signaling proteins excreted comparable amounts of K during the (PKCa, GRK2) as well as potassium in SS refeeding period. However plasma K was
43 2019 APS ALDOSTERONE AND ENAC IN HEALTH AND DISEASE: THE KIDNEY AND BEYOND ABSTRACTS OF INVITED AND VOLUNTEERED PRESENTATIONS substantially higher in AS-/- (6.5 ± 0.7 mM) Glomerular filtration rate was measured from compared with AS+/+ (3.5 ± 0.5 mM). The creatinine clearance and fractional sodium and amount of cleaved γENaC was elevated in both potassium excretion (FKE) were estimated from genotypes, although the increase was smaller in the sodium and potassium measures from urine the knockouts (AS+/+: 2.9 ± 0.3 fold; AS-/- : 2.1± and serum. Results: The animals of both groups 0.2 fold). These results suggest that elevated developed an intense hyperkalemia 3 hours aldosterone secretion facilitates excretion of a K after KCl administration (KCl + VEH = 8,1mM [K+]; load in WT mice, at least in part through KCl + SL = 8,4mM [K+]; ns). These K+ levels processing and activation of ENaC. In the reduced equally in both groups at the 6th hour absence of aldosterone, ENaC can still be period (KCl + VEH = 5,3mM [K+]; KCl + SL = upregulated by through a mechanism that 5,5mM [K+]; ns). However, in the 9th and 12th requires much higher levels of extracellular K. hour, K+ remained higher in SL treated group (KCl + VEH = 4,5mM [K+]; KCl + SL = 5,4mM [K+]; 6.12 p<0,05, at 9th hour and KCl + VEH = 4,0mM [K+]; Mineralocorticoid receptor antagonism in acute KCl + SL = 4,8mM [K+]; p<0,05, at 12th hour). hyperkalemia: is aldosterone a must-have? Aldosterone followed a similar pattern, Frederico Fazan1, Eduardo Colombari2 increasing in the SL treated rats at 9th and 12th 1Physiology, Federal Univ. of São Paulo; hour after KCl administration (KCl + VEH = 2Pathology and Physiology, UNESP/FOAr 126mg/dL [Aldo]; KCl + SL = 366mg/dL [Aldo]; Introduction: Hyperkalemia is one of the most p<0,05, at 9th hour and KCl + VEH = 42mg/dL potent ways to increase aldosterone levels in [Aldo]; KCl + SL = 423mg/dL [Aldo]; p<0,05, at the blood. A large number of scientific evidence 12th hour). FKE showed strong positive has shown that aldosterone increases K+ correlation with urine output (Pearson excretion by the kidneys and therefore is correlation = 0,99, p<0,001, during all collection essential in potassium homeostasis. The periods). There was no difference in the increase in extracellular K+ also promotes natremia between groups; however, both intense urinary flow that is associated with high groups increased Na+ content in serum after 6th K+ clearance. However, aldosterone is also hour of KCl administration (KCl + VEH or SL = usually associated with Na+ retention and 134,2mM [Na+] at 3rd hour; KCl + VEH or SL = reduced urinary flow. Hence, the real role of 142mM [Na+] at 9th hour, p<0,05). Conclusion: aldosterone in the severe hyperkalemia context The 2,3M KCl gavage promotes rapid is yet to be determined: is high aldosterone a hyperkalemia independent of SL treatment. mere consequence of hyperkalemia or is this However, previous treatment with SL reduced hormone actively fighting against potassium the homeostatic drive for extracellular K+, overload? Objective: To analyze the time- maintaining higher levels in the serum after 6h. dependent role of aldosterone in the acute This can explain why aldosterone levels were hyperkalemia induced by intragastric KCl higher and increased at the later periods of the injection. Methods: Male Holtzman rats weighing protocol. Interestingly, even with SL, K+ was 300-320g received a pretreatment of 80mg.kg-1 eliminated in the urine with a strong correlation s.c. injection of spironolactone (SL) dissolved in with urine output, questioning the role of sunflower seed oil or only oil (VEH). Three hours aldosterone in intense hyperkalemia as serum after pretreatment, all animals received 3,5 mL K+ above 5,5mM promoted no differences of 2.3M KCl solution through gavage. For the among the groups. Finally, the high urinary next 12 hours, three random animals (n=3 in all output possibly increased free water clearance, results) from both groups were placed inside concentrating Na+ in the serum. Further work is metabolic cages for urine analysis from hours 0- needed in order to elucidate the role of 3; 3-6; 6-9; and 9-12 respectively after KCl aldosterone hyperkalemia. Financial aid: administration. After the urine collection, the FAPESP (2018/02194-0), CNPq animals were anesthetized for blood analysis.
44 2019 APS ALDOSTERONE AND ENAC IN HEALTH AND DISEASE: THE KIDNEY AND BEYOND ABSTRACTS OF INVITED AND VOLUNTEERED PRESENTATIONS
6.13 improved, joints tophacae decreased in size, Probiotics effectively restore the function and normalized creatinine levels (under 90 μmol/L) structure of damaged kidney in gout and uric acid levels (under 250 μmol/L) in all Rostyslav Bubnov1, Mykola Spivak1 patient after focused probiotic administation. 1Interferon, Zabolotny Institute of Microbiology Most signs of gouty nephropathy improved, and Virology, National Academy of Sciences of namely increasing in kidneys size and Ukraine parenchyma, decreased IR under 0.7, decreased Introduction Gout is extremely spread pathology petrifications, small cystic lesions; fibrotic involving kidney, liver and joints, is met more changes retained in parenchyma after short- often in men. Gout has deep interplay with term treatment. In 6 patients microsplenia was metabolic syndrome (MetS), including obesity detected, size improved to norm after treatment. and leads to chronic renal failure in every fourth Conclusion Short-term individualized probiotic patient. Sonography can clearly determine therapy is effective to treat the signs of MetS symptoms of gouty nephropathy also at early and hyperuricemia and can successfully restore stages, can be used as main method for the function and structure of damaged kidney in screening and monitoriung treatment. gout. Modulating gut microbiome has great potential to improve metabolic health in gout. The Aim: 6.14 was to study the efficacy of individualized Sex-dependent protection from high fat diet- probiotic intervention on signs of gout and MetS induced metabolic disease in mice lacking by monitoring sonographic diagnostic markers normal levels of TrpC6 and/or ASIC2 and β of gouty nephropathy. Material and methods We ENaC proteins included twelve patients (age 32-67 years) with Heather Drummond1, Zachary Mitchell1, Emily BMI>30, waist circumference (WC)>110, who met Hildebrandt1, David Stec1 criteria of MetS with hyperuricemia (the levelsof 1Physiology and Biophysics, Univ. of Mississippi uric acid over 400 μmol/L) and increased blood Medical Center preassure. All patients underwent extensive Degenerin proteins (β ENaC and ASIC2) and general clinical, lab tests; multiparameter TrpC6 have been implicated in cardiovascular ultrasound (US) of liver, kidneys, joints, function. However, their roles and potential measuring visceral fat (FV). The typical interaction in metabolic disease has not been symptoms of gouty nephropathy in sonographic studied. To begin to assess this interaction, we display were documented. Patients were given evaluated the impact of a high fat diet (HFD) in probiotics (B. animalis VKB / B. animalis VKL mice lacking normal levels of TrpC6 (TrpC6-/-) strains at a dose 108 CFU daily during 10 days); and/or ASIC2 (ASIC2-/-) and β ENaC (β in cases of liver fibrosis - L. delbrueckii subsp. ENaCm/m) on a mixed genetic background. bulgaricus IMV B-7281, B. ?nimalis VKB, L. casei Twenty week old male and female mice were IMV B-7280 (considered individually according placed on a 60% HFD for 12 weeks. Body weight to the knowledge on treatment mechanics was measured weekly and body composition obtained from in vivo in vitro studies and (non-invasive ECHO MRI) and fasting blood existing evidence). Results. We registered glucose were measured at 0, 4, 8 and 12 weeks. ultrasound signs of symptoms of gouty Data are presented as mean ± SEM; order of nephropathy in all patients, namely detection of data presentation is ASIC2-/-/β ENaCm/m, small hyperechoic inclusions in parenchyma ASIC2-/-/β ENaCm/m/TrpC6-/- vs wildtype (WT). (chalk-stone), increase in resistive index (RI) in At 20 weeks of age, female ASIC2-/-/β segmental vessels over 0.7, thinning of ENaCm/m (n=9), but not ASIC2-/-/β parenchyma (less than 13 mm), fibrotic changes ENaCm/m/TrpC6-/- (n=12) mice, weighed less in parenchyma, hilly kidney margins, anaechoic than WT (n=9)(22.3± 0.5, 23.7± 0.8 vs 25.5± 0.6 strips under the capsule. Weight, BMI, WC and g). Both ASIC2-/-/β ENaCm/m and ASIC2-/-/β VF decreased, liver structure, blood pressure ENaCm/m/TrpC6-/-gained less weight than WT
45 2019 APS ALDOSTERONE AND ENAC IN HEALTH AND DISEASE: THE KIDNEY AND BEYOND ABSTRACTS OF INVITED AND VOLUNTEERED PRESENTATIONS after 12 weeks HFD (13.4± 0.8, 12.8± 1.5 vs. 20.8± 6.15 0.8 g). Total body fat (16.2± 0.5, 17.8± 1.2 vs Effects of dietary potassium restriction and K+ 23.5± 0.7 g) and lean body masses (20.2± .04, loading on blood pressure and renal tubular Na+ 19.5± 1.3 vs 24.1± 0.6 g) were reduced in female transport ASIC2-/-/β ENaCm/mand ASIC2-/-/β Cary Boyd-Shiwarski1, Rebecca Beacham1, Claire ENaCm/m/TrpC6-/-mice. In contrast, male Weaver1, Daniel Shiwarski2, Kelly Connolly1, ASIC2-/-/β ENaCm/m(n=10), but not ASIC2-/-/β Allison Marciszyn1, Arohan Subramanya1 ENaCm/m/TrpC6-/-(n=6), mice had lower body 1Dept. of Medicine, Renal/Electrolyte Division, weight compared to WT (n=11) at 20 weeks Univ. of Pittsburgh; 2Dept. of Biomedical (30.5± 0.4, 34.1± 0.8 vs. 34.4± 1.1 g). Body weight Engineering, Carnegie Mellon Univ. gain in male ASIC2/β ENaCm/m and ASIC2-/-/β It is well documented that dietary potassium ENaCm/m/TrpC6-/- mice were not quite different intake inversely correlates with blood pressure. than WT (11.7± 1.6, 12.4± 1.5, vs. 16.1± 1.5 g). Male Yet, it is unclear how potassium restriction leads total body fat (14.6± 1.1, 16.8± 1.0 vs. 18.6± 0.8 g) to hypertension, or how potassium excess and lean body masses (28.5± 0.7, 29.7± 0.6 vs. causes a natriuresis despite elevated 32.5± 0.5 g) were less in ASIC2-/-/β aldosterone levels. Our goal was to study the ENaCm/mand ASIC2-/-/β ENaCm/m/TrpC6-/- effects of dietary potassium on blood pressure, compared to WT. Fasting blood glucoses were acid/base balance, and ion transport in wild-type also lower in female (154± 4, 172± 6 vs. 201± 6 SV129 mice. These mice were fed either 1) K+ mg/dL) and male (147± 25, 181± 3 vs. 228± 11 deficient, 2) control, 3) high K+ basic, or 4) high mg/dL) ASIC2-/-/β ENaCm/mand ASIC2-/-/β KCl diets for 10 days. We monitored BP using ENaCm/m/TrpC6-/-mice compared to WT. In radiotelemetry probes, urine electrolyte females, liver (0.94± 0.04, 0.85± 0.04 vs. 2.18± excretion via metabolic cages, and transporter 0.37 g) and liver fat masses (0.035± 0.009, expression via immunofluorescence, western 0.016± 0.007 vs.0.341± .109 g), as well as blots, and diuretic challenges. Interestingly, percent liver fat (3.5± 0.7, 1.5± 0.9 vs. 13.8± despite the induction of hypokalemia, extreme 2.5%), were reduced in ASIC2-/-/β ENaCm/m K+ depletion had no effect on blood pressure. In and ASIC2-/-/β ENaCm/m/TrpC6-/-mice after contrast, K+ loading augmented blood pressure HFD. In males, liver mass was lower in ASIC2-/- by ~10mmHg. To determine whether these /β ENaCm/m and ASIC2-/-/β ENaCm/m/TrpC6-/- unexpected effects were dependent on NaCl mice (1.67± 0.14, 1.77± 0.13 vs. 2.48± 0.17 g). intake, we challenged mice with 1% saline. The However, liver fat mass (0.23± 0.08, 0.17± .06 vs. K+ deficient mice developed an increase in 0.46± .06 g) and percent liver fat were only blood pressure (~8 mmHg), whereas K+ replete lower in ASIC2-/-/β ENaCm/m/TrpC6-/-mice (11.1± mice exhibited no significant change in blood 3.4, 8.8± 2.6 vs. 17.6± 1.6%). These highly novel pressure with saline challenge. Notably, just 10d findings suggest that ASIC2, β ENaC and/or their of K+ restriction was associated with diabetes interaction with TrpC6, robustly protects against insipidus, evidenced by polyuria and a decrease HFD induced-metabolic disease in female mice. in AQP2 expression. This was associated with The mechanisms underlying this response will an increase in sodium transporters in the be examined in future studies. This work was upstream tubule, likely the cause of salt supported by NIH P01HL051971, P20GM104357, sensitivity. The elevated blood pressure on the P20GM121334 and R01HL136684 sources. K+ loaded diet correlated with elevated aldosterone levels and increased ENaC activation. During K+ loading, the type of anion (basic vs. chloride-rich) had a considerable effect on key transporters along the tubule, despite no differences on blood pressure. In our model, the effect of dietary K+ on blood pressure was coupled to NaCl intake, and the
46 2019 APS ALDOSTERONE AND ENAC IN HEALTH AND DISEASE: THE KIDNEY AND BEYOND ABSTRACTS OF INVITED AND VOLUNTEERED PRESENTATIONS inverse relationship between dietary K+ intake ENaC likewise reduce PIP2-dependent Na+ and blood pressure was only observed in the entry into cells transfected with ENaC. These setting of NaCl loading. Our data strongly results are consistent with PIP2 binding to ENaC suggest that AQP2 expression should be closely playing a structural role in stabilizing the open monitored to accurately interpret such results. conformation of channel via moderate and weak Further, the accompanying anionic content electrostatic interactions within the cytoplasmic should be taken into consideration when termini. This work is supported by NIH/HHLBI modeling the physiologic effect of K+ intake on T32 HL07446 and NIH/NIDDK R01 DK113816 to tubular salt transport and blood pressure. JDS.
6.16 6.17
PIP2 regulates ENaC through multiple Effects of dietary habits on hypertension among electrostatic interactions medical students Crystal Archer1, Chase Carver1, Benjamin Suyog Mhamankar1 Enslow1, Yinghua Chen2, James Stockand1 1Biochemistry, Spartan Health Science Univ. 1Cellular and Integrative Physiology, UT Health Hypertension is one of the leading public health San Antonio; 2Biophysics and Physiology, Case problems in most countries. This study was Western Reserve Univ. conducted to find out how dietary patterns can Phosphatidylinositol 4,5-bisphosphate (PIP2) affect or cause hypertension and its prevalence regulates the activity and gating of many among the population on medical school different ion channels to include the epithelial campus. The cross-sectional study involved a Na+ channel (ENaC). To date, understanding of survey of 90 respondents aged 16 to 57 years the biophysical interaction between ENaC and using the random sampling technique. Google PIP2 has been limited to indirect observation forms were used to collect the data and data using functional assays. The cryoEM structure of analysis was done using Microsoft excel sheets. trimeric ENaC containing α, β and γ subunits The results were based majorly on two factors was recently elucidated. However, the i.e. diet and lifestyle. Considering the analysis on cytoplasmic amino and carboxy termini, which these factors, only 4.5% participants had high are believed to bind PIP2, were not resolved in cholesterol intake, whereas 16.6% participants earlier structural analyses. Thus, structural take heavy amounts of sugars and only 3.3% insight about how intracellular ENaC termini participants had high caffeine intake. Prevalence might interact with PIP2 remains obscure. In this of smoking was 11.1% and heavy alcohol study, we used steady state intrinsic consumption was 4.07%. Contradictory to the fluorescence and microscale thermophoresis to expected results, only 6.7% population was compare the binding affinities of PIP2 and its found to be diagnosed by hypertension while analogues to synthetic peptides corresponding 40-45% of people among the selected to putative PIP2 binding sites within the amino population were at risk of developing and carboxy termini of ENaC. We found that the hypertension. 30.7% population did not have extreme amino terminus of the β ENaC subunit any exercise habits which is a risk factor leading has the strongest electrostatic interaction with to hypertension. the PIP2 headgroup (Kd ~4-10 uM). We also identified two other much weaker PIP2 binding sites within the cytoplasmic termini of γ and β ENaC subunits. Using the CIBN-CRY2 optogenetic dimerization system to modify cellular levels of PIP2, we showed that removal of the 5’ phosphoryl group from PIP2 reduces Na+ entry into cells transfected with ENaC; and that mutation to the PIP2 binding sites in γ and β
47 2019 APS ALDOSTERONE AND ENAC IN HEALTH AND DISEASE: THE KIDNEY AND BEYOND ABSTRACTS OF INVITED AND VOLUNTEERED PRESENTATIONS
6.18 CFTR occurs in the sweat duct, a normally Cl- A mechanism to reduce cysts in autosomal absorbing epithelium. Conclusion: The data dominant polycystic kidney disease by inducing suggest that VX-809 reduces cyst size in the fluid absorption via CFTR, NHE3 and ENaC. PC1-null mice by promoting an absorptive Liudmila Cebotaru1, Muraly Yanda2 phenotype. Given that administration of VX-809 1Medicine, Johns Hopkins Univ.; 2Physiology, is safe, this drug potentially offers a new way to Johns Hopkins Univ. treat patients with ADPKD. Autosomal dominant polycystic kidney disease (ADPKD), caused by malfunction of either PC1 or 7: SYMPOSIUM 3A: SIGNALING AND 2, is associated with progressive enlargement of REGULATION OF ENAC AND OTHER cysts, leading to a decline in function and renal EPITHELIAL CHANNELS/ failure. We demonstrated previously that VX- TRANSPORTERS 809, a CFTR corrector, conventionally used to manage cystic fibrosis, reduces cyst growth in 7.1 mouse models. To address the mechanism of ENaC regulation by the mTORC2-SGK1 signaling how this occurs, we used proximal tubule- module derived, cultured Pkd1-knockout cells and the David Pearce1, Catherine Gleason2, Mads Pkd1fl/fl; Pax8rtTA; TetO-cre mouse model Sørensen3, Bidisha Saha4 which, when treated with doxycycline, allows the 1Medicine, Univ. of California, San Francisco; ablation of PC1 in renal tubular epithelial cells. 2Cell Biology, Circle Pharma; 3Biomedicine, These mice, when injected with doxycycline Physiology, Health, Aarhus Univ.; 4Medicine, develop multiple large cysts. We found that Univ. of California, San Francisco cysts are reduced when the mice are treated Aldosterone and ENaC play central roles in with VX-809 and renal function improved. VX- potassium (K+) homeostasis, and maintaining 809 treatment of cultured Pkd1-knockout cells normal blood K+ concentration by controlling increased by two-fold the activity of the sodium the driving force for K+ secretion in principal proton exchanger, NHE3, (as measured by the cells of the distal nephron. SGK1, a key rate of change in intracellular pH induced by intermediary between aldosterone and ENaC, is changes in extracellular Na+). To study this under dual regulation: the aldosterone-regulated further we assessed the location of NHE3 in the mineralocorticoid receptor stimulates SGK1 gene cystic kidneys using confocal microscopy. In transcription, while the master kinase, mTORC2 mice treated with doxycycline, NHE3 was (the type 2 mTOR complex), phosphorylates present in large cysts but was primarily SGK1 to stimulate its activity. The physiologically colocalized with Rab11, a marker of recycling relevant regulators of mTORC2 remain poorly endosomes. In the mice treated with both characterized. We have found that in principal doxycycline and VX-809, NHE3 now colocalized cells, both K+ itself and angiotensin II act directly with the plasma membrane marker E-cadherin in distal nephron principal cells to activate the consistent with the increase in NHE3 activity. mTORC2-SGK1 signaling module and thereby Similarly, VX-809 also increased the apical stimulate ENaC. The K+ effect requires localization of ENaC. In mice treated with both basolateral Kir4.1 channels (possibly in the form doxycycline and VX-809, CFTR protein levels of Kir4.1/5.1 heteromultimers) but does not increased. When mice were treated with depend on changes in aldosterone, or on doxycycline and large cysts developed CFTR enhanced distal delivery of Na+ from upstream was colocalized with calnexin, the ER marker, nephron segments. The angiotensin II effect is and with E-cadherin but when mice were treated mediated by AT1R and requires PKC activity, with doxycycline and VX-809, surprisingly CFTR which induces a change in subcellular colocalization with the Na+/K+ ATPase), a localization of mTORC2. Mechanisms of marker for the basolateral membrane, increased specificity and differences between angiotensin 2 fold. Interestingly, basolateral localization of II and K+ will be discussed.
48 2019 APS ALDOSTERONE AND ENAC IN HEALTH AND DISEASE: THE KIDNEY AND BEYOND ABSTRACTS OF INVITED AND VOLUNTEERED PRESENTATIONS
7.2 17~92 cluster (specifically miR-19). Upregulation Role of microRNAs in aldosterone signaling and of these miRs after extended aldosterone ENaC regulation stimulation in mCCD cells and in mouse kidney Michael Butterworth1 (24-96hrs) results in reduced expression of the 1Dept. Cell Biology, Univ. of Pittsburgh mineralocorticoid receptor (MR) and the serum MicroRNAs (miRs) are non-coding RNAs and glucocorticoid kinase (SGK1). By chronically between 18-23 nucleotides long and function inhibiting proteins that are needed for primarily to degrade target mRNA and prevent aldosterone signal transduction in the CCD, the protein synthesis. The role of miRs is miRs act as a rheostat to prevent overactivity increasingly being realized in many aspects of that would be associated with extended kidney physiology, from development, ion aldosterone stimulation. By preventing the homeostasis and in disease. Aldosterone upregulation of these miRs we can demonstrate regulates miR expression in the aldosterone- that MR and SGK1 expression is increased and sensitive distal nephron where they act in that the negative feedback by aldosterone- concert with long established protein induced miRs is blocked. A significant increase participants to fine-tune aldosterone signaling. in both ENaC-mediated transport and Aldosterone regulated miRs serve two primary aldosterone sensitivity of the mCCD cells functions. First, miRs can alter the expression of results. The action of the miRs is reminiscent of channels, transporters and associated proteins other physiological pathways and constitutes a to regulate Na+ reabsorption across the distal form of aldosterone escape that prevents nephron epithelial cells. Our previous studies excessive sodium reabsorption. The miR identified 2 regulatory proteins that serve this pathways involved in altering sodium transport purpose. This first involved upregulation of the directly or acting as negative feedback scaffold protein Ankyrin-G (ANK3). ANK3 regulators will be presented. Funding source: facilitated the delivery of ENaC to the apical NIH-NIDDK R01: DK102843 surface of CCD cells to increase Na+ transport. Follow-up studies investigated the miRs that 7.3 were induced by aldosterone and we identified AMPK regulates ENaC via a tripartite inhibitory a cluster of miRs that were increased in complex response to aldosterone stimulation, miRs- Kenneth R. Hallows1, Pei-Yin Ho1, Hui Li1, Lei 23,34,27. These miRs targeted the endocytic Cheng2, Robert A. Fenton2, Vivek Bhalla3 accessory protein intersectin 2 (Itsn2) to inhibit 1Medicine - Nephrology and Hypertension, Univ. ENaC endocytosis. A reduction in Itsn2 of Southern California; 2Biomedicine, Aarhus expression reduced the formation of clathrin Univ.; 3Medicine - Nephrology, Stanford Univ. coated pits that endocytose ENaC resulting in The metabolic sensor AMP-activated protein increased ENaC residency at the apical surface kinase (AMPK) inhibits ENaC, a key regulator of of mouse cortical collecting duct cells (mCCDs). salt reabsorption by the kidney and thus total Both pathways increase ENaC expression at the body volume and blood pressure (1). Earlier work apical surface of CCD cells resulting in demonstrated that AMPK regulates ENaC by increased Na+ transport in line with the promoting the association of the E3 ubiquitin characterized role of aldosterone. The second ligase Nedd4-2 with ENaC with subsequent function of the miRs is to act as local negative ENaC ubiquitination and degradation in various feedback regulators of aldosterone signaling. epithelial tissues (2). Recently, we found that Several miR groups are upregulated by long- AMPK promotes the association of the PAK- term aldosterone stimulation to feedback and interacting exchange factor β 1Pix, 14-3-3 inhibit elements of the renin-angiotensin- proteins, and the ubiquitin ligase Nedd4-2 into a aldosterone signaling cascade (RAAS). In mice complex that inhibits ENaC by enhancing this includes members of the miR-466 family, Nedd4-2 binding to ENaC and ENaC and in mouse and humans it involves the miR- degradation. Functional β 1Pix is required for
49 2019 APS ALDOSTERONE AND ENAC IN HEALTH AND DISEASE: THE KIDNEY AND BEYOND ABSTRACTS OF INVITED AND VOLUNTEERED PRESENTATIONS
ENaC inhibition by AMPK and promotes the Na+channel (ENaC) in blood pressure regulation AMPK-dependent phosphorylation of Nedd4-2 has been well studied, however, we recently at a site critical for Nedd4-2 stability in mouse found that dendritic cells (DCs) in response to kidney cortical collecting duct (CCD) cells (3). increases in extracellular [Na+] exhibit an ENaC- New findings reveal that AMPK also directly dependent activation of NADPH-oxidase, phosphorylates β 1Pix in vitro. Among several superoxide production, isolevuglandin (IsoLG)- AMPK phosphorylation sites on β 1Pix detected protein adduct formation, and cytokine secretion by tandem mass spectrometry, one site was which promote hypertension. In this study, we validated as functionally significant. Compared hypothesized that elevated Na+activates the to wild-type β 1Pix, overexpression of a NLRP3 inflammasome in antigen presenting phosphorylation-deficient β 1Pix mutant cells (APCs) through an ENaC-dependent significantly attenuated ENaC inhibition and the mechanism. To test this hypothesis, we isolated AMPK-activated interaction of both β 1Pix and human monocytes from 11 volunteers and Nedd4-2 to 14-3-3 proteins in CCD cells. cultured them in either normal salt (150mM NaCl) Overexpression of a β 1Pix deletion-tract mutant or high-salt (190 mM NaCl) for 72 hours. Using unable to bind 14-3-3 proteins decreased the high-throughput sequencing of cDNA (RNAseq), interaction between Nedd4-2 and 14-3-3 we found that high-salt treatment markedly proteins, suggesting that 14-3-3 binding to β 1Pix increased inflammasome component NLRP3 is critical for the formation of a β 1Pix/Nedd4- (1759 ± 260.8 vs 2994 ± 487.6 relative 2/14-3-3 complex. With expression of a general expression, p<0.05), pyroptotic and apoptotic peptide inhibitor of 14-3-3-target protein caspases, and pro-inflammatory cytokines, interactions (R18), binding of both β 1Pix and including IL-1β (1144 ± 350.2 vs 4160 ± 785.0 Nedd4-2 to 14-3-3 proteins were reduced, and relative expression, p<0.01) transcription in AMPK-dependent ENaC inhibition was also human-derived monocytes. In additional attenuated. Altogether, our results demonstrate experiments, we cultured mouse splenocytes in the importance of AMPK-mediated normal-salt or high-salt media with or without co- phosphorylation of Nedd4-2 and β 1Pix, which treatment with the ENaC inhibitor, amiloride (20 promotes 14-3-3 interactions with β 1Pix and mM). Using flow cytometry, we found that high- Nedd4-2 to form a tripartite ENaC inhibitory salt increased both monocyte and DC complex, in the mechanism of ENaC regulation production of IL-1β, which was confirmed by AMPK. through an ELISA assay detecting release of IL- 1β (2.131± 0.733 vs 12.75 ± 1.108 pg/mL, 7.4 p<0.0001) into the culture media. Treatment with Elevated sodium activates the NLRP3 amiloride prevented production of IL-1β (12.75 ± inflammasome in antigen presenting cells 1.108 vs 1.905 ± 0.3495 pg/mL, p<0.0001) in through an ENaC-dependent mechanism both monocytes and DCs. Our findings suggest Ashley Pitzer1, Natalia Barbaro1, Annet Kirabo1 a role for ENaC-dependent NLRP3 1Clinical Pharmacology, Vanderbilt Univ. inflammasome activation in APCs in response to Medical Center high-salt diet, which may represent a promising Hypertension is a major public health issue due approach to treatment of salt-induced to the profound risk of developing hypertension. cardiovascular (CVD) diseases, including stroke, heart failure, and kidney disease. Approximately 50% of hypertensive patients are salt-sensitive, and reducing dietary sodium (Na+) decreases both blood pressure and CVD risk. The precise mechanisms of how elevated Na+ leads to hypertension are still not well defined. The role of the renal amiloride-sensitive epithelial
50 2019 APS ALDOSTERONE AND ENAC IN HEALTH AND DISEASE: THE KIDNEY AND BEYOND ABSTRACTS OF INVITED AND VOLUNTEERED PRESENTATIONS
7.5 there were no significant differences in Postprandial effects on ENaC-mediated sodium expression of NCC, NKCC2, or NKA. absorption Additionally, using mass spectrometry-based Christine A. Klemens1, Gregory Blass1, Michael analysis, we interrogated different metabolites W. Brands2, Oleg Palygin1, Alexander of RAAS signaling between the stimulated and Staruschenko1 fasted rats and found no significant changes in 1Physiology, Medical College of Wisconsin; aldosterone or angiotensin metabolite levels. 2Physiology, Augusta Univ. These results demonstrate that acute Recent studies have validated that postprandial hyperinsulinemia following a meal increases increases in insulin directly influence renal Na+ ENaC activity independent of the RAAS transport to reduce urinary Na+ excretion signaling cascade. Our study demonstrates that following a meal. While there are a number of ENaC regulation via insulin is a likely contributor renal transporters that respond to insulin to an evolutionary mechanism for preserving stimulation, which of these transporters are sodium and volume loss after a meal, and that involved in this Na+ sparing mechanism under this regulation is distinct from classical ENaC normal physiological conditions is not fully regulation by RAAS. established. The epithelial sodium channel (ENaC) is the rate limiting step for sodium 8: SYMPOSIUM 3B: SIGNALING AND reabsorption in the distal nephron and its REGULATION OF ENAC AND OTHER regulation by insulin is well established; EPITHELIAL CHANNELS/ however, the regulatory effect of acute TRANSPORTERS II hyperinsulinemia (as would result from a meal) on ENaC under normal conditions was 8.1 previously unknown. This study hypothesized WNK kinases and cell volume regulation that increased ENaC activity in the postprandial Arohan Subramanya1 time frame contributes to Na+ reclamation to 1Medicine, Univ. of Pittsburgh prevent Na+ wasting from the osmotic load of a When challenged by hypertonicity, dehydrated meal. To test this, we gave fasted Sprague cells recover their volume through regulatory Dawley a high carbohydrate supplement to volume increase (RVI). In most cells, this process simulate a meal and analyzed changes to single- is mediated by the bumetanide-sensitive channel ENaC activity and protein expression. cotransporter NKCC1, which is phosphorylated Additionally, we assessed protein expression of and activated by the WNK-SPAK/OSR1 pathway. other renal transporters involved in sodium How cells sense a reduction in volume and homeostasis known to be regulated by insulin transduce the RVI signal despite a strengthened signaling. As ENaC is classically regulated by ionic milieu that favors low WNK activity remains the renin-angiotensin-aldosterone system unclear. Here, I will describe our efforts to (RAAS) hormones, we also interrogated unravel this mechanism using a combination of circulating levels of RAAS metabolites to high-speed live cell imaging, optogenetic, and exclude them as contributing to changes in biochemical approaches in gene-edited cells. ENaC activity. We found that 4 hours post We have found that during cell shrinkage and carbohydrate stimulus, ENaC open probability macromolecular crowding caused by reduced increased relative to controls in split-open cell water, WNK1 dynamically condenses into isolated collecting duct tubules (0.34 ± 0.06 vs. punctate liquid droplets, leading to pathway 0.70 ± 0.07, n≥5, p<0.01), while the number of autoactivation, cytosolic exchange, and NKCC1 channels at the surface and total ENaC α- and β phosphorylation within the RVI time course. The -subunit levels remained unchanged. We also formation of WNK1 condensates was driven by examined expression levels of the Na+-Cl- its large intrinsically disordered C-terminus, cotransporter (NCC), Na+-K+-2Cl- cotransporter whose strong regional disorder tendency was (NKCC2), and Na+-K+-ATPase (NKA); however, highly conserved despite poor sequence
51 2019 APS ALDOSTERONE AND ENAC IN HEALTH AND DISEASE: THE KIDNEY AND BEYOND ABSTRACTS OF INVITED AND VOLUNTEERED PRESENTATIONS homology across evolution. This disorder- SPLUNC1-ENaC interactions in the CF encoded phase behavior allows WNK kinases to environment and (ii) neutrophil-elastase- react to a crowded cytosol, bypass the ionic mediated degradation of SPLUNC1 in CF ASL. strength problem caused by cellular However, therapeutic inhibition of ENaC may dehydration, and rescue cell volume via mass serve to restore ASL hydration and mucociliary action. Thus, our findings indicate that in clearance, potentially reversing and preventing addition to their well-appreciated role as progression of airway obstructive disease in CF. chloride sensors, WNK kinases are physiological We have developed a SPLUNC1 peptidomimetic, crowding sensors that phase separate to called SPX-101, that is resistant to degradation coordinate a conserved hyperosmotic stress by neutrophil elastase. Inhaled SPX-101 does not response. I will wrap up by discussing how cross the epithelial boundary and hence, has crowding-induced phase transitions in the WNK little effect on ENaC in the kidney. Further, this signaling pathway might play a role in DCT peptide increases ASL height in CF airway physiology, where the WNK-SPAK/OSR1 cultures, causes an increase in mucus clearance pathway forms WNK bodies – membraneless rates in sheep treated with CFTR inhibitors and electron-hypodense condensates that amplify prolongs survival in bENaC overexpressing the potassium stress response via KS-WNK1, a mice. In this talk, we shall discuss the functional disordered scaffold for WNK kinases. progression from SPLUNC1 to S18 to SPX-101 , as Funding: NIH R01DK098145, R01DK119252, well as preclinical data in CF patients with Class K08DK118211, P30DK079307 1 CFTR mutations, that are not responsive to Vertex compounds will also be discussed. SPX- 8.2 101 is currently being evaluated in CF patients in From SPLUNC1 to SPX-101, novel ongoing clinical trials. Funded by the CFF, peptidomimetics to treat sodium Emily’s Entourage, the NCBC, the NIH and hyperabsorption in the CF lung Spyryx Biosciences. Robert Tarran1 1Cell Biology & Physiology, Univ. of North 8.3 Carolina Direct effect of potassium on ENaC regulation CF airways are characterized by abnormal and potassium secretion in collecting duct cells: anion/cation transport that promote airway role of mTORC2/SGK1 signaling surface liquid (ASL) dehydration, causing a Bidisha Saha1, Mads Vaarby Sørensen2, Peng detrimental reduction in mucus clearance. Wu3, Catherine E. Gleason1, Wen-Hui Wang3, Impaired mucociliary clearance promotes mucus David Pearce1 plugging, bacterial colonization and chronic 1Dept. of Medicine, Division of Nephrology, Univ. inflammation. Restoration of ASL volume to of California, San Francisco; 2Dept. of physiological levels can be achieved by Biomedicine, Aarhus Univ.; 3Dept. of reducing Na+ absorption via inhibition of the Pharmacology, New York Medical College epithelial Na+ channel (ENaC). We have The balance between potassium (K+) intake and previously shown that the secreted airway excretion is crucial in maintaining electrolyte protein, short palate lung and nasal epithelial homeostasis in mammals. In the principal cells of clone 1 (SPLUNC1), can inhibit ENaC via direct the distal nephron, electrogenic Na+ transport binding of SPLUNC1’s N-terminal S18 region to by the epithelial sodium channel (ENaC) ENaC subunits. SPLUNC1 is an allosteric generates the electrical driving force for K+ inhibitor of ENaC. Indeed, SPLUNC1’s S18 region secretion across the apical membrane. The role binds extracellularly to ENaC leading to rapid, of aldosterone in the control of ENaC regulation NEDD4-2-dependent ubiquitination and has been well studied. Aldosterone stimulates internalization of ENaC. SPLUNC1-mediated the gene transcription of the ENaC-regulatory ENaC inhibition is diminished in CF patients, kinase, SGK1 which subsequently undergoes both due to (i) pH-sensitive inhibition of phosphorylation and activation by mTOR
52 2019 APS ALDOSTERONE AND ENAC IN HEALTH AND DISEASE: THE KIDNEY AND BEYOND ABSTRACTS OF INVITED AND VOLUNTEERED PRESENTATIONS complex 2 (mTORC2). However, the possibilities 8.4 that ENaC can also be regulated in an High mobility group box-1 protein regulates lung aldosterone independent mechanism have been epithelial sodium channel activity via the demonstrated by several studies. In this study, receptor for advanced glycation end products we identified a direct effect of K+ in cortical Garett J. Grant1, Theodore G. Liou1, Robert Paine collecting duct principal cells to stimulate ENaC III1, My N. Helms1 and enhance K+ secretion, and investigated the 1Internal Medicine, Univ. of Utah underlying mechanism. In mice an acute K+ load Background and Significance: The epithelial enhances benzamil-inducible Na+ excretion (a sodium channel (ENaC) plays an important role reflection of ENaC activity) in parallel with in maintaining proper airway hydration and has benzamil suppressible kaliuresis indicating been shown to be hyperactive in the cystic increased K+ excretion prior to significant fibrosis (CF) lung. In CF, ENaC dysfunction leads change in plasma aldosterone level; moreover, to dehydrated airways, chronic inflammation, this early effect on ENaC is mineralocorticoid and a progressive decline in lung function. High receptor (MR) independent. Whole cell patch mobility group box-1 (HMGB-1) is a nuclear and clamp studies in intact, isolated mouse cytoplasmic protein known to play an important collecting duct, and electrophysiological studies role in activating inflammatory responses and in cultured collecting duct cells reveal a rapid has also been shown to be elevated in the CF alteration in ENaC activity in response to lung. Our aim was determine if a causal changes in extracellular [K+] on the blood side of connection exists between elevated HMGB-1 the cells, involving basolateral membrane K+- and hyperactive ENaC in the CF lung. Successful channel activity. The stimulation of ENaC activity completion of our studies can lead to new by increased extracellular K+ is mediated by therapeutic strategies for CF. Hypothesis: We mTORC2/SGK1 signaling pathway. The K+- hypothesize that HMGB-1 signals via the induced increase in SGK1 phosphorylation by receptor for advanced glycation end products mTORC2 is selective and highly dependent on (RAGE) to increase ENaC activity and WNK1, but interestingly, without the requirement inflammation in the airways. Methods and of WNK1 kinase activity, which is distinct from Results: We measured equivalent short circuit the regulation of NCC by WNK1. Taken together, current (Isc) and single channel ENaC open our data strongly support the idea that K+ is probability (Po) in human small airway epithelial sensed locally by principal cells to activate cells (SAEC) in the presence or absence of WNK1-mTORC2-SGK1 signaling cascade to human HMGB-1 peptide (40 nM). HMGB-1 activate ENaC and to stimulate its own significantly increased amiloride-sensitive ENaC secretion. We propose that this local effect acts Isc by -6.5± 0.93 μA/cm2;p<0.01 after 5 minutes, in concert with aldosterone and increased Na+ while pretreatment with 1 μM FPS-ZM1 (a RAGE delivery from upstream nephron segments to inhibitor) attenuated HMGB-1 effects. HMGB-1 sustain K+ homeostasis. Acknowledgements increased ENaC Po from 0.15± 0.03 to 0.28± This research was supported by grants from NIH 0.04;p<0.01. Pretreatment with FPS-ZM1 for 1 (R01-DK56695 to DP, R01-DK54983 to WHW, hour inhibited the HMGB-1 mediated increase in the James Hilton Manning and Emma Austin Po. Mean absolute total immune cell counts Manning Foundation to DP, Collaborative were significantly higher in bronchoalveolar Projects between AIAS and the Aarhus Univ.'s lavage fluid (BALF) from mice intraperitoneally Research Foundation to IBSJ. MVS was co- (IP) injected with HMBG-1 (27.4×103± 8.8×103 funded by Aarhus Univ.'s Research Foundation cells) vs vehicle injected mice (5.2×103± 1.3×103 and the European Union’s 7th Framework cells);p<0.05. Masson’s Trichrome labeling Program under grant agreement No. 609033. showed that IP injected HMGB-1 significantly increased pulmonary fibrosis. IP injection of HMBG-1 and FPS-ZM1 did not lead to an increase in pulmonary fibrosis. Flow cytometric
53 2019 APS ALDOSTERONE AND ENAC IN HEALTH AND DISEASE: THE KIDNEY AND BEYOND ABSTRACTS OF INVITED AND VOLUNTEERED PRESENTATIONS analysis of HMGB-1 and vehicle injected mice furosemide treatment were enriched in distal showed significant increases in IL-1β, IL-10, IL-6, nephron segments. For example Pkd1, Akt, IL-27, IL-17A, IFN-β, and GM-CSF;p<0.05. Foxo3 were significantly and differentially Confocal microscopic imaging and analysis of expressed in hypertrophied distal nephron SAECs indicate that CF cells have more segments. We also mapped a gradient of cytoplasmic HMGB-1, along with a larger nucleus transcriptional changes congruent with (277± 8.7 μm2) than normal SAEC (187± 5.7 enhanced distal sodium transport specific to μm2);p<0.01. Treating CF SAEC with 1 μM each of four cell clusters. Collectively, we amiloride for 24 hours significantly decreased demonstrate cell type-specific forms of tubular cytoplasmic HMBG-1 and nucleus size to 198± remodeling including selective hyperplasia with 6.8 μm2;p<0.01. Conclusions: HMGB-1 signals growth of DCT cells, progressive hypertrophy via RAGE to increase ENaC Po and inflammation and expression of gene signatures related to in the CF lung. HMGB-1 activation of ENaC may cell growth in the distal nephron, death of TAL play an important role in osmotic stress, nuclear cells, and heterogeneity of differentially swelling, and increases translocation of nuclear expressed genes across cell clusters. These proteins. Outcomes from this study proposes findings highlight novel mechanisms of diuretic that HMGB-1 signaling to RAGE plays a critically resistance, provide a repository of important role in perpetuating lung ENaC transcriptional responses to a common drug, dysfunction and inflammation in CF patients. and expand the implications of long-term loop Funding This work was supported by NIH 1 R01 diuretic use for human disease. HL137033-01A1 awarded to MH. 9.2 9: SYMPOSIUM 4A: ENAC BIOGENESIS Regulation of renal ion transport by AND TRAFFICKING ubiquitylation and phosphorylation networks Olivier Staub1 9.1 1Dept. of Pharmacology & Toxicology, Univ. of Novel mechanisms of diuretic resistance Lausanne revealed by single cell analysis The kidney plays a major role in ion Vivek Bhalla1 homeostasis, and is able to maintain constant 1Medicine/Nephrology, Stanford Univ. School of plasma concentration of ions such as sodium, Medicine potassium and chloride. Thereby, signaling Furosemide, a widely prescribed diuretic for pathways involving ubiquitylation and edema-forming states, inhibits sodium phosphorylation have been shown to play a reabsorption in the thick ascending limb (TAL) of major role in the control of ion balance. the nephron. Tubular adaptation to diuretics has Ubiquitin-protein ligases such as NEDD4-2 or been observed but the range of mechanisms KLHL3/CUL3 have been shown to have major along the nephron has not been fully explored. impact on the activity and the trafficking of ion Using morphometry, we found that furosemide channels such as ENaC or transporters such as induces renal tubular epithelial hyperplasia the Na,Cl-cotransporter NCC. These selectively in distal nephron segments. In ubiquitylation events can be controlled by contrast, we found progressive cellular protein kinases, as is the case for SGK1 that hypertrophy in both proximal and distal nephron controls the action of NEDD4-2; on the other segments. Using single cell RNA sequencing we hand the WNK kinases WNK1 and WNK4 are detected furosemide-induced apoptosis and negatively controlled by KLHL3/CUL3. I will ~50% fewer TAL cells in mice and significant discuss these mecanisms in my presentation. down-regulation in human sections. We also detected an increase in DCT cells. By differential expression in specific cell types, changes in hypertrophic and ion transport genes with
54 2019 APS ALDOSTERONE AND ENAC IN HEALTH AND DISEASE: THE KIDNEY AND BEYOND ABSTRACTS OF INVITED AND VOLUNTEERED PRESENTATIONS
9.3 subunit. Specifically, we found that intersubunit Regulation of ENaC by the ER lumenal, interactions between ENaC transmembrane molecular chaperone, GRP170 domains are required to block Lhs1-dependent Teresa Buck1, Stephanie Mutchler2, Evan Ray3, degradation [4]. We confirmed that the Allison Marciszyn3, Thomas Kleyman3, Jeffrey mammalian homolog of Lhs1, GRP170, also Brodsky1 targets αENaC for ERAD. Conversely, when all 1Biological Sciences, Univ. of Pittsburgh; three ENaC subunits are present GRP170 2Pharmacology and Chemical Biology, Univ. of promotes forward trafficking and the surface Pittsburgh; 3Dept. of Medicine, Univ. of expression of ENaC. Next to study the role of Pittsburgh GRP170 in regulating ENaC in the mammalian The epithelial sodium channel (ENaC) is kidney, we generated an inducible, kidney- responsible for sodium reabsorption in a variety tubule specific, GRP170 knock-out (KO) mouse. of epithelia. In the kidney ENaC is expressed in We hypothesized that in the absence of GRP170 the distal nephron where it regulates salt and ENaC surface expression would be reduced. water homeostasis and, therefore, blood Preliminary data suggest that GRP170 is required pressure. ENaC gain of function mutations result for efficient salt-reabsorption and water in Liddle syndrome and high blood pressure, retention. In addition, the GRP170 KO mice whereas loss of function mutations results in display elevated aldosterone levels, reduced Pseudohypoaldosteronism Type 1 (PHA1), salt- plasma Na+ levels and increased plasma K+ wasting and low blood pressure. In the lung levels, all of which are consistent with ENaC ENaC function is linked to the pathology of dysregulation. The role of GRP170 in the quality cystic fibrosis and ENaC is a drug target for control, trafficking and regulation of other ion cystic fibrosis patients. ENaC is a member of the channels and transporters is unknown and is the degenerin family of ion channels and is a subject of both ongoing but future studies by heterotrimeric channel composed of an α, β our group. Supported by Pittsburgh Center for and γENaC subunit. Each subunit is composed Kidney Research (DK79307) and R01DK117126 of two transmembrane domains, a large (PI: Buck). 1. Buck and Brodsky, Curr Opin extraceullar loop and short N- and C- termini. Nephrol Hypertens. 2018 2. Buck et al., Mol Biol While the ENaC subunits are ~30-40% identical Cell. 2010 3. Buck et al. J Biol Chem. 2013 4. they are subject to differential post-translational Buck et al., Biochem J. 2017 modifications and cellular regulatory pathways [1]. ENaC subunits are synthesized and assemble 9.4 in the endoplasmic reticulum (ER). However, A conserved region in the N-terminus of α-ENaC ENaC subunits that fail to assemble are targeted regulates proteolytic processing during for degradation by the ER associated anterograde transport degradation (ERAD) pathway. ERAD substrates Pradeep Kota1, Hong He1, M. Jackson Stutts1 are recognized by molecular chaperones, 1Cystic Fibrosis Research Center, Univ. of North ubiquitinated, retrotranslocated to the cytosol Carolina, Chapel Hill and degraded by the cytosolic 26S proteasome. Expression, function and regulation of epithelial To characterize the ENaC degradation pathway, sodium channels (ENaC) is required for salt we developed a yeast ENaC expression system, homeostasis. The α-, β - and γ-ENaC subunits which we used to identify a unique subset of assemble into heteromers in the endoplasmic molecular chaperones that are required for reticulum (ER), which are further processed and ENaC degradation [2]. For example, the ER proteolyzed in the Golgi/TGN en route to the lumenal Hsp70-like chaperone, Lhs1, is required plasma membrane. Surprisingly, the molecular for αENaC degradation, but is dispensable for determinants involved in anterograde transport the ERAD of the β or γENaC subunit [3]. In of ENaC from the ER to the plasma membrane contrast, assembly of the ENaC heterotrimer remain largely uncharacterized. We have blocks Lhs1 targeted degradation of the α- recently demonstrated that a splice variant of
55 2019 APS ALDOSTERONE AND ENAC IN HEALTH AND DISEASE: THE KIDNEY AND BEYOND ABSTRACTS OF INVITED AND VOLUNTEERED PRESENTATIONS the rat α-ENaC (deletion of aa 34-82) reaches myeloid-specific MR knockout (MyMR-KO) mice the plasma membrane in a form that is not and their littermate controls (MyMR-intact) were processed by furin in Xenopus oocytes. Here, exposed to Sugen5416(Su)/hypoxia to induce we show that a similar sequence in the human PH or normoxia for 4 weeks. We measured right α-ENaC, when excised, reaches the mammalian ventricular (RV) hemodynamics, cardiomyocyte cell surface in a predominantly uncleaved form. size, pulmonary vascular muscularization and Using deletion mutagenesis, we have identified collagen deposition, and perivascular lung a 13-amino acid stretch in the N-terminus of inflammation. In vitro, human pulmonary human α-ENaC that regulates processing by adventitial fibroblasts (hFibs) were treated with furin-like convertases. Using cell surface conditioned medium from MyMR-KO MØs (vs. biotinylation and whole-cell INa measurements, MyMR-intact) and expression of profibrotic we have determined that ENaC lacking the 13 genes was measured. Results: SU/hypoxia amino acid stretch reach the cell surface as produced similar increases in RV systolic efficiently as WT ENaC and could be activated pressure and RV mass in both MyMR-KO and by exogenous protease treatment. Hughey and MyMR-intact mice. However, MyMR-KOs showed colleagues have identified a C-terminal region in attenuated pulmonary vascular remodeling α-ENaC that regulates exit from the ER. Our compared with MyMR-intact mice with results suggest a role for the N-terminus of α- decreased muscularization and arterial wall ENaC in a distal compartment of the collagen deposition. RV cardiomyocyte area was anterograde transport pathway. Identification of also reduced in MyMR-KO mice. Moreover, lung trafficking branch points that mediate inflammation was attenuated in MyMR-KO mice impairment of furin-mediated processing will with down-regulation of TNFα expression and help illuminate the anterograde transit routes for reduced infiltration of Galectin-3 (Gal-3) positive surface expression of ENaC. cells in the lungs compared to MyMR-intact mice. When exposed to conditioned medium 11: POSTER SESSION 2 from MyMR-KO MØs, cultured hFibs significantly decreased mRNA expression of profibrotic 11.1 genes: collagen Iα1 (Col IA1), transglutaminase 2 Myeloid mineralocorticoid receptor contributes (TG2) and α-smooth muscle actin (α-SMA), but to lung inflammation and vascular remodeling in not Ki67, a proliferative gene. Conclusions: Our experimental pulmonary hypertension results suggest that MR in MØs contributes to Guanming Qi1, Rod R. Warburton1, Krishna C. experimental PH via increased accumulation of Penumatsa1, Mary E. Moss2, Joshua Man2, Qing Gal-3 positive inflammatory cells in the Lu2, Seung Kyum Kim2, Nicholas S. Hill1, Iris Z. perivascular area, followed by production of Jaffe2, Ioana R. Preston1 paracrine factors that activate adventitial 1Pulmonary, Critical Care and Sleep Division, fibroblasts towards a profibrotic phenotype. Tufts Medical Center; 2Molecular Cardiology Research Institute, Tufts Medical Center 11.2 Objective: Previous studies have indicated that Endothelial mineralocorticoid receptor deletion mineralocorticoid receptor (MR) regulates protects female mice from obesity-induced systemic vascular function and contributes to endothelial dysfunction and modulates vascular cardiovascular disease by promoting cell TRPV4 expression and function proliferation and fibrosis, but the underlying Lauren Biwer1, Brigett Carvajal1, Qing Lu1, Iris mechanism remains unclear. We hypothesize Jaffe1 that myeloid-MR plays a pivotal role in the cross- 1Molecular Cardiology Research Institute, Tufts talk between macrophages (MØs) and Medical Center pulmonary fibroblasts, promoting pulmonary Background: The incidence of obesity is inflammation-mediated vascular remodeling of increasing, particularly among women. One of pulmonary hypertension (PH). Methods: Male the earliest vascular consequences of obesity is
56 2019 APS ALDOSTERONE AND ENAC IN HEALTH AND DISEASE: THE KIDNEY AND BEYOND ABSTRACTS OF INVITED AND VOLUNTEERED PRESENTATIONS endothelial cell (EC) damage, resulting in colocalized in caveolin-rich domains of the impaired arterial dilation. Extracellular calcium plasma membrane, where eNOS is unable to be entry via transient receptor potential vanilloid 4 activated by calcium. eNOS mRNA expression (TRPV4) channels can activate endothelial nitric was increased in vessels from EC-MR-KO oxide (NO) synthase (eNOS) and/or calcium- females on control diet and this effect was activated potassium (K) channels to mediate abrogated by obesity. Total caveolin protein microvessel dilation. Levels of the expression was not changed by exposure to mineralocorticoid hormone aldosterone are obesity, but is significantly decreased in obese elevated in obesity and our lab recently EC-MR-KO mice. Summary: Taken together, demonstrated that deletion of the these results indicate that EC-MR may directly mineralocorticoid receptor (MR) from EC in mice modulate the expression, activity and/or rescues endothelial dysfunction specifically in composition of plasma membrane signaling obese females by inducing a switch from K domains of vascular TRPV4 channels specifically channel to NO-mediated dilation. The combined in obese females. This research gives novel role of EC-MR and TRPV4 in regulating this insights to the pathophysiology of vascular process has not been explored in females or dysfunction in obesity and the potential during obesity. Methods: Female EC-MR importance of sex and obesity-specific wildtype or knockout (EC-MR-KO) littermates treatments, including MR antagonists, for were fed control or high fat diet for 12 weeks to microvascular dysfunction. Grant support: NIH produce a model of diet-induced obesity with T32HL007609(LAB), R25GM066567(BVC), elevated fasting glucose. Mesenteric vessels R01HL095590(IZJ) were isolated to quantify mRNA by QRT-PCR or protein by immunoblotting. Mesenteric 11.3 microvessel function was measured by wire Long extracellular beta strand in ENaC gating myography. Results: Obesity alone did not affect Shaohu Sheng1, Jingxin Chen1, Xueqi Wang1, expression of TRPV4, however mesenteric Thomas Kleyman1 vessel TRPV4 mRNA increased significantly in 1Medicine, Univ. of Pittsburgh obese EC-MR-KO mice compared to obese MR- Epithelial Na+ channel (ENaC) activity is intact mice. Conversely, TRPV4 protein modulated by Na+ self-inhibition which reflects expression was significantly decreased in an open probability reduction initiated vessels from obese EC-MR-KO females. TRPV4 specifically by extracellular Na+. While many function in mesenteric microvessels was extracellular factors affect ENaC activity through measured by direct TRPV4 activation with hampering or boosting Na+ self-inhibition, the GSK101 (30nM) or inhibition with GSK219 allosteric mechanisms of this inhibition remain (100nM) during wire myography. In MR-intact unresolved. Previous studies have implicated females, the peak GSK101-induced vasodilation extracellular finger, knuckle, thumb and palm was significantly increased by obesity (69± 5.2% domains as well as the junctions between in obesity vs 44± 5.2% on normal chow, extracellular domains and transmembrane p=0.006) and this change was lost in EC-MR-KO domains in the Na+ self-inhibition. In this study, mice. TRPV4 inhibition significantly increased we investigated the role of the palm domain b10 endothelial-dependent vasodilation to strand traversing nearly the entire extracellular acetylcholine in vessels from obese MR-intact domain at the trimeric symmetry axis. All twelve females, but again, had no effect in obese EC- b10 residues of mouse aENaC were individually MR-KO littermates. Downstream of TRPV4- mutated to a Cys residue. Through comparing mediated calcium entry, K channel activation WT and mutant mouse ENaCs expressed in results in vasodilation. However, the degree of Xenopus oocytes, we identified two mutations vasodiliation to the IK/SK channel activator (aV508C and aY517C) that reduced and three NS309 (1μM) was unaffected by EC-MR-KO or mutations (aS507C, aT509C and aN510C) that obesity. TRPV4, MR, and eNOS can also increased the magnitude of Na+ self-inhibition.
57 2019 APS ALDOSTERONE AND ENAC IN HEALTH AND DISEASE: THE KIDNEY AND BEYOND ABSTRACTS OF INVITED AND VOLUNTEERED PRESENTATIONS
Other seven Cys mutants show a similar Na+ levels of endothelial MR compared to males, self-inhibition response to WT channels. Both which predisposes females to endothelial aV508C and aY517C mutants showed an mineralocorticoid receptor-induced endothelial approximately 50% greater amiloride-sensitive dysfunction. In addition, we have shown that current than WT, however, the difference did not leptin-induced aldosterone-mediated reach statistical significance (p>0.05), which endothelial dysfunction in female mice is might be attributed to a limited number of ablated by amiloride, an inhibitor of the examined cells (n=12-13 in each group). epithelial sodium channel (ENaC). Recent Interestingly, aV508 of mouse ENaC is reports indicate that endothelial MR activation homologous to aV481 of human ENaC. aV481M induces dysfunction in endothelial cells and that was previously reported as a gain-of function amiloride-sensitive aldosterone-induced Na+ variant (PMID: 27582106). We also examined influx can promote endothelial stiffness in accessibility of the introduced sulfhydryl groups female mice. However, whether control female to positively changed MTSET in the first seven mice are more sensitive to ENaC-like channel- mutants (S507 through K512). Only the aS507C mediated endothelial dysfunction compared to and aK512C mutants were modified by males has not been directly tested. Therefore, externally applied MTSET, evidenced by an we hypothesized that agonism of the α subunit irreversible decrease in currents. Our results of ENaC, induced by the TIP peptide, could suggest that the beginning and ending b10 induce moderate endothelial dysfunction in residues in a subunit may contribute to the control female mice. We infused male and mechanism of Na+ self-inhibition whereas the female Balb/C wild-type mice with TIP peptide majority residues in the middle of the longest b (50 mg/dose, 3x) over a period of 7 days. strand are not involved in the Na+ inhibition. Endothelial-dependent acetylcholine-induced Instead, the middle segment of b10 may indeed vasorelaxation responses in aorta of female function as a scaffold as previously suggested mice were moderately impaired following TIP for ASIC (PMID: 22842900), to maintain the core treatment (2-way ANOVA with repeated structure of the extracellular domain facilitating measures, *P<0.05), however, no impairment other peripheral and mobile regions to change was induced in male mice. No significant conformations in response to various signals. differences were observed with TIP treatment in (Supported by DK079307, DK051391 and endothelial-independent (sodium nitroprusside) HL147818) relaxation or phenylephrine-induced constriction responses with TIP peptide treatment in either 11.4 male or female mice. To test whether ENaC-α ENaC-α agonism can induce endothelial agonism is downstream of the effects of dysfunction in control female mice only endothelial MR activation, we infused TIP independently of endothelial mineralocorticoid peptide into female mice with selective deletion receptors of endothelial MR. We observed that endothelial Jessica Faulkner1, Simone Kennard1, Iris Jaffe2, MR deletion did not protect female mice from Rudolf Lucas1, Eric Belin de Chantemèle1 ENaC-α agonist-induced endothelial dysfunction 1Vascular Biology Center, Medical College of (*P<0.05). Neither sodium nitroprusside or Georgia at Augusta Univ.; 2Molecular phenylephrine responses were affected by TIP Cardiology Research Institute, Tufts Medical peptide in female mice with deletion of Center endothelial MR. Collectively, these data indicate Emerging clinical evidence indicates that that ENaC-α agonism induces moderate women have a more pronounced cardiovascular endothelial dysfunction, independently of benefit to mineralocorticoid receptor (MR) endothelial MR expression, in control female antagonism compared to men. Published data mice exclusively. This indicates that endothelial from our group demonstrates that female mice MR-induced increases in ENaC activity may be a and humans endogenously express higher pathway for endothelial impairment in females
58 2019 APS ALDOSTERONE AND ENAC IN HEALTH AND DISEASE: THE KIDNEY AND BEYOND ABSTRACTS OF INVITED AND VOLUNTEERED PRESENTATIONS under these specific conditions, as other reports below baseline levels in both groups. Plasma K+ indicate positive effects of endothelial ENaC-a decreased after salt-loading in both groups and activation during bacterial infection. increased significantly (p<0.05) after salt+amiloride in HT. Plasma Renin Activity (PRA) 11.5 was lower among NT at baseline but increased Effect of amiloride on epithelial sodium channel significantly (p<0.05) following salt-loading; among adult Nigerian subjects there was a reduction following salt+amiloride Simiat Elias1, Olusoga Sofola2,3, Smith Jaja2 (p<0.05). Aldosterone was lower (p<0.001) 1Physiology, Lagos State Univ. College of among NT and reduced below baseline in both Medicine; 2Physiology, College of Medicine, NT (p<0.001) and HT with salt+amiloride. We Univ. of Lagos; 3Physiology, Eko Univ. of conclude that amiloride reversed the pressor Medical and Health Sciences and other effects of enhanced ENaC activity in Polymorphisms of the β -ENaC subunit has been adult Nigerians. We acknowledge the Tertiary recorded among Nigerians. Amiloride has been Education Trust Fund (TETFUND) for funding this shown to reduce blood pressure especially work. References: Elias et al., (2014). Clin & Exp among Blacks though with varied results. This Hypertension 41(2):144-151 Pratt JH (2005). J Am study was designed to determine the effect of Soc Nephrol 16:3154-3159 Baker EH et al., amiloride on ENaC activity after salt ingestion (2002). Hypertension 40: 13-17 among adult Nigerians. Age-matched normotensive (NT, n=38) and hypertensive (HT, 11.6 n=52) subjects participated in the study. Ethical Epithelial sodium channel modulation by acid clearance (CM/COM/8/Vol. XXI) was obtained and anions in salt taste from the College of Medicine, Univ. of Lagos. All Alan Ryan1, Daniel Collier2, Peter Snyder1 subjects gave informed written consent and 1Internal Medicine, Univ. of Iowa; tests were carried out in accordance with 2Pharmaceutical Sciences, Univ. of Tennessee Helsinki Declaration. After baseline Health Science Center measurements, subjects were salt-loaded with The epithelial sodium channel (ENaC) functions 200mmol Na+/day for 5 days. After one week in the maintenance of extracellular volume wash-out period, subjects ingested a balance. In the tongue, ENaC functions as a salt combination of the salt-load and 5mg amiloride taste receptor, where ENaC is exposed to wide tablets daily for 5 days in subjects with blood fluctuations in ionic content. For example, many pressure >110 mmHg (systolic) and >60 mmHg foods and beverages that we ingest are highly (diastolic) only. Repeat BP measurements and acidic. To test the effects of acid on ENaC, we blood tests were carried out after each measured ENaC current. Exposure of ENaC to intervention. Data were compared with ANOVA acid (pH 2.5) increased ENaC current more than and Neuman-Keuls post-hoc tests using two-fold. We found that this increase in current GraphPad Prism version 5. Significance was resulted from an increase in open-state accepted at p<0.05. Significant pressor probability. In human subjects, we found that responses to salt-loading were recorded in NT exposure of the tongue to acid enhanced salt and HT subjects (p<0.01). Salt+amiloride resulted taste. Foods and beverages can also vary in in significant decreases in systolic and diastolic their anion composition, and in previous work, blood pressures compared with baseline and we identified anion binding sites in the salt-loading in NT (p<0.05; p<0.01 respectively) extracellular domain of ENaC. In the presence of and HT (p<0.001; p<0.001 respectively) subjects. Cl-, we found that acid enhanced ENaC current. At baseline and after salt-loading, plasma Na+ Likewise, acid stimulated ENaC when Cl- was was higher (p<0.05) while urine Na+ excretion replace with Br-, phosphate, gluconate, or a was significantly lower (p<0.05) in HT subjects number of other anions. In contrast, when Cl- compared with NT. Following ingestion of was replaced with sulfate, acid failed to increase salt+amiloride, plasma Na+ was reduced to ENaC current. To determine a dose-response
59 2019 APS ALDOSTERONE AND ENAC IN HEALTH AND DISEASE: THE KIDNEY AND BEYOND ABSTRACTS OF INVITED AND VOLUNTEERED PRESENTATIONS relationship for sulfate, we tested mixtures of Cl- conductance and depolarized DCT membrane. and sulfate. Half-maximal inhibition occurred at Moreover, the expression of pNCC/tNCC and ~5 mM sulfate, a concentration which is present thiazide-induced natriuresis were significantly in some foods and beverages. Through site- inhibited in the double KO mice. However, directed mutagenesis, we identified a potential deletion of Nedd4-2 or double deletion of Kir4.1/ anion binding site at the interface between the β Nedd4-2 in the kidney increases the expression and γ subunits that was necessary for the of full-length and cleaved ENaCα. Finally, Ks- response to sulfate. In summary, we found that Kir4.1/Nedd4-2 double KO mice have significant ENaC activity is modulated by acid and by renal K+ wasting and were hypokalemic while sulfate. This modulation may alter our Ks-Nedd4-2 KO mice were normokalemic. Thus, perception of salt in foods and beverages. hypokalemia and renal K+ wasting in the double KO mice were reminiscent to the Ks-Kir4.1 KO 11.7 mice. Conclusion We conclude that Nedd4-2 The stimulation of the basolateral Kir4.1/Kir5.1 in regulates the activity of basolateral Kir4.1/Kir5.1 distal convoluted tubule is involved in in the DCT and that the deletion of Nedd4-2- augmenting NCC activity of Nedd4-2-deficient induced stimulation of the basolateral K mice. channels is at least in part responsible for Peng Wu1, Xiaotong Su2, Zhong-Xiuzi Gao1, increasing NCC activity. Olivier Staub3, Wenhui Wang1, Daohong Lin1 1Pharmacology, New York Medical College; 11.8 2Nephrology and Hypertension, Oregon Health MLP regulates ENaC activity in renal distal & Science Univ.; 3Pharmacology & Toxicology, convoluted tubule cells Univ. of Lausanne Chang Song1, Janet Le2, Qiang Yue1, Monisha Background: We have previously demonstrated Mistry1, Otor Al-Khalili1, Brandi Wynne1, Douglas that Nedd4-2 inhibits Kir4.1/Kir5.1 by increasing Eaton1 Kir4.1 ubiquitination. The aim of the present 1Medicine, Emory Univ. School of Medicine; study is to test hypothesis that deletion of 2Biology, Univ. of Texas Nedd4-2-induced stimulation of NCC is in part The epithelial sodium channel (ENaC) is achieved by increasing Kir4.1/Kir5.1 activity in the regulates blood pressure by fine-tuning distal DCT. Methods: We have used electrophysiology, nephron sodium reabsorption. Our previous immunoblotting, immunostaining and renal work has shown that ENaC gating is regulated clearance to study the basolateral Kir4.1/Kir5.1 by anionic phospholipid phosphates including channel activity in the DCT and to examine the phosphatidylinositol 4,5-bisphosphate (PIP2). activity of thiazide-sensitive Na-Cl cotransporter The PIP2-dependent regulation of ENaC is (NCC) in WT, Ks-Nedd4-2 knockout (KO) and Ks- mediated by the myristoylated alanine-rich Kir4.1/Nedd4-2 double KO mice. Results: protein kinase C substrate-like protein-1 (MLP-1). Deletion of Nedd4-2 increased the activity and MLP-1 is a reversible source of PIP2 at the expression of basolateral Kir4.1 in the DCT and plasma membrane. Both MARCKS and ENaC are hyperpolarized DCT membrane. Also, the regulated by proteolysis. Our previous work has expression of phosphorylated NCC (pNCC) / shown that ENaC proteins are present in distal total NCC (tNCC) and thiazide-induced convoluted tubule (DCT) cells. We examined natriuresis was significantly increased in Ks- MLP-1 regulation of ENaC in DCT-15 cells. We Nedd4-2 KO mice. To test the possibility that wanted to show if ENaC is regulated by MLP-1 Nedd4-2 deletion-induced stimulation of DCT cleavage. The properties of DCT-15 cells are hyperpolarization may be involved in stimulating similar to those of in situ cells of the DCT-2 NCC activity in Ks-Nedd4-2 KO mice, we nephron segment. We used monolayers of DCT- generated Ks-Kir4.1/Nedd4-2 double KO mice. 15 cells on permeable polyester supports to The double deletion of Nedd4-2 and Kir4.1 in the measure transepithelial voltage, resistance, and kidney largely abolished the basolateral K+ current. Our results show that DCT-15 cells had
60 2019 APS ALDOSTERONE AND ENAC IN HEALTH AND DISEASE: THE KIDNEY AND BEYOND ABSTRACTS OF INVITED AND VOLUNTEERED PRESENTATIONS relatively low resistances (206 ± 5.71 Ω-cm2, R01 DK-110409 to DCE and K01 DK-115660 and n=36) and small, but measurable amiloride- ASN Gottschalk AWARD to BMW sensitive currents (2.87 ± 0.237 μA, n=36), showing that these cells contain functional 11.9 ENaC. 100 nM aldosterone significantly The differential effects of aldosterone and high increased mean DCT-15 cell current (from 2.87 ± salt on kidney injury 0.237, n=36, to 5.33 ± 0.395μA, n=8). Wild-type Stephanie Mutchler1, Mahpara Hasan2, Roderick MLP-1 ran as a doublet at apparent MW of 40 Tan1, Thomas Kleyman1 and 33 KDa despite having 21 KDa predicted 1Renal-Electrolyte, Univ. of Pittsburgh; MW. To further examine MLP in DCT-15 cells, we 2Biological Sciences, Carnegie Mellon Univ. constructed several MLP mutants. WT: a full The steroid hormone aldosterone (aldo) is length wild-type protein; S3A: 3 substitutions in responsible for mediating the restoration of fluid the effector domain to prevent phosphorylation; and electrolyte homeostasis. While it plays this S3D: 3 substitutions in the effector domain to important physiological function, inappropriate mimic constitutive phosphorylation; GA: one chronic elevation of aldo has been shown to substitution to prevent myristoylated. Each increase renal injury and inflammation. However, mutant was tagged with either N-terminal many of the studies characterizing aldo’s role in 3XFLAG or C-terminal m-Cherry. Transfection kidney injury have been conducted in mice with MLP mutants modified current in DCT-15 provided high sodium via either food or water, cells: current was highest in S3A and lowest in with full factorial experiments not being S3D; the current after transfection with either performed to separately analyze the construct was significantly different from WT. In independent effects of aldo and sodium. To Western blots, when transfected with 3XFLAG further our understanding of how aldo and tagged MLP mutants, the expression of the full sodium act alone and in synergy to cause renal length of MLP at 52 KDa) increased in mutant damage, we examined four groups of mice: a S3A-MLP transfected DCT-15 cells and control group receiving standard 0.4% NaCl decreased in S3D-MLP transfected DCT-15 cells. chow, a high salt diet (HSD) group receiving 8% Whereas the expression of the full length MLP NaCl chow for 4 weeks, an aldo group given a was not significantly different fromS3A-MLP and subcutaneous minipump implant delivering aldo S3D transfected DCT-15 cells when tranfected at 240mg/kg/day for two weeks, and a with cherry-tagged MLP mutants. A small combination group receiving both HSD for 4 molecular weight protein about 25 kDa was weeks and aldo administration during the final detected in 3X-tagged-MLP-mutant-tranfected two weeks. Urinary analysis revealed that both DCT cells, and the expression of this possible aldo alone and the combination treatment fragment of MLP in S3D tranfected cells was caused an increase in urinary protein excretion. significantly higher than in S3A transfected cells. However, qPCR showed that while transcript In cell transfected with mCherry mutants, full levels of the kidney injury markers KIM1 and length protein was detected with products NGAL were increased approximately 10-fold with corresponding to the WT bands at 40 and 33 aldo alone, these levels were increased 100-fold KDa (when corrected for mCherry’s size) and a with combination treatment. Furthermore, third band at lower MW (about 28 KDa). The sum histology revealed that while aldo alone caused of the small Flag-tagged (N-terminal fragment) some tubule injury, combination treatment led to and the small mCherry-tagged (C-terminal tubular dilation and indications of fibrosis Fragment) is almost equal to the the size of the exceeding those of aldo treatment alone. At a full-length protein suggesting that the two bands transcript level, genes associated with represent two cleaved pieces ogf MLP-1. Our inflammation, including those producing IL-1b, results suggest a complicated role for MCP-1, TNFa, and IL-6, were most elevated with proteolytic processing in MLP-1 regulation. combination treatment as compared to either Support or Funding Information Supported by HSD or aldo alone. Together, these data
61 2019 APS ALDOSTERONE AND ENAC IN HEALTH AND DISEASE: THE KIDNEY AND BEYOND ABSTRACTS OF INVITED AND VOLUNTEERED PRESENTATIONS emphasize that while aldo alone does lead to 11.11 renal injury and inflammation, there is a Epithelial sodium channel (ENaC) in endothelium synergistic effect when it is administered with modulates vascular reactivity with a high-salt HSD, therefore experiments should be careful to diet separate the effects of these stressors acting Stephanie Mutchler1, Thomas Kleyman1 alone versus in combination. 1Renal-Electrolyte, Univ. of Pittsburgh The epithelial sodium channel (ENaC) is best 11.10 known for its role in fluid and electrolyte Characterization of mice with a hypomorphic balance, however, it has more recently been ENaC γ subunit shown to be involved in pressure- and flow- Evan C. Ray1, Jenna Barbour1, Austin Lieber1, induced responses in endothelial cells. In cell Aaliyah Winfrey1, Stephanie Mutchler1, Allison culture, endothelial ENaC is upregulated in Marciszyn1, Yaakov Barak2, Harry Blair3, Thomas response to high aldosterone and extracellular Kleyman1 [Na+]. While in vivo experiments have been 1Renal-Electrolyte, Univ. of Pittsburgh; performed examining the role of endothelial 2Obstetrics, Gynecology, and Reproductive ENaC under physiological conditions and in Sciences, Magee-Women’s Research Institute, animals given a Western diet, the in vivo effects 3Pathology, Univ. of Pittsburgh of endothelial ENaC in the setting of high salt ENaC is expressed in numerous epithelia. In diet (HSD) and aldosterone are unclear. To test kidney, complete channels include α, β , and γ this, we divided mice into four treatments: the subunits and participate in Na retention during first group received control chow (0.4% NaCl), periods of extracellular fluid volume depletion. the second group received four weeks of 8% Reports have suggested the α and γ subunits NaCl diet, the third group received two weeks of are expressed in bone periosteum, where the aldosterone administration via a subcutaneous channel has been postulated to contribute to minipump (240mg/kg/day), and the fourth group osteoblast activity. Additionally, renal tubular received both high salt diet and aldosterone. ENaC activity could influence bone architecture Using pressure myography, we found that none and density through alteration of divalent cation of the treatments significantly reduced handling. Perinatal mortality of γ subunit knock- acetylcholine (Ach) responsiveness in the out mice has hindered examination of the thoracodorsal artery (TDA) of mice, but when subunit’s physiologic roles. We have developed amiloride was added to the lumen of the vessels a viable mouse γ subunit global hypomorph to to understand the role ENaC in vasodilation, assist in exploring the requirement for this responses were varied among the different subunit in various tissues. In these mice, plasma treatments. The greatest difference in Ach aldosterone levels are elevated, and blood responsiveness between control and amiloride pressures are lower, relative to controls. We treated vessels was noted in the high salt diet observe increased blood K on a high K diet. treated animals. In the majority of the vessels Bone parameters, including bone mineral taken from mice treated for four weeks with high density, cortical thickness, trabecular thickness, salt diet, amiloride treatment shifted the Ach inter-trabecular spacing, bone tissue dose response curve to the right, suggesting volume/total volume, surface/volume, and that ENaC was promoting vasodilation in these connectivity density did not differ as compared vessels. However, when this high salt regimen to litter-mate controls. These results provide no was extended to eight weeks, amiloride addition evidence for a role of this subunit in bone to the lumen of vessels shifted the Ach-dose physiology. On-going studies are exploring the response to the left, suggesting that ENaC was role of ENaC’s γ subunit in other extra-renal limiting vasodilation at this time point. At both tissues. time points, serum aldosterone was decreased as compared to control treated animals as measured by ELISA. Together these data
62 2019 APS ALDOSTERONE AND ENAC IN HEALTH AND DISEASE: THE KIDNEY AND BEYOND ABSTRACTS OF INVITED AND VOLUNTEERED PRESENTATIONS suggest that aldosterone may not be required subunits. The four ENaC subunits arose from for ENaC’s expression and activity in endothelial three gene duplication events. First, a single cells. They additionally demonstrate that gene diverged to an α/∆ precursor and a β /γ endothelial ENaC has a role in promoting the precursor. Shortly after, the β /γ precursor vascular dysfunction seen with chronic high salt diverged to β and γ subunits. These two events intake, but that this role may stem from a occurred in time for the emergence of jawless compensatory mechanism as ENaC seems to fishes, as lampreys have α, β, and γ subunits. promote vasodilation in animals given a high salt Later, but prior to the terrestrial migration, α and diet for shorter periods of time. Ongoing studies ∆ diverged from the α/∆ precursor; the lobe- are examining the role of nitric oxide and eNOS finned coelacanth is the first known species to phosphorylation status in the endothelial have all four subunits. We observed that the response to a HSD. activating cleavage sites in the α and γ subunits appeared around the transition to terrestrial life. 11.12 Among the fishes, only the Australian lungfish γ Epithelial Na+ channel activation by cleavage subunit had two cleavage sites, and all tetrapod coevolved with the terrestrial migration of α and γ subunits had two apparent cleavage vertebrates sites. We ran nested likelihood models in Xueping Wang1, Deidra M. Balchak2, Clayton BayesTraits and found a significant relationship Gentilcore1, Nathan L. Clark3, Ossama B. between terrestrial status and having two Kashlan1,3 cleavage sites in the α and γ subunits. Both sites 1Dept. of Medicine, Univ. of Pittsburgh; 2Division showed a statistically significant coevolutionary of Hematology, Children's Hospital of pattern with the terrestrial state (proximal site, p Pittsburgh; 3Computational and Systems = 0.004; distal site, p = 0.006). The sites also Biology, Univ. of Pittsburgh showed a coevolutionary pattern with each The epithelial Na+ channel (ENaC) is critical to other (p = 1x10-7). In contrast, the C-terminal PY regulating extracellular fluids through its activity motif, which is important for aldosterone in several epithelia, including the aldosterone- regulation through Sgk1/Nedd4-2, did not show sensitive distal nephron and colon, and the a dependence on terrestrial status (p = 0.58). airway and alveoli. Human ENaC subunits are Furthermore, reconstruction of ancestral ENaC subject to activating proteolysis. During subunits predict that neither site was present at processing in the trans-Golgi network, furin key gene duplication events (proximal site, p = cleaves the α subunit twice. This liberates an 0.01; distal site, p = 0.003), suggesting that each imbedded 26-mer inhibitory tract, moderately of the cleavage sites in the α and γ subunits activates the channel, and leaves 30 kDa and arose independently. The vertebrate terrestrial 65 kDa fragments as parts of the channel migration is associated with many changes, complex. Furin cleaves the γ subunit once. This including accessibility to dietary Na+, differences event has no effect on channel activity, but in osmotic stress, and the development of lungs. leaves 18 kDa and 75 kDa fragments as parts of One or a combination of these factors may have the channel complex. Once at the cell surface, provided the selection pressure to develop this one of a number of proteases may cleave the γ form of channel regulation. subunit in a region ~35-50 residues distal to the furin cleavage site. This second cleavage event of the γ subunit also releases an imbedded inhibitory tract, 43-residues when prostasin is the second protease, and greatly activates the channel. To examine the evolution of ENaC cleavage sites, we generated a phylogenetic tree of ENaC and ENaC-like subunits to identify key branch points in the evolution of ENaC
63 2019 APS ALDOSTERONE AND ENAC IN HEALTH AND DISEASE: THE KIDNEY AND BEYOND ABSTRACTS OF INVITED AND VOLUNTEERED PRESENTATIONS
11.13 11.14 Wnt4(+) cell mapping and changes in ENaC Angiotensin and aldosterone receptor regulatory genes in furosemide-treated mice antagonism attenuates angiotensin II- induced George Kunkel1, Steven Schaffert2, Aram hypertension in sprague dawley rats Krauson1, Yanli Qiao1, Purvesh Khatri2, Vivek Stacy Tletlepantzi1 Bhalla1 1Natural Sciences, Univ. of California, Merced 1Medicine / Nephrology, Stanford Univ. School Stacy A. Tletlepantzi, Debye Conte, Ruben of Medicine; 2Medicine / Division of Biomedical Rodriguez, Mouhamed S. Awayda*, and Rudy M. Informatics Research, Stanford Univ. School of Ortiz Dept. of Molecular & Cell Biology, School Medicine of Natural Sciences, Univ. of California, Merced, Furosemide, a commonly prescribed loop *Dept. of Physiology & Biophysics, State Univ. of diuretic to treat hypertension and edema- New York, Buffalo, NY forming states, increases sodium excretion by The inappropriate activation of the renin- inhibiting reabsorption via the sodium-potassium angiotensin-aldosterone system (RAAS) 2 chloride cotransporter (NKCC2) in the thick contributes to hypertension and cardiovascular ascending limb. Chronic use of loop diuretics damage. The blockade of the Ang II receptor can lead to resistance and the need for higher (via ARB) robustly ameliorates hypertension, doses and/or additional classes of diuretics with more so than the antagonism of the adverse effects. Using tubular morphometry and mineralocorticoid receptor (via eplerenone) single cell RNA sequencing of vehicle- and alone. Moreover, a consequence of chronic MR furosemide-treated mice, we found that blockade (MRb) is exacerbation of plasma furosemide induces significant nephron aldosterone levels, which may contribute to the segment-specific heterogeneity in tubular failure of MRb to completely ameliorate remodeling (hyperplasia, hypertrophy, hypertension. However, the combined effects of apoptosis) and in gene expression. On ARB and MRb simultaneously on arterial examination of differential expression between pressure and renal Na+ regulation are not well- vehicle and furosemide treated mice, we defined during Ang II-induced hypertension. demonstrate that a small cluster of Wnt4- While we expected ARB alone and in enriched cells harbor large relative gene combination would completely ameliorate the expression changes. These cells were markedly Ang II-induced hypertension, we hypothesized enriched for several transcripts from the Wnt that the addition of MRb would have the added signaling system: Wnt4, Wnt7b, and Wnt 9b, benefit of increased UNaV. To test our compared to CCD principal cells. In pseudotime hypothesis, SBP and urinary Na+ were plots, Wnt4(+) cells preceded CCD principal cells measured in five groups of Sprague- Dawley and multiple other cell types, supporting prior male rats: (1) untreated controls, (2) Ang II observations that Wnt4(+) cells are a infused (80 ng/min x 28d), (3) Ang II + AT1 development precursor of tubular epithelia. With receptor blocker (ARB; 10 mg losartan/kg/d x 21 furosemide treatment, these cells decreased d), (4) Ang II + MRb (Epl; 100 mg eplerenone/d x expression of their identifying markers (Wnt4, 21d), and (5) Ang II + ARB + Epl (combo; 21 d). Wnt7b) and increased markers of principal cells Both the ARB and combo group attenuated Ang (Scnn1b, Scnn1g, Hsd11b2, and Nr3c1). Ongoing II- induced hypertension, while the Epl treatment studies seek to understand the functional only prolonged the onset of AngII- induced consequences of these changes toward a hypertension. The Epl treatment exacerbated principal cell gene programme. In conclusion, urinary aldosterone excretion over the 21 days furosemide-induced changes in Wnt4-enriched (Epl: 275 ± 119; Control: 64 ± 18 pmol/d), which cells may represent transdifferentiation, a novel was confirmed by plasma aldo on day 28. mechanism of tubular remodeling and diuretic However, the exacerbation of aldo was resistance. abolished with the co-treatment with ARB. Despite some group effects in UNaV early in the
64 2019 APS ALDOSTERONE AND ENAC IN HEALTH AND DISEASE: THE KIDNEY AND BEYOND ABSTRACTS OF INVITED AND VOLUNTEERED PRESENTATIONS study, by day 28, there were no detectable up-regulating ENaC in the uterus for pro- group effects. This lack of a change in UNaV at inflammatory shift in term and preterm labor. the end of the study was consistent with a lack of a change in g-ENaC content; however, the 11.16 combination of both receptor blockers Primary aldosteronism decreases insulin decreased the expression of a- ENaC by 42% secretion and increases insulin clearance in when compared to control. These results humans suggest that during Ang II-dependent James Luther1, Gillian Murray2, Adina Turcu3, hypertension, the contribution of aldosterone to Nian Hui4, Chang Yu4, Dungeng Peng1, Gail the volume-dependent effects of the Adler2 hypertension appears to be minimal especially 1Medicine, Vanderbilt Univ. Medical Center; as it pertains the ENaC-mediated component of 2Medicine, Brigham and Women’s Hospital; renal Na+ regulation. 3Medicine, Univ. of Michigan; 4Biostatistics, Vanderbilt Univ. 11.15 Primary aldosteronism is associated with an Serum and glucocorticoid‐regulated kinase 1 increased risk of diabetes, but the mechanism is (SGK1) up-regulates ENaC in the uterus for pro- uncertain. We enrolled 9 patients with primary inflammatory shift in term and preterm labor aldosteronism and assessed insulin secretion Ye Chun Ruan1 and insulin sensitivity beforte and after 3-12 1Dept. of Biomedical Engineering, The Hong months after either adrenalectomy (n-6) or Kong Polytechnic Univ. medical treatment (n=3). Plasma 8AM We have recently demonstrated in humans and Aldosterone (p=0.02) and 11- mouse models that uterine ENaC together with deoxycorticosterone (p=0.02) decreased after its downstream inflammatory signaling plays an treatment, whereas cortisol was unchanged essential role in term and preterm labor (EMBO (p=0.57). Systolic blood pressure (131.1± 3.6 vs Mol Med 2018). The surge in cortisol levels, a 124.3± 7.7; p=0.43), serum potassium (3.7± 0.1 vs major glucocorticoid in humans, is observed in 4.1± 0.1 mEq/L; p=0.063), and creatinine (1.1± 0.1 pregnant women as approaching toward the vs 1.2± 0.1 mg/dL; p=0.35) were similar after end of pregnancy. SGK1 is phosphorylated and treatment. During hyperglycemic clamps, we activated by glucocorticoids and promotes infused glucose to increase plasma glucose to a ENaC expression in many other systems. We target of 200mg/dL. The insulin response therefore investigated possible involvement of (218.6± 39.8 vs 403.1± 42.0 pM insulin mean at SGK1 in regulation of ENaC in the uterus at term 90-120 min, p=0.004) and L-arginine stimulated and preterm labor. Our results showed that insulin response increased compared to Pre- uterine SGK1 is activated at labor in mice. Treatment assessment. C-peptide results were Placental SGK1 expression is upregulated in similar to insulin alterations. Comparison to women with spontaneous preterm labor as control subjects matched for age, race, sex, and compared to those with term labor. Analysis of BMI indicated that insulin secretion after the human transcriptome database reveals treatment was comparable to healthy significant and positive correlation of SGK1 with individuals. During hyperinsulinemic-euglycemic ENaC alpha subunit (ENaCα) as well as labor- clamps, insulin clearance (steady state insulin associated pro-inflammatory factors in labored divided by insulin infusion rate) increased after birth groups (both term and preterm), but not the treatment (855± 68 vs 615± 57 mL/min, p=0.031). non-labored group. In human endometrial cells Insulin sensitivity decreased after treatment in vitro, treatment with cortisol enhances ENaCα (30.7± 2.1 vs 18.5± 1.6 M/I; p=0.016), but and COX-2 expression, which is significantly disposition index was unchanged (acute insulin attenuated by SGK1 knockdown. Therefore, response x M/I; p=0.81). Insulin secretion these results have suggested a role of SGK1 in increases after treatment for primary aldosteronism. This effect was explained in part
65 2019 APS ALDOSTERONE AND ENAC IN HEALTH AND DISEASE: THE KIDNEY AND BEYOND ABSTRACTS OF INVITED AND VOLUNTEERED PRESENTATIONS by decreased insulin clearance. These changes (1.00 ± 0.09 vs. 0.80 ± 0.22, NK vs. HK are accompanied by a decrease in insulin respectively, p = 0.04). Chronic HK-fed mice that sensitivity and no change in the disposition were not fasted had a higher plasma potassium index. (4.22 ± 0.16 vs. 3.79 ± 0.13, p < 0.05) and lower pNCC abundance compared to NK-fed mice 11.17 (1.00 ± 0.85 vs. 0.24 ± 0.08, NK vs. HK Differential effects of acute vs. chronic dietary respectively, p = 0.03). Chronic HK-fed mice had potassium intake on plasma potassium a trend toward a lower sodium excretion in concentration and NCC phosphorylation and response to NCC inhibition than mice on a NK expression diet (0.23 ± 0.02 vs. 0.16 ± 0.01 umol/min, NK vs. Jonathan Nizar1, Tim Davie1, Vivek Bhalla2 HK respectively, p = 0.10). In conclusion, chronic 1Internal Medicine, Nephrology, Univ. of Iowa; high potassium diets reduce total NCC 2Internal Medicine/Nephrology, Stanford Univ. expression which correlates with decreased Diets high in potassium are associated with natriuretic sensitivity to NCC inhibition. In short-term blood pressure reductions and long- contrast, acute dietary potassium can increase term cardiovascular benefits in humans. Dietary plasma potassium levels and decrease NCC potassium may regulate blood pressure via its phosphorylation, but does not correlate with effects on NCC-mediated sodium excretion. NCC activity, as measured by natriuretic Over the short-term in humans, and over days to sensitivity. weeks in mice, potassium intake has been reported to affect plasma potassium 11.18 concentration which regulates NCC Age and sex-specific reference ranges are phosphorylation and protein expression. We needed for the aldosterone/renin ratio investigated the differential effect of acute vs. Jun Yang1,2,4; Pravik Solanki1,2; Stella May Gwini3; chronic dietary potassium intake on plasma Peter J Fuller1,4; Jimmy Shen1,4; James CG levels and NCC phosphorylation and expression Doery2; Kay Weng Choy1 in which dietary potassium intake was varied 1Dept. of Endocrinology, Monash Health; 2Dept. over weeks and again over 4 hours in a 2x2 of Medicine, Monash Univ.; 3Univ. Hospital comparison. Mice were fed either a 5% KCl Geelong, Barwon Health; 4Hudson Institute of (high, HK) or 0.7% (normal, NK) diet for 18-35 Medical Research days, fasted 4 hours before sacrifice, and Background: The aldosterone/renin ratio (ARR) gavaged with 5% of their daily K intake (gavage) is the standard screening test for primary or water (vehicle) 30 minutes before sacrifice. aldosteronism (PA). Current guidelines define Plasma potassium was increased by acute ARR>70 (pmol/L)/(mIU/L) as abnormal regardless dietary potassium (gavage) in the HK compared of age or sex. However, fluctuations in female to NK group (5.94 ± 0.43 vs. 4.08 ± 0.12 mmol/L hormones over the menstrual cycle influence respectively, p < 0.01). However, it was no the ARR. Objective: To characterise variations in different in HK compared to NK mice that did not the ARR according to age and sex. Methods: A receive the KCl gavage (vehicle) (3.81 ± 0.07 vs. retrospective analysis of 466 clinically indicated 3.93 ± 0.03 mmol/L respectively, p = 0.11). ARRs at Monash Health from December 2016 – Concordant with plasma potassium levels, June 2018 was conducted. Patients who took dietary potassium decreased phospho-Thr53 spironolactone, oral contraceptive pill, were NCC only when gavaged (1.00 ± 0.18 vs. 0.16 ± pregnant or had a known adrenal condition 0.06, NK vs. HK respectively, p < 0.05) and was (including untreated PA) were excluded. no different in acute vehicle-gavaged, chronic Results: Among patients aged 20-39 years HK-fed compared to NK-fed mice (1.00 ± 0.10 (N=74), females had significantly higher median vs. 0.93 ± 0.27, NK vs. HK respectively, p = aldosterone (373.5 vs 231 pmol/L), lower median 0.82). Total NCC abundance was decreased in renin (16.5 vs 23.5 mIU/L), and higher median chronically HK-fed compared to NK-fed mice ARR (20.75 vs 10.49) than males. However,
66 2019 APS ALDOSTERONE AND ENAC IN HEALTH AND DISEASE: THE KIDNEY AND BEYOND ABSTRACTS OF INVITED AND VOLUNTEERED PRESENTATIONS females had lower median systolic (135 vs 145 12.2 mmHg, p=0.021) and diastolic (89 vs 96.5 Sex differences in the role of the smooth muscle mmHg, p=0.007) blood pressure (BP). These sex cell mineralocorticoid receptor in cardiovascular differences were not observed in the 40 – 59 aging years (n=161) or 60 – 79 years (n=157) age Jennifer DuPont1, Seung Kim1, Rachel Kenney1, groups. When females were divided into pre- Qing Lu1, Iris Jaffe1 and post-menopausal groups (at an arbitrary 1Molecular Cardiology Research Institute, Tufts age cut-off of 45 years), those ≤45 had Medical Center significantly higher median aldosterone levels Aging is universal and is the most significant risk (364 vs 273 pmol/L, p=0.047), lower systolic BP factor for cardiovascular disease. Clinical data (139 vs 148 mmHg, p=0.002), but higher diastolic supports that the time course for developing BP (89.5 vs 80 mmHg, p=0.001). aging-associated cardiovascular disease occurs differentially in males vs. females. We previously Conclusion: Our findings highlight the need for demonstrated that male mice with the age- and sex-specific ARR reference ranges, mineralocorticoid receptor deleted from smooth particularly for younger women who have a muscle cells (SMC-MR-KO) are protected from significantly higher ARR that is not associated cardiovascular aging. The purpose of this study with higher BP. was to determine whether there are sex differences in the role of SMC-MR in 12: SYMPOSIUM 5A: MR IN THE cardiovascular aging. Vascular MR mRNA VASCULATURE expression increases at 12 months of age in males but not until 18 months of age in females. 12.1 We therefore compared microvascular Sex-specific mechanisms of resistance vessel contractile function and large vessel structure in endothelial dysfunction in obesity male and female mice aged 3 months (young Ana Davel1 adult), 12 months (middle aged) and 18 months 1Dept. of Structural and Functional Biology, Univ. (old) with MR-intact to SMC-MR-KO littermates. of Campinas We previously identified miR-155 as a regulator The epidemic of obesity that affects more of the angiotensin II type 1 receptor (AT1R) that is women than men worldwide is one of the major negatively regulated by MR in the aging male risk factors for cardiovascular disease. The vasculature. We now confirm that the rise in MR mechanisms underlying obesity-induced in aging vessels is accompanied by a endothelial dysfunction and blood pressure concomitant down-regulation of vascular increase have been suggested to be sex- microRNA (miR)-155 at 12 months in males and at specific. In this talk, I will present data on the 18 months in females, however, this is prevented pathophysiological role of endothelial by SMC-MR-KO in males only. Functionally, we mineralocorticoid receptor (MR) activation for find that AT1R-mediated vasoconstriction of the endothelial dysfunction in obese females. I mesenteric resistance vessels is increased at 12 will also show how the perivascular adipose months in males and at 18 months in females tissue might contribute for the obesity-induced and was prevented in SMC-MR-KO mice of both endothelial dysfunction in males. The sexes. We next explored aging-related changes differences in the molecular mechanisms in vascular structure by measuring aortic pulse triggering endothelial dysfunction in male and wave velocity to quantify vascular stiffness, a female obesity support the need for a sex- parameter that rises with age in humans and specific therapeutic approach for patients at correlates with risk of cardiovascular disease cardiometabolic risk. and death. Vascular stiffness increases significantly by 12 months in males but not until 18 months in females and that is prevented by SMC-MR-KO. Carotid fibrosis increased at 12
67 2019 APS ALDOSTERONE AND ENAC IN HEALTH AND DISEASE: THE KIDNEY AND BEYOND ABSTRACTS OF INVITED AND VOLUNTEERED PRESENTATIONS months in males vs. 18 months in females, and is area was significantly decreased in MyMR-KO attenuated by SMC-MR-KO in males only. vs. MR-intact mice (p=0.028), but there was no Cardiac function declines at 18 months in both difference in the percent of the plaque males and females, but is attenuated by SMC- composed of neutral lipid, necrotic core, or MR-KO in females only. In summary, the time collagen content by Oil Red O and Masson’s course and mechanisms of cardiovascular aging trichrome staining. Immune cell populations are distinct between males and females with within aortic arch plaques were then quantified SMC-MR playing a differential role in males by flow cytometry. The numbers of versus females. Furthermore, these studies macrophages and T cells in the aortic arch were suggest that sex-specific therapies may be significantly decreased in MyMR-KO vs. MR- essential to improve CVD outcomes in the aging intact mice (p=0.007, p=0.041 respectively), population. while the other populations (granulocytes, monocytes, B cells) were not different by 12.3 genotype. Although females had larger plaques, Role of the myeloid mineralocorticoid receptor they were less inflamed with significantly fewer in vascular inflammation in atherosclerosis plaque myeloid and T cells compared to male Joshua Man1,2, Iris Jaffe1 mice (p=0.013, p=0.008 respectively). 1Molecular Cardiology Research Institute, Tufts Interestingly, the ratio of Ly-6Chi:Ly-6Clo Medical Center; 2Sackler School of Graduate monocytes was significantly increased in MyMR- Biomedical Sciences, Tufts Univ. School of KO vs. MR-intact mice (p<0.0001), but this was Medicine driven by a reduction in Ly-6Clo monocytes in Background: Elevated plasma aldosterone is MyMR-KO mice (p=0.020). Myeloid MR knockout associated with increased risk of heart attack did not affect hematopoiesis in the bone marrow and stroke, and mineralocorticoid receptor (MR) or spleen, suggesting that differences in plaque antagonists reduce cardiovascular-related inflammation observed between MyMR-KO and mortality. Heart attack and stroke are caused by MR-intact mice are likely due to changes in rupture of atherosclerotic plaques with recruitment, proliferation, and/or survival of abundant pro-inflammatory leukocytes. In immune cells. Intravital microscopy data studies mouse atherosclerosis models, MR antagonists will quantify leukocyte rolling along the reduce plaque size and inflammation, endothelium in vivo in response to TNFα to particularly the proportion of pro-inflammatory assess the role of MyMR in leukocyte Ly-6Chi myeloid cells. Deficiency of MR in recruitment. Summary and Conclusions: endothelial but not smooth muscle cells Deficiency of myeloid MR attenuates attenuates plaque inflammation, but neither atherosclerotic plaque size and inflammation reproduced the decrease in pro-inflammatory and reduces plaque macrophages and T cells. myeloid cells observed with systemic MR Female mice have larger plaques but blockade. Here we test the hypothesis that significantly fewer plaque myeloid and T cells myeloid MR contributes to atherosclerotic compared to male mice. This study suggests plaque inflammation by modulating the myeloid that a decrease in plaque inflammation from cell phenotype in vivo. Methods and Results: blocking myeloid MR may contribute to the LysM-Cre/MRfl/fl mice were crossed with ApoE- improved patient outcomes observed in clinical /- mice to generate atheroprone mice with MR trials with MR antagonists. Funding Sources: specifically deleted from myeloid cells (MyMR- This work was supported by grants from the KO). PCR amplification of the floxed MR locus National Institutes of Health HL095390 (to IZJ) confirmed that the MR was specifically deleted and the American Heart Association EIA in macrophages by at least 50% and only in 18290005 (to IZJ). MyMR-KO mice. Two month old male and female MyMR-KO and MR-intact littermates were fed high-fat diet for 8 weeks. Aortic root plaque
68 2019 APS ALDOSTERONE AND ENAC IN HEALTH AND DISEASE: THE KIDNEY AND BEYOND ABSTRACTS OF INVITED AND VOLUNTEERED PRESENTATIONS
12.4 administered by continuous infusion (Alzet Aldosterone and angiotensin II increase aortic osmotic pump) to 9 month old wild type (WT) stiffness and endothelial dysfunction via an and EnNaC alpha subunit KO mice for four action of oxidative stress on the endothelial weeks. The antioxidant tempol (2 mM/L) was sodium channel added to the drinking water in a cohort of Ang II James Sowers1, Guanghong jia2, Sun Zhe3, Yan treated mice. Both Aldo and Ang II increased Yang4, Javad Habibi2, Guido Lastra2, Camila whole cell sodium current in ECs, on patch Manrique2, Vincent Demarco5, Adam Whaley- clamp that was temporally- related to aortic EC Connell2, Michael Hill6, Frederic Jaisser7, stiffness on atomic force microscopy and Annayya Aroor2 impaired aortic relaxation. These abnormalities 1Diabetes and Cardiovascular Research Center were associated with increased oxidative stress. and Medicine, Medical Pharmacology and Interestingly EnNaC alpha subunit deletion Physiology, Research Service Harry S Truman prevented Aldo induced aortic stiffness and Memorial Hospital, and Dalton Cardiovascular impaired NO dependent aortic relaxation both of Research Center, Univ. of Missouri; 2Diabetes which were associated with a decrease in and Cardiovascular Research Center and oxidative stress suggesting role of oxidative Medicine, and Research Service Harry S stress in ECMR/ EnNaC mediated aortic Truman Memorial Hospital, Univ. of Missouri; stiffening and endothelial dysfunction. 3Dalton Cardiovascular Research Center, Univ. Moreover, antioxidant mediated improvement in of Missouri; 4Dalton Cardiovascular Research endothelial/arterial stiffness and endothelial Center, Univ. of Missouri; 5Diabetes and dysfunction induced by Ang II were similar in Cardiovascular Research Center and Medicine, EnNaC alpha KO treated with tempol and Ang II Medical Pharmacology and Physiology, and compared to EnNaC KO mice infused with Ang II Research Service Harry S Truman Memorial without tempol. These results suggest a role for Hospital, Univ. of Missouri; 6Dalton oxidative stress as both an upstream regulator Cardiovascular Research Center, and Medical and a downstream mediator of EnNaC signaling Pharmacology and Physiology, Univ. of in Ang II mediated aortic stiffening and Missouri; 7Cordliers Research Center, Sorbonne endothelial dysfunction. In summary, these data Univ. demonstrate a critical role for oxidative stress in Renin-angiotensin-aldosterone system activation Aldo and Ang II mediated EnNaC activation, promotes accelerated arterial stiffening in obese endothelial cell stiffness, impaired endothelium- men and women. Importantly, arterial stiffening dependent vascular relaxation and heightened increases the risk of cardiovascular and chronic arterial stiffness. kidney disease. There is emerging evidence that activation of the epithelial sodium channel in endothelial cells (ECs) (EnNaC) promotes endothelial stiffness and impaired nitric oxide (NO) production, which, in turn, contributes to arterial remodeling, inflammation and stiffening. However, the role that mineralocorticoid receptor and angiotensin II (Ang II) receptor type 1 signaling leads to EnNaC activation in arterial stiffening is not fully understood. Thereby, we hypothesized that deletion of the alphsubunit of EnNaC would decrease both aldosterone (Aldo) and Ang II- mediated EnNaC activation with consequent improvement in EC stiffening and NO mediated relaxation. Aldo (250 ng/kg/day)and Ang II (500 ng/kg/day) were
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12.5 treatment with FIN, we confirmed increased The novel non-steroidal MR antagonist expression of UCP-1 and PGC1-alpha in FIN- finerenone improves metabolic parameters via treated adipocytes, as observed in vivo. ATGL-mediated lipolysis of brown adipose Furthermore, we analyzed mitochondrial tissue in high-fat diet fed mice dynamics (fission/fusion), a process potentially Vincenzo Marzolla1, Alessandra Feraco1, Stefania affecting mitochondrial abundance and Gorini1, Caterina Mammi1, Carmen Marrese1, efficiency. In particular, we observed a reduction Carla Boitani2, Peter Kolkhof3, Marc Lombes4, of mitochondrial fission with concomitant Andrea Armani1, Massimiliano Caprio1 reduction of mitophagy in FIN-treated T37i cells, 1Laboratory of Cardiovascular Endocrinology, suggesting that FIN is able to preserve the IRCCS San Raffaele Pisana; 2Dept. of integrity of mitochondria. We hypothesize that Anatomical, Histological, Forensic and free fatty acids, derived from increased ATGL- Orthopedic Sciences, Sapienza Univ. of Rome; mediated lipolysis, could increase fatty acids 3Dept. of Cardiovascular Research, Bayer AG; oxidation and energy expenditure, determining 4ac Med Paris Sud, Univ. Paris Sud, INSERM favorable metabolic effects in high-fat diet fed UMR_S U1185 mice. Herein, we evaluated metabolic effects of the novel non-steroidal MR antagonist Finerenone 13: SYMPOSIUM 5B: MR STRUCTURE AND (FIN) on adipose tissues in a mouse model of ROLE IN THE HEART AND LUNGS diet-induced obesity (HFD, with 60% kcal from fat). After 3 months of normal diet (ND) or HFD+/- 13.1 FIN (0.1 g/kg of diet), C57BL6 male mice fed Enhanced endothelium epithelial sodium HFD showed increased body weight and channel signaling prompts left ventricular impaired glucose tolerance. HFD+FIN group did diastolic dysfunction in obese female mice not show changes in body weight gain, Guanghong Jia1, Javad Habibi1, Annayya R compared with HFD group. FIN treatment Aroor1, Vincent DeMarco1, Yan Yang2, Guido improved glucose tolerance, as shown by a Lastra1, Camila Manrique1, Michael A Hill3, Adam significant reduction of the area under curve of Whaley-Connell4, Frederic Jaisser5, James R intraperitoneal glucose tolerance tests at 20 min Sowers1 after glucose injection. Histological analysis of 1Medicine-Endocrinology, Univ. of Missouri brown adipose tissue (BAT) showed increased School of Medicine; 2Dalton Cardiovascular multilocularity in the HFD+FIN group, as Research Center, Univ. of Missouri School of compared to the HFD group. Differently, FIN did Medicine; 3Dalton Cardiovascular Research not affect adipocyte size in white adipose Center, Univ. of Missouri; 4Medicine, Univ. of depots, suggesting a BAT-specific effect. We Missouri School of Medicine; 5Cordeliers then analyzed mRNA and protein expression of Research Center, Sorbonne Univ. UCP-1 and PGC1-alpha, showing a significant Increased activation of membrane epithelial increase of both markers in BAT of the HFD+FIN sodium channels in endothelial cells (ECs) group. Interestingly, we observed a significant (EnNaC) occurs in states of cardiovascular increased phosphorylation of AMPK at T172 in oxidative stress, increased vascular stiffness and BAT of the HFD+FIN group. Such increase was impaired nitric oxide mediated cardiovascular associated with activation of adipose triglyceride relaxation. Our previous studies in female mice lipase (ATGL) phosphorylation at S404 and a fed a Western diet (WD) have shown that concomitant inhibitory phosphorylation of activation of EC mineralocorticoid receptor hormone sensitive lipase (HSL) at S565. In order signaling promotes expression and translocation to better understand FIN effects in BAT, we of EnNaC to the surface of ECs contributing to wondered if FIN was able to affect adipose arterial pro-fibrotic and inflammatory responses differentiation of T37i brown adipocytes. In this as well as macrophage infiltration and M1 cell line, after 7 days of differentiation along with polarization. Emerging information also supports
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EnNaC involvement in high salt/DOCA-induced 13.2 cardiac fibrosis in non-hypertensive rodents. Structural determinants of activation of the However, a specific role for EnNaC signaling in mineralocorticoid receptor: an evolutionary the development of cardiac stiffness and perspective diastolic dysfunction has not been examined in Peter Fuller1 the setting of diet-induced obesity (DIO). 1Centre for Endocrinology and Metabolism, Accordingly, we hypothesized that inhibition of Hudson Institute of Medical Research EnNaC activation with amiloride or endothelium The mineralocorticoid receptor (MR) has been specific deletion of the α subunit of EnNaC highly conserved across vertebrate evolution. (αEnNaC-/-) would prevent cardiac stiffening and the zebra fish (Danio rerio) (z)MR for instance diastolic dysfunction with DIO in female mice. shares ~50% identity with the human (h)MR. In Female αEnNaC KO (EnNaC-/-), wild-type terrestrial vertebrates the MR mediates the littermate and C57BL6/J females were fed a WD regulation of sodium homeostasis by containing high fat (46%), sucrose (17.5%), and aldosterone (aldo) and also acts as a receptor high fructose corn syrup (17.5%) for 12-16 weeks. for cortisol (or corticosterone in rodents). The C57BL6/J females were treated with or without MR also binds progesterone (prog) which acts as a low dose of amiloride (1mg/kg/day). Low dose an antagonist of the hMR; the commonly used amiloride administration attenuated WD-induced MR antagonist, spironolactone (spiro), is based impairment of left ventricular initial filling rate, on the prog molecule. A curious feature of relaxation time, and cardiac relaxation as corticosteroid receptor evolution is that although measured by high resolution magnetic the MR is present in teleosts (ray-finned fish), resonance imaging (MRI). Further, this DIO these lack aldo synthesis. Despite this, teleost induced cardiac diastolic dysfunction was MR respond to aldo and cortisol but in contrast associated with increases in cardiac oxidative to the hMR, the teleost MR is activated by both stress, activation of pro-fibrotic signaling TGF-β prog and spiro. We sought to define the 1, and interstitial fibrosis. Amiloride molecular basis of these divergent responses of administration prevented WD-induced increases the zMR and hMR to spiro and prog using MR in endothelium permeability and this effect of chimeras and molecular modeling. A series of amiloride was associated with decreased zMR:hMR chimerias coupled with reciprocal site- expression of claudin-5 and occluding and a directed mutagenesis of putative critical amino reduction of total MΦ recruitment (CD11b) and acids in the two MR was screened using a M1 polarization (CD11c). Furthermore, αEnNaC-/- conventional transactivation assay in CV-1 cells prevented DIO-induced increases in inward and then confirmed in a zebra fish cell line (ZF-4 sodium currents in isolated pulmonary ECs. The cells incubated at 28C). Each MR DIO induced abnormalities occurred in chimera/mutant was examined using aldo or conjunction with in vitro cardiomyocyte spiro/prog alone, or both together. The stiffening (Atomic force microscopy) as well as structural consequences of the critical increased left ventricular filling pressure and differences were assessed using molecular myocardial performance index examined by in dynamic simulation (MD) based on the crystal vivo Doppler in αEnNaC wild-type littermates on structures of the MR ligand-binding domain. a WD. Collectively, these findings support the Substitution of a leucine (amino acid 856 in the notion that DIO related EnNaC activation zMR) by threonine (amino acid 870 in the hMR) mediates coronary artery endothelium in helix 8 of the ligand-binding domain of the dysfunction which, in turn, promotes cardiac zMR confers the antagonist response to oxidative stress and proinflammatory responses, prog/spiro. This leucine is conserved across fish resulting in cardiomyocyte stiffness, followed by species whereas threonine (serine in rodents) is cardiac fibrosis and diastolic dysfunction in conserved in terrestrial vertebrate MR. This females. difference is not a product of the heterologous system in that an agonist response to prog with
71 2019 APS ALDOSTERONE AND ENAC IN HEALTH AND DISEASE: THE KIDNEY AND BEYOND ABSTRACTS OF INVITED AND VOLUNTEERED PRESENTATIONS the zMR is also seen in a homologous system, adopts a similar quaternary arrangement after the ZF-4 cells. MD identified an interaction of the receptor activation. To that end we used a leucine in helix 8 with a highly conserved fluorescence imaging technique, Number & leucine in helix 1 that stabilises the agonist Brightness (N&B), in a system where receptor- conformation including the interaction between DNA interaction can be studied in live cells in helices 3 and 5, an interaction which has real time. Our results show that MR adopts a previously been characterised. This switch in the tetrameric organization in the nucleoplasm upon MR coincides with the evolution of terrestrial agonist binding (aldosterone or corticosterone), vertebrates and of aldosterone synthesis. It was suggesting that unlike GR, MR does not need perhaps mandatory if the appearance of DNA binding to form tetramers. MR interaction aldosterone as a specific mediator of the with a gene target promotes quaternary homeostatic salt retention was to be tolerated. complexes with an average of 7 receptor units. The conformational changes also provide novel Interestingly, an MR antagonist, spironolactone, insights into the structural basis of agonism induced dimerization, but not tetramerization in versus antagonism in steroid receptors, with the nucleoplasm. With corticosterone, GR can potential implications for drug design in this incorporate to MR complexes partially displacing important therapeutic target. In addition we have MR monomers. The structural determinants of confirmed that this agonist response to prog in MR oligomerization were studied using different the zMR is observed in a homologous system mutants. Truncation of the LBD only partially which argues that prog is indeed a physiological reverts higher order oligomerization, consistent agonist for the teleost MR with a corollary that with previously reported data showing that studies in fish using spiro as an MR antagonist similar mutants retain residual activity. Mutant should be interpreted with caution. P654R, analogous to mutant P481R mimicking DNA binding in GR, increases the number of MR 13.3 monomers forming a complex in the The quaternary structure of the nucleoplasm after aldosterone stimulation, but mineralocorticoid receptor depends on ligand not at HREs. Our results indicate that the nature and DNA binding of the ligand (agonist or antagonist) and specific Diego Alvarez de la Rosa1,2, Gregory Fettweis2, binding to HRE determines the final quaternary Diana A. Stavreva2, Thomas A. Johnson2, structure of active MR. Gordon L. Hager2 1Dept. of Physiology, Univ. de La Laguna; 13.4 2Laboratory of Receptor Biology and Gene Vascular cell-specific roles of mineralocorticoid Expression, National Cancer Institute receptors in pulmonary hypertension The mineralocorticoid and glucocorticoid Ioana Preston1, Divya Padmanabhan Menon1, receptors (MR and GR) are evolutionary related Guanming Qi1, Seung Kyum Ki2, M. Elizabeth nuclear receptors with high sequence Moss2, Krishna C Penumatsa1, Rod W conservation in the ligand- and DNA-binding Warburton1, Deniz Toksoz1, Jamie Wilson1, domains (LBD and DBD). Both receptors are Nicholas S Hill1, Iris Z Jaffe2 activated by glucocorticoids, but MR can be 1Pulmonary, Critical Care and Sleep, Tufts selectively activated by aldosterone. In addition, Medical Center; 2Molecular Cardiology both receptors bind a shared hormone response Research Institute, Tufts Medical Center element (HRE) with the same DNA consensus Abnormalities that characterize pulmonary sequence. MR and GR physically and arterial hypertension (PAH) include an functionally interact, potentially contributing to impairment in the structure and function of cell type-specific corticosteroid signaling. It has pulmonary vascular endothelial (EC) and smooth recently been proposed that DNA binding muscle cells (SMC). Aldosterone levels are promotes liganded GR self-association as elevated in human PAH and in experimental tetramers. Here we investigated whether MR pulmonary hypertension (PH), while inhibition of
72 2019 APS ALDOSTERONE AND ENAC IN HEALTH AND DISEASE: THE KIDNEY AND BEYOND ABSTRACTS OF INVITED AND VOLUNTEERED PRESENTATIONS the aldosterone-binding mineralocorticoid preserved ejection fraction (HFpEF) and receptor (MR) attenuates PH in multiple animal spironolactone is the drug of choice in patients models. This study explored the specific role of with resistant hypertension . The use of these s- MR in ECs and SMCs in PH using cell specific MRAs has however been limited by the MR knockout mice exposed to sugen/hypoxia- occurrence of hyperkalemia (HK). Despite a induced PH. MR inhibition with spironolactone, class 1 indication in international guidelines for at a dose that does not impact systemic blood patients with HFrEF the s-MRAs are often not pressure, significantly reduces right ventricular initiated due to the fear of inducing HK and (RV) systolic pressure. However, this is not when initiated are often discontinued within the reproduced by MR deletion in either SMCs or first several due to HK. New nonsteroidal MRAs ECs. Similarly, MR inhibition attenuates the (ns-MRAS) such as Finerenone are under increase in RV cardiomyocyte area independent investigation and appear to be at least as of vascular MR with no effect on RV weight or effective as the s-MRAs in patients with HFrEF interstitial fibrosis. RV perivascular fibrosis is as well as those with diabetes mellitus (DM) and significantly decreased by spironolactone and is or chronic kidney disease (CKD) but with a lower reproduced by specific deletion of MR from ECs. incidence of HK. These ns-MRAs if shown to be MR inhibition significantly reduces pulmonary effective in patients with HF, DM and or CKD arteriolar muscularization, independent of EC- could further reduce cardiovascular mortality. MR or SMCMR. Finally, the degree of Alternatively new potassium binding agents perivascular inflammation in pulmonary vessels such as patiromer or lokalma may be used in is attenuated by MR antagonism and is fully conjunction with the s-MRAs to treat and or reproduced by SMC-specific MR deletion. These prevent HK. The potassium binder sodium studies demonstrate that, in the sugen/hypoxia polystyrene sulfate (SPS) has been available for PH model, systemic-MR blockade significantly many years but its use has been limited by the attenuates the disease and vascular MR has cell- occurrence of bowel necrosis and the and tissue-specific effects, with EC-MR occurrence of edema and loss of blood pressure contributing to RV perivascular fibrosis and control in patients with hypertension due to the SMC-MR stimulating pulmonary perivascular fact that it exchanges potassium for sodium. The inflammation. The role of MR in pulmonary new potassium binding agents have recently vascular muscularization and RV cardiomyocyte become available for clinical use and are not hypertrophy may be mediated by MR in other associated with bowel necrosis. There are cell types such as cardiomyocytes and/or however important differences between the new inflammatory cells. potassium binders –patiromer and lokalma – in that patiromer exchanges potassium for calcium 14: CLINICAL PLENARY LECTURE and lokalma exchanges potassium for sodium , although less than SPS. The availability of the 14.1 new potassium binders along with the likely The role of non- steroidal MR antagonists and availability of new ns-MRAs holds the promise of new potassium binders for the treatment of more effective blood pressure control in patients cardiovascular disease with resistant hypertension with DM and or CKD, Bertram Pitt1 prevention of the progression of CKD, as well as 1School of Medicine, Univ. of Michigan a further reduction in mortaity and The steroidal mineralocorticoid receptor hospitalizations for heart failure in patients with antagonists (s- MRAs) spironolactone and HFrEF and HFpEF. eplerenone have been shown to be effective in reducing mortality and hospitalizations for heart failure in patients with heart failure and a reduced ejection fraction (HFrEF), are likely effective in patients with heart failure and a
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15: SYMPOSIUM 6A: NORMAL AND These findings demonstrate that activation of PATHOGENIC REGULATION OF fasciculata Gq signaling is sufficient to ALDOSTERONE BIOSYNTHESES disorganize normal adrenocortical CYP11B2 expression and suggest that the Gq pathway 15.1 plays an important role in differentiation of Development of an inducible mouse model of glomerulosa to fasciculata. This transgenic aldosteronism mouse also provides an inducible and reversible William Rainey1, Mathey Taylor1 in vivo model of hyperaldosteronism for the 1Molecular and Integrative Physiology, Univ. of testing of primary aldosteronism therapeutics Michigan (including inhibitors of CYP11B2). Adrenal steroid production relies on regulated expression of five cytochrome P450s that direct 15.2 steroid production toward mineralocorticoids or A gain of function mutation in CLCN2 chloride glucocorticoids. Differential expression of the channel gene causes primary aldosteronism steroid biosynthetic enzymes, including the Fabio Fernandes-Rosa1 P450s, determines the zone-specific production 1PARCC, INSERM U970 of steroid hormones. The zona glomerulosa Primary aldosteronism is the most common and produces aldosterone due to its sole expression curable form of secondary arterial hypertension. of aldosterone synthase (CYP11B2), while the Germline mutations in genes coding for ionic zona fasciculata produces cortisol due to channels were associated to familial and early- expression of the closely related 11β - onset forms of PA, but the pathophysiology of a hydroxylase (CYP11B1). Normally CYP11B2 and large proportion of cases is still unknown. To aldosterone production are under tight control identify new genes involved in primary of the renin-angiotensin II (AngII) system. aldosteronism we performed whole exome Dysregulated expression of adrenal CYP11B2 sequencing in germline DNA from 12 patients and aldosterone production leads to the most with young onset hypertension and PA common adrenal disease, primary diagnosed before age 25 ys. Two index cases aldosteronism. Primary aldosteronism results were investigated together with their parents from renin-independent expression of CYP11B2. and unaffected sibling to search for de novo Because of its clinical significance, we have variants. A de novo heterozygous focused on defining the normal and pathologic c.71G>A/p.Gly24Asp mutation was identified in mechanisms controlling CYP11B2 expression. the CLCN2 gene, coding for the voltage-gated The predominant hormonal regulator of ClC-2 chloride channel, in a patient with CYP11B2 is AngII, which acts through hypertension and hypokalemia due to primary glomerulosa-specific Gq signaling pathways. aldosteronism diagnosed at age 9 ys. CLCN2 is Our working hypothesis has been that Gq expressed in the human and mouse adrenal signaling is the dominant regulator of cortex and patch-clamp analysis of glomerulosa glomerulosa differentiation and its capacity to cells of mouse adrenal gland slices revealed express CYP11B2. Recently, we generated hyperpolarization-activated Cl- currents that transgenic mice with adrenal cortex expression were abolished in Clcn2-/- mice. The ClC-2 of Gq-coupled designer receptors exclusively p.Gly24Asp mutation, located in a well activated by designer drugs (DREADDs). conserved inactivation domain of the chloride Adrenal-wide ligand activation of Gq DREADD channel, abolished the voltage- and time- triggered a disorganization of adrenal functional dependent gating of ClC-2. The resulting almost zonation with induction of CYP11B2 in CYP11B1- ohmic currents strongly increased the Cl- expressing fasciculata cells. This resulted in conductance at resting potentials. Expression of renin-independent aldosterone production and ClC-224Asp in adrenocortical H295R-S2 cells hypertension. All parameters were reversible led to increased aldosterone production, which following termination of DREADD Gq activation. is paralleled by increased expression of
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CYP11B2, coding for aldosterone synthase. This biosynthesis. miRNAs are a class of non-coding involves plasma membrane depolarization, small RNAs, 21–23 nucleotides in length, that opening of voltage-gated calcium channels and bind the 3’ untranslated region of target increased calcium influx through voltage-gated messenger RNAs, resulting in down-regulation calcium channels. Conversely, knock-down of of gene expression through inhibition of endogenous ClC-2 by shRNA diminished translation or promotion of messenger RNA aldosterone production and reduced CYP11B2 degradation. In higher eukaryotes, miRNA expression. In conclusion, a gain-of-function impact on gene expression may be comparable mutation in the ClC-2 chloride channel underlies to that of transcription factors, and they have a genetic form of secondary arterial been proposed to act as ‘genetic hormones’, hypertension. Increased Cl- currents induced by acting on tissues far from their site of the ClC-2 p.Gly24Asp mutation depolarize the production. Finally, their release into the zona glomerulosa cell membrane, thereby circulation, has led to their emergence as opening voltage-gated calcium channels which biomarkers for certain conditions. We propose trigger autonomous aldosterone production by that circulating miRNAs represent a minimally- increasing intracellular Ca2+ concentrations. invasive biomarker that will enable accurate These results highlight for the first time the diagnosis of PA. The ENS@T-HT project is important role of chloride in aldosterone designed to define specific ‘omics’ signatures biosynthesis and identify ClC-2 as the foremost for endocrine hypertension. Our focus has been chloride conductor of resting glomerulosa cells. the profiling of circulating miRNAs in plasma, in order to identify those of diagnostic value and 15.3 also highlight novel biological pathways relevant Circulating MicroRNAs as diagnostic biomarkers to PA. Initial miRNA profiling is now complete of primary aldosteronism and preliminary analysis confirms differential Eleanor Davies1, Josie van Kralingen1, Casper miRNA expression in PA, essential Larsen2, Scott M MacKenzie1, Stacy Robertson1, hypertensives and normotensive subjects. John D McClure1, Parminder S Reel3, Smarti Confirmation of the diagnostic miRNA signature Reel3, Emily R Jefferson3, Maria-Christina is now being tested in a prospective study Zennaro 2 population. Our findings indicate the potential 1ICAMS, Univ. of Glasgow; 2Paris Cardiovascular for circulating miRNAs as diagnostic biomarkers Research Centre, INSERM UMRS_970; 3Health for PA. As we identify those miRNAs that show Informatics Centre, Univ. of Dundee. Members the most marked and consistent dysregulation in of the ENSAT-HT Consortium PA we will turn our attention to their biological Aldosterone is a key cardiovascular hormone roles in order to gain insight into its pathology. which, when present in excess, accounts for This will complement our previous studies of the substantial cardiovascular morbidity. Its adrenal cortex, which have shown that overproduction in primary aldosteronism (PA) is components of the aldosterone biosynthetic detected in ~10% of hypertensives although this pathway are directly targeted by miRNAs. By figure may increase significantly if simpler, less combining our studies of tissue and circulating expensive, non-invasive diagnostic techniques miRNAs, we hope to gain fresh insight into the were available. Timely and accurate detection of development of PA and identify potential targets aldosterone excess, combined with a for therapeutic intervention. comprehensive understanding of the mechanisms underlying aldosterone biosynthesis are therefore key to preventing the adverse cardiovascular effects. MicroRNAs (miRNAs) have emerged as key regulators of human gene expression, affecting numerous biological processes including aldosterone
75 2019 APS ALDOSTERONE AND ENAC IN HEALTH AND DISEASE: THE KIDNEY AND BEYOND ABSTRACTS OF INVITED AND VOLUNTEERED PRESENTATIONS
15.4 structure and function, we have explored the The retinoic acid receptor a contributes to the adrenal phenotype of 12 and 52 weeks old male development of primary aldosteronism by and female Rarα knockout mice. Inactivation of regulating adrenal cortex structure through Rarα in mice led to major structural and interactions with Wnt and Vegfa signaling functional disorganization of the adrenal cortex Sheerazed Boulkroun1,2, Rami M El Zein1,2, in both sexes, with modifications of the vessel Audrey H Soria1,2, Jose Felipe Golib Dzib3, architecture and extracellular matrix and Amanda J. Rickard1,2, Fabio L Fernandes-Rosa1,2, reduced expression of steroidogenic genes. Benoit Samson-Couterie1,2, Isabelle Giscos- These abnormalities were due to increased Douriez1,2, Angelique Rocha1,2, Marko Poglitsch4, proliferation, decreased Vegfa expression and Celso E Gomez-Sanchez5, Laurence Amar1,2,6, modifications in extracellular matrix Norbert B Ghyselinck7, Arndt Benecke3, Maria- components. Rarα inactivation reduces non- Christina Zennaro1,8 canonical Wnt signaling, without affecting the 1PARCC, INSERM U970; 2Univ. de Paris; 3Centre canonical Wnt pathway nor PKA signaling. National de la Recherche Scientifique (CNRS), Adrenal cortex disorganization persisted with Institut des Hautes Etudes Scientifiques; aging, while molecular abnormalities affecting 4Attoquant Diagnostics; 5Division of Wnt signaling and Vegfa or steroidogenic gene Endocrinology, G.V. (Sonny) Montgomery VA expression regressed. Conclusions: Our study Medical Center and Univ. of Mississippi Medical suggests that Rarα contributes to normal Center; 6Assistance Publique-Hôpitaux de Paris, adrenal cortex development, by modulating Wnt Hôpital Européen Georges Pompidou, Unité and Vegfa signaling. We propose a model in Hypertension artérielle; 7Institut de Génétique et which interaction between retinoic acid, Wnt and de Biologie Moléculaire et Cellulaire (IGBMC), Vegf signaling pathways is required for normal Département de Génétique Fonctionnelle et development of the adrenal cortex. Cancer; CNRS, UMR7104; INSERM U1258, Univ. Dysregulation of this interaction may contribute de Strasbourg (UNISTRA); 8Assistance Publique- to abnormal cell proliferation and APA Hôpitaux de Paris, Hôpital Européen Georges development in human, creating a propitious Pompidou, Service de Génétique environment for the emergence of specific Rationale: Primary aldosteronism (PA) is the driver mutations in APA. most frequent form of secondary arterial hypertension. While somatic mutations in 16: SYMPOSIUM 6B: INTEGRATED aldosterone producing adenomas (APA) are REGULATION OF RENAL ION responsible for increased aldosterone TRANSPORT production, adenoma formation may involve additional mechanisms. The identification of new 16.1 determinants of APA development may offer The role of ENaC in hyperoxia-induced preterm new clues for the care of patients with lung injury. secondary hypertension. Objective: To identify My Helms1, Garett Grant1, Matthew Gaffrey2, mechanisms involved in the development of PA Tong Zhang2, Wei-Jun Qian2 and, in particular, specific signaling pathways 1Internal Medicine, Univ. of Utah; 2Division of responsible for abnormal cell proliferation and Biological Science, Pacific Northwest National APA formation. Method and Results: We have Laboratory performed a transcriptome analysis on 48 APA Significance: Oxygen supplementation is an and investigated correlations of gene important component of intensive care for expression with their genetic, morphological and preterm infants and many patients with water in functional characteristics. We identified retinoic the lungs. Preterm lungs have 25% more water acid receptor alpha (RARα) signaling as a central in the lungs than term infants. ENaC serves as molecular network involved in APA formation. the rate limiting step for net salt and water To understand how RARα modulates adrenal transport across an epithelium, and therefore,
76 2019 APS ALDOSTERONE AND ENAC IN HEALTH AND DISEASE: THE KIDNEY AND BEYOND ABSTRACTS OF INVITED AND VOLUNTEERED PRESENTATIONS plays an important role in resolving water in the significantly decreased alveolar fluid clearance lungs. Our lab has shown that during high O2 and increased lung injury. A more pronounced exposure, the level of glutathione disulfide decrease in the levels of S-glutathionylation in (GSSG) is significantly increased, and that GSSG response to hyperoxic conditions were significantly decreases ENaC activity. Herein, we observed in Scnn1b mice than those from WT. investigate whether chronic O2 treatment Conclusion: Scnn1b over-expression in mouse contributes to lung injury via S-glutathionylation lung increases ENaC activity, which translates dependent down-regulation of ENaC using a into improved fluid clearance rates and mouse model of preterm lung injury. Results protection from hyperoxia-induced injury. S- from our studies could lead to new treatment glutathionylation plays a potential significant options for O2-induced lung injury, and role in such protection. Future studies will addresses the unmet clinical needs of preterm evaluate the role of S-glutathionylated proteins infants. Hypothesis: We hypothesize that S- in the regulation of lung fluid clearance, and glutathionylation dependent down-regulation of evaluate the therapeutic value of reversing lung ENaC activity plays an important role in GSH/GSSG redox potentials in hyperoxia- hyperoxia-induced lung injury. Methods: We induced lung injury. Funding: R01HL137033-01 employed the term mouse model of hyperoxia- induced lung injury, and a gain-of-ENaC function 16.2 C57Bl6 mouse model which overexpresses the Regulation of renal ion transport and blood Scnn1b gene (referred to as Scnn1b hereafter). pressure by the CRL3-WNK-SPAK pathway Scnn1b and WT litter mates were maintained on James McCormick1 85% or 21% O2 from birth up to postnatal days 1Medicine (Nephrology and Hypertension), (PN) 11-15. A subset of normoxic WT C57Bl6 mice Oregon Health and Science Univ. were nasally insufflated with 400 μM GSSG from The rare disease Familial Hyperkalemic PN 1-11. Lung injury and development were Hypertension (FHHt) primarily involves assessed by morphometric analysis (radial dysregulation of the thiazide-sensitive Na+-Cl- alveolar counts) of Masson’s trichrome stained Cotransporter (NCC), expressed along the distal tissue; hydroxyproline assays quantified fibrosis. convoluted tubule (DCT) in the kidney. The Evan’s blue assay was used to measure rate Identification of the genes mutated that cause of alveolar fluid clearance. Quantitative redox FHHt has revealed a complex NCC regulatory proteomic characterization of S-glutathionylation pathway. NCC is activated by phosphorylation in lung tissue was conducted using mass through the With-No-Lysine [K] (WNK) kinases spectrometry. Results: Our studies show that and Ste20-related Proline Alanine Rich kinase newborn mice maintained on 85% O2 from birth (SPAK) pathway. WNK-SPAK activity is in turn up to PN 11-15 can be used as a biologically regulated by the Cullin3 Ring Ligase (CRL3) relevant model for O2-induced lung injury since ubiquitin protein-ligase complex, which chronic hyperoxia correlated with increased modulates WNK abundance. Our studies using fibrosis and attenuated lung development. genetically modified mice have provided several Scnn1b mice were protected from O2 injury; insights into CRL3-WNK-SPAK function in vivo. Masson’s Trichrome staining showed less We showed that disruption of SPAK, which fibrotic tissue in hyperoxic Scnn1b mice directly phosphorylates NCC, mimics the human compared to hyperoxic WT littermates. Radial disease Gitelman syndrome caused by alveolar counts (a reliable measure of lung inactivating NCC mutations (Cell Metab 2011). maturation) increased from 8.1± 0.29 in We identified a kidney-enriched truncated form (hyperoxia) WT pups to 9.9± 0.35 in (hyperoxia) of SPAK (KS-SPAK) which lacks the kinase Scnn1b pups. Overexpression of lung ENaC, domain and competitively inhibits activity of under both normoxic and hyperoxic conditions, SPAK and the closely related kinase Oxidative correlated positively with increased alveolar Stress Response 1 (OSR1) (Cell Metab 2011 and fluid clearance. Nasal insufflation of GSSG Am J Physiol Renal Physiol 2013). In vivo we
77 2019 APS ALDOSTERONE AND ENAC IN HEALTH AND DISEASE: THE KIDNEY AND BEYOND ABSTRACTS OF INVITED AND VOLUNTEERED PRESENTATIONS found that KS-SPAK is expressed along the renal spectrometry to measure WNK kinase domain thick ascending limb where it may inhibit OSR1- thermal stability and autophosphorylation in vitro stimulated activity of the Na+-K+-2Cl- in the presence of varying concentrations of Cotransporter 2 (NKCC2). We also potassium. We examined the activity of DmWNK demonstrated that SPAK and OSR1 play critical (Drosophila WNK) and mammalian WNKs in the roles in potassium homeostasis (J Physiol 2016) Drosophila Malpighian tubule, using as components of the DCT plasma [K+] we phosphorylation of transgenically expressed collaboratively identified (Cell Metab 2015 and J kinase-dead rat SPAK as a readout. We Am Soc Nephrol 2017). Our recent work has developed baths with varying extracellular focused on the mechanisms by which FHHt- potassium and fixed intracellular chloride causing disrupt CRL3 function. We showed that concentrations (measured using the transgenic the FHHt-causing Cullin3 mutant inappropriately sensor ClopHensor). We measured intracellular degrades the WNK-binding CRL3 substrate potassium in the tubule using inductively adaptor KLHL3 (J Clin Invest 2014) and does not coupled plasma mass spectrometry. We simply cause FHHt by inducing its own measured transepithelial ion flux in Malpighian degradation (JCI Insight 2017). Funding: NIH tubules in which DmWNK was knocked down (NIDDK) R01DK098141 References Cell Metab. and replaced by mammalian WNK3 or WNK4, 2011 Sep 7;14(3):352-64 Am J Physiol Renal with or without Mo25. Results Potassium directly Physiol. 2013 Dec 15;305(12):F1687-96 J Clin binds to the kinase domain of DmWNK Invest. 2014 Nov;124(11):4723-36 Cell Metab. (Drosophila WNK) and human WNK1 in vitro, as 2015 Jan 6;21(1):39-50 J Physiol. 2016 Sep assayed by differential scanning fluorimetry, and 1;594(17):4945-66 J Am Soc Nephrol. 2017 inhibits autophosphorylation and activation of Jun;28(6):1814-1825 JCI Insight. 2017 Dec DmWNK and HsWNK3 (human WNK3) kinase 21;2(24) J Physiol. 2016 Sep 1;594(17):4945-66 domains. In the tubule, high potassium bath, which increases intracellular potassium 16.3 concentration, inhibits DmWNK and mammalian WNK regulation of ion transport in the WNK activity in both 16 mM and 30 mM malpighian tubule intracellular chloride conditions, and also inhibits Aylin Rodan1, Sima Jonusaite1, John Pleinis1, chloride-insensitive mammalian WNK activity. Feng Zhang1, Jason Sosa-Pagan1, John HsWNK3 rescues ion transport in DmWNK Humphreys2, Radha Akella2, Haixia He2, knockdown tubules in both basal and stimulated Elizabeth Goldsmith2 conditions. MmWNK4 (mouse WNK4) is not able 1Internal Medicine, Univ. of Utah; 2Biophysics, to rescue unless co-expressed with Mo25 in Univ. of Texas Southwestern Medical Center basal conditions, and chloride-insensitive Background and Objective Mutations in WNK MmWNK4 co-expressed with Mo25 rescues (With No Lysine (K)) kinases in mice and humans DmWNK knockdown in stimulated conditions. result in abnormal serum potassium Rescue was achieved with RnWNK1 (rat WNK1) in concentration and blood pressure. WNKs basal conditions, but not in stimulated phosphorylate and activate SPAK and OSR1 conditions, regardless of Mo25 co-expression or kinases, which phosphorylate SLC12 sodium mutating the chloride-binding site. Conclusion chloride and sodium-potassium-2-chloride Our studies uncover novel regulatory cotransporters. Our lab has shown that this mechanisms for WNK pathway signaling, cascade is conserved in the Drosophila including direct effects of potassium and Malpighian (renal) tubule, and that chloride and differential effects of Mo25 on mammalian WNK the scaffold protein Mo25 regulate pathway paralogs, which may further increase the activity 1,2. Here, we examine roles for dynamic range of WNK signaling in different potassium and Mo25 in regulating Drosophila tissues. and mammalian WNKs. Methods We used differential scanning fluorimetry and mass
78 2019 APS ALDOSTERONE AND ENAC IN HEALTH AND DISEASE: THE KIDNEY AND BEYOND ABSTRACTS OF INVITED AND VOLUNTEERED PRESENTATIONS
16.4 of current was considerably higher in the DCT2 Interleukin 6 activation of the epithelial sodium (10.04 vs.1.98 fold change/baseline), as channel in the distal convoluted tubule and compared to the CCD. Further, we found that cortical collecting duct via Rac1 with Rac1 KD, there was a complete inhibition of Oishi Paul1, Auriel Moseley1, Chang Song1, Otor IL-6 induced Na+current (-1.16 fold Al-Khalili1, Celso Gomez-Sanchez2, Douglas change/baseline) and that IL-6 did not further Eaton1, Brandi Wynne1 increase current in cells with Rac1 OE. Our data 1Medicine, Renal Division, Emory Univ.; are the first to investigate ENaC activity in the 2Pharmacology and Toxicology, Univ. of DCT2. Additionally, we demonstrate that IL-6 is Mississippi Medical Center a strong stimulus for ENaC activity in both the Hypertension (HTN) is a primary global health DCT2 and CCD. Our data suggest that Rac1 may concern. HTN is an inflammatory disease that is play an important role in IL-6 mediated characterized by excessive sodium (Na+) activation of the MR in the DCT2. Our data reabsorption and increased pro-inflammatory reveals a potential mechanism of MR activation cytokines, such as interleukin 6 (IL-6). during HTN, without increased Aldo levels. Aldosterone (Aldo) is the primary ligand for the mineralocorticoid receptor (MR), which is PHOTOGRAPH/VIDEO RECORDING: expressed along the distal nephron referred to Photo or video capture of any scientific as the aldosterone-sensitive distal nephron presentation, whether an oral or poster (ASDN). Although studies demonstrate a presentation in whole or in part, is expressly beneficial effect of MR inhibition during HTN, prohibited. Recording or taking photography Aldo levels are not always increased, of another person without their explicit suggesting an alternate MR activation pathway. permission is prohibited. The epithelial sodium channel (ENaC) is located in both the late distal convoluted tubule (DCT2) Individuals observed photographing or and cortical collecting duct (CCD), and is major videotaping any presentation, in whole or in Na+ transport protein. Data from our laboratory part, will be asked to leave the conference have shown that IL-6 can activate the MR in immediately, forfeiting the registration fee. vitro, and that pharmacological inhibition of Rac reduces mineralocorticoid response element (MRE) activation. We then hypothesized that IL-6 activates the MR via the small GTPase Rac1 in the distal nephron, increasing ENaC activity. To investigate this, we measured transepithelial voltage and resistance using a voltohmmeter (EVOM), and calculated current in both murine distal convoluted tubule (mDCT15) and cortical collecting duct (mpkCCD) cells. Cells were transfected with a vector containing wild-type (WT), Rac1 knock-down (KD) or constitutively active Rac1 (overexpression, OE) and then treated with vehicle, Aldo [100nM] or IL-6 [100ng/mL]. Amiloride was applied to confirm current was ENaC-dependent. Data are shown as a fold change of current before and after treatments (1hr). We observed a significant increase in amiloride-sensitive current in both mDCT2 and mpkCCD cells following both Aldo and IL-6 treatments. Interestingly, the magnitude
79
2019 APS ALDOSTERONE AND ENAC IN HEALTH AND DISEASE: THE KIDNEY AND BEYOND AUTHOR INDEX
*Indicates Invited Speaker
Cebotaru, Liudmila, 6.18 8.3 Kirabo, Annet, 7.4 A Chen, Chao, 2.3 Goldsmith, Elizabeth*, 16.3 Klemens, Christine, 6.2, Adler, Gail*, 3.1, 11.16 Chen, Jingxin, 11.3 Golib Dzib, Jose Felipe, 7.5 Akella, Radha*, 16.3 Chen, Yinghua, 6.16 15.4 Kleyman, Thomas*, 5.1, Al-Khalili, Otor, 11.8, 16.4 Cheng, Lei*, 7.3 Gomez-Sanchez, Celso, 6.3, 9.3, 11.10, 11.11, 11.3, Alvarez de la Rosa, Diego, Cherezova, Alena, 3.3 15.4, 16.4 11.9 13.3 Clark, Nathan L., 11.12 Gorini, Stefania, 12.5 Knöpp, Fenja, 4.3 Amar, Laurence, 15.4 Clere, Nicolas, 3.4 Grant, Garett*, 8.4, 16.1 Kolkhof, Peter, 12.5 Antonova, Galina, 3.4 Collier, Daniel, 4.5, 11.6 Korbmacher, Christoph*, Archer, Crystal, 6.16 Colombari, Eduardo, 6.12 H 5.2 Armani, Andrea, 12.5 Connolly, Kelly, 6.15 Habibi, Javad*, 12.4, 13.1 Kota, Pradeep, 9.4 Aroor, Annayya*, 6.5, 12.4, Hager, Gordon L., 13.3 Krauson, Aram*, 11.13 13.1 D Hallows, Kenneth R.*, 7.3 Kunkel, George*, 11.13 Ashley, Zoe, 6.9 Davel, Ana*, 12.1 Harwood, Daisy, 3.4 Auchus, Richard, 2.5 Davie, Tim, 11.17 Hasan, Mahpara, 11.9 L Awumey, Emmanuel, 6.10 Davies, Eleanor, 15.3 Hatcher, Katherine, 3.3 Larsen, Casper, 15.3 DeMarco, Vincent*, 12.4, He, Haixia*, 16.3 Lastra, Guido*, 12.4, 13.1 B 13.1 He, Hong, 9.4 Lazo-Fernandez, Yoskaly, Baconguis, Isabelle, 4.1 Denton, Jerod, 6.2 Helms, My*, 8.4, 16.1 2.3 Balchak, Deidra M., 11.12 Doery, James CG, 11.18 Hildebrandt, Emily, 6.14 Le, Janet, 11.8 Baldin, Jan-Peter, 4.3 Drummond, Heather, 6.14 Hill, Michael*, 6.5, 12.4, Leader, Catherine, 6.9 Bandulik, Sascha, 5.3 DuPont, Jennifer*, 12.2 13.1 Levchenko, Vladislav, 2.4 Banki, Eszter, 5.3 Hill, Nicholas, 11.1, 13.4 Li, Hui*, 7.3 Barak, Yaakov, 11.10 E Ho, Pei-Yin*, 7.3 Lieber, Austin, 11.10 Barbaro, Natalia, 7.4 Eaton, Douglas, 2.3, 4.4, Hodges, Matthew, 2.4 Lin, Daohong, 11.7 Barbour, Jenna, 11.10 11.8, 16.4 Hui, Nian*, 11.16 Liou, Theodore G., 8.4 Barth, Daniel, 4.3 El Zein, Rami M, 15.4 Hummel, Scott, 2.5 Liu, Xuefei, 6.7 Bazzell, Brian, 2.5 Elias, Simiat, 11.5 Hummler, Edith*, 6.4, 6.6 Loffing, Johannes, 5.3 Beacham, Rebecca, 6.15 El-Meanawy, Ashraf, 6.2 Humphreys, John*, 16.3 Lombes, Marc, 12.5 Belin de Chantemèle, Eric Enslow, Benjamin, 6.16 Lu, Qing*, 11.1, 11.2, 12.2 *, 3.2, 3.4, 11.4 I F Lucas, Rudolf, 11.4 Benecke, Arndt, 15.4 Isaeva, Elena, 6.2 Luther, James*, 11.16 Bhalla, Vivek*, 7.3, 9.1, Faulkner, Jessica, 3.4, 11.4 11.13, 11.17 Faure, Sebastien, 3.4 J M Bharadwaj, Arpita, 4.1 Fazan, Frederico, 6.12 Jaeger, Yannick*, 6.6 MacKenzie, Scott, 15.3 Biwer, Lauren, 11.2 Fedoriuk, Mykhailo, 6.2 Jaffe, Iris*, 11.1, 11.2, 11.4, Mamenko, Mykola, 3.3 Blair, Harry, 11.10 Fenton, Robert A.*, 7.3 12.2, 12.3, 13.4 Mammi, Caterina, 12.5 Blass, Gregory, 7.5 Feraco, Alessandra, 12.5 Jaisser, Frederic*, 12.4, Man, Joshua, 11.1, 12.3 Bohovyk, Ruslan, 6.2 Fernandes-Rosa, Fabio*, 13.1 Manis, Anna, 2.4 Boitani, Carla, 12.5 15.2, 15.4 Jaja, Smith, 11.5 Manrique, Camila*, 12.4, Bollag, Wendy, 3.4 Fettweis, Gregory, 13.3 Jefferson, Emily, 15.3 13.1 Bonventre, Joseph*, 1.1 Forst, Anna-Lena, 5.3 Jia, Guanghong*, 6.5, Marciszyn, Allison*, 6.15, Boulkroun, Sheerazed, Frateschi, Simona*, 6.4, 12.4, 13.1 6.3, 9.3, 11.10 15.4 6.6 Johnson, Thomas A., 13.3 Marrese, Carmen, 12.5 Boyd-Shiwarski, Cary, 6.15 Frindt, Gustavo*, 6.1, 6.11 Jonusaite, Sima*, 16.3 Marzolla, Vincenzo, 12.5 Fronius, Martin, 4.3, 6.9 Brands, Michael, 3.3, 7.5 K May-Gwini, Stella, 11.18 Brodsky, Jeffrey*, 9.3 Fujita, Toshiro, 2.1 McClure, John, 15.3 Kashlan, Ossama*, 4.2, Bruttini, Marco, 2.5 Fuller, Peter*, 13.2, 11.18 McCormick, James*, 16.2 11.12 Bubnov, Rostyslav, 6.13 G McDonald, Fiona, 6.9 Buck, Teresa*, 9.3 Katsurada, Kenichi, 6.7 Mechakra, Asma*, 6.6 Gaffrey, Matthew*, 16.1 Kellenberger, Stephan*, Buncha, Vadym, 3.3 Menon, Divya Butterworth, Michael*, 7.2 Gao, Zhong-Xiuzi, 11.7 6.6 Padmanabhan, 13.4 Gautschi, Ivan*, 6.6 Kennard, Simone, 11.4 Byrd, J. Brian, 2.5 Mérillat, Anne-Marie*, 6.6 Gentilcore, Clayton, 11.12 Kenney, Rachel*, 12.2 Mhamankar, Suyog, 6.17 C Ghyselinck, Norbert B, Khatri, Purvesh*, 11.13 Mistry, Monisha, 11.8 15.4 Khedr, Sherif, 2.4 Caprio, Massimiliano, 12.5 Mitchell, Zachary, 6.14 Carattino, Marcelo, 6.3 Giscos-Douriez, Isabelle, Ki, Seung Kyum, 13.4 Montalbetti, Nicolas, 6.3 Carvajal, Brigett, 11.2 15.4 Kim, Seung*, 12.2 Moseley, Auriel, 16.4 Carver, Chase, 6.16 Gleason, Catherine*, 7.1, Kim, Seung Kyum, 11.1
81 2019 APS ALDOSTERONE AND ENAC IN HEALTH AND DISEASE: THE KIDNEY AND BEYOND AUTHOR INDEX
*Indicates Invited Speaker
Moss, M. Elizabeth, 13.4 S Wang, Yanhua, 2.3 Moss, Mary E., 11.1 Saha, Bidisha*, 7.1, 8.3 Warburton, Rod R., 11.1 Mugloo, Sama, 6.9 Sammut, Ivan, 6.9 Warburton, Rod W., 13.4 Murray, Gillian*, 11.16 Samson-Couterie, Benoit, Warth, Richard, 5.3 Mutchler, Stephanie*, 6.3, 15.4 Weaver, Claire, 6.15 9.3, 11.10, 11.11, 11.9 Schaffert, Steven*, 11.13 Weigert, Maria, 5.3 Sergi, Chloe*, 6.6 Weng-Choy, Kay, 11.18 Wengi, Agnieszka, 5.3 N Shalygin, Alexey, 6.2 Shen, Jimmy, 11.18 Whaley-Connell, Adam*, Nizar, Jonathan, 11.17 Sheng, Shaohu, 11.3 12.4, 13.1 Noreng, Sigrid, 4.1 Shi, Shujie, 6.3 Williams, Gordon*, 3.1 O Shiwarski, Daniel, 6.15 Williford, Noah, 4.5 Wilson, Jamie, 13.4 Okechukwu, Charles, 6.10 Snyder, Peter, 4.5, 11.6 Sofola, Olusoga, 11.5 Winfrey, Aaliyah, 11.10 P Solanki, Pravik, 11.18 Wu, Peng, 8.3, 11.7 Paine III, Robert, 8.4 Song, Chang, 11.8, 16.4 Wynne, Brandi, 4.4. 11.8, Palmer, Lawrence*, 6.1, Sørensen, Mads*, 7.1, 8.3 16.4 6.11 Soria, Audrey H, 15.4 Y Palygin, Oleg, 2.4, 6.2, 7.5 Sosa-Pagan, Jason*, 16.3 Yanda, Muraly, 6.18 Sowers, James*, 6.5, 12.4, Patel, Kaushik, 6.7 Yang, Jun, 11.18 13.1 Paul, Oishi, 4.4, 16.4 Yang, Lei*, 6.1, 6.11 Pearce, David*, 7.1, 8.3 Spivak, Mykola, 6.13 Yang, Yan*, 6.5, 12.4, 13.1 Staruschenko, Alexander, Peng, Dungeng*, 11.16 Yoshioka, Craig, 4.1 2.4, 6.2, 7.5 Penton Ribas, David, 5.3 Yu, Chang*, 11.16 Penumatsa, Krishna, 11.1, Staub, Olivier*, 9.2, 11.7 Yue, Qiang, 2.3, 4.4, 11.8 13.4 Stavreva, Diana A., 13.3 Peterson, Zerubbable, 4.5 Stec, David, 6.14 Z Peyrollaz, Thibaud*, 6.4, Stockand, James, 6.16 Zennaro, Maria-Christina, 6.6 Stutts, M. Jackson, 9.4 15.3, 15.4 Pham, Truyen, 2.3 Su, Xiaotong, 11.7 Zhang, Feng*, 16.3 Pitt, Bertram*, Subramanya, Arohan*, Zhang, Tong*, 16.1 Pitzer, Ashley, 7.4 6.15, 8.1 Zhe, Sun, 12.4 Pleinis, John*, 16.3 Sullivan, Jennifer, 3.4 Zheng, Hong, 6.7 Poglitsch, Marko, 15.4 T Zocco, Davide, 2.5 Pojoga, Luminita*, 3.1 Posert, Richard, 4.1 Tan, Roderick, 11.9 Preston, Ioana, 11.1, 13.4 Tarran, Robert*, 8.2 Taylor, Mathey*, 15.1 Q Thumova, Monika, 2.3 Qi, Guanming, 11.1, 13.4 Tletlepantzi, Stacy, 11.14 Qian, Wei-Jun*, 16.1 Toksoz, Deniz, 13.4 Qiao, Yanli*, 11.13 Turcu, Adina*, 11.16 R V Rainey, William*, 2.5, 15.1 Vaidya, Anand*, 2.2 Ray, Evan*, 9.3, 11.10 van Kralingen, Josie, 15.3 Reel, Parminder, 15.3 Verlander, Jill, 2.3 Reel, Smarti, 15.3 Vijayan, Deepika, 6.4 Ren, Yilin, 4.4 Vohra, Twinkle, 5.3 Rickard, Amanda J., 15.4 W Robertson, Stacy, 15.3 Rocha, Angelique, 15.4 Walker, Rob, 6.9 Rodan, Aylin*, 16.3 Wall, Susan, 2.3, 4.4 Romero, Cesar, 2.3, 4.4 Wang, Hong, 4.4 Romero, Jose*, 3.1 Wang, Wenhui, 11.7 Rossier, Bernard, 5.4 Wang, Wen-Hui, 8.3 Ruan, Ye Chun, 11.15 Wang, Xueping, 11.12 Ryan, Alan, 4.5, 11.6 Wang, Xueqi, 6.3, 11.3
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Meetings and Conferences Ad - October 2019 8.5x11.indd 1 9/6/2019 11:10:19 AM
Addendum to the Program Book
Program Updates:
Thursday, October 3, 2019 8:00 a.m. – 2.1 Aldosterone-independent activation of mineralocorticoid receptor in salt- 8:30 a.m. sensitive hypertension and glomerular diseases Toshiro Fujita, The Univ. of Tokyo New Title: Aldosterone-independent MR activation in salt-sensitive hypertension
Saturday, October 5, 2019 8:00 a.m. – 12.0 Symposium 5A: MR in the vasculature 10:00 a.m. Pavilion Theater, 1st Floor Chairs: Frederic Jaisser, INSERM *Jun Yang, Monash Univ. (Replacing Iris Jaffe, Tufts Medical Center)
9:30 a.m. – 12.5 The novel non-steroidal MR antagonist finerenone improves metabolic 9:45 a.m. parameters via ATGL-mediated lipolysis of brown adipose tissue in high-fat diet fed mice Massimiliano Caprio, San Raffaele Roma Univ. Replacing Vincenzo Marzolla, IRCCS San Raffaele Pisana
Sunday, October 6, 2019 8:00 a.m. – 15.0 Symposium 6A: Normal and pathogenic regulation of aldosterone biosynthesis 10:00 a.m. Pavilion Theater, 1st Floor Chairs: Eleanor Davies, Univ. of Glasgow *Iris Jaffe, Tufts Medical Center (*Replacing Jun Yang, Monash Univ.)
10:15 a.m. – 16.0 Symposium 6B: Integrated regulation of renal ion transport 12:00 p.m. Pavilion Theater, 1st Floor Chairs: Daniela Rotin, Hospital for Sick Children and Univ. of Toronto (Replacing David Ellison, Oregon Health Science Univ.) Peter Snyder, Univ. of Iowa (Replacing Johannes Loffing, Univ. of Zurich)
11:45 a.m. – 16.4 Interleukin 6 activation of the epithelial sodium channel in the distal convoluted 12:00 p.m. tubule and cortical collecting duct via Rac1 Oishi Paul, Emory Univ. Talk has moved to Session 2A, Thursday, October 3, 2:45 – 3:00 p.m.