Volume 84, Issue 3, Pages 501-508 (September 2013) Intrarenal ghrelin receptors regulate ENaC-dependent sodium reabsorption by a cAMP- dependent pathway  Brandon A. Kemp, Nancy L. Howell, John J. Gildea, Susanna R. Keller, Shetal H. Padia  Kidney International  Volume 84, Issue 3, Pages 501-508 (September 2013) DOI: 10.1038/ki.2013.187 Copyright © 2013 International Society of Nephrology Terms and Conditions

Figure 1 Intrarenal ghrelin receptors (GRs) are localized to the collecting duct. High-power(× 400) confocal micrographs of Sprague–Dawley (SD) rat kidney labeled with antibodies to marker proteins specific for each segment: villin for proximal tubules, Tamm–Horsfall protein (THP) for thick ascending limb, calbindin-D28K (CAL) for distal convoluted tubule, and aquaporin-2 (AQP-2) for the collecting duct. As shown by the gold color in a, GRs (red) colocalize with the collecting duct marker AQP-2 (green); however, no overlap in staining is observed with distal tubule marker CAL (blue). (b) GRs (red) are completely separated from the proximal tubule marker villin (green). (c) GRs (red) do not colocalize with the thick ascending limb marker THP (green) and confirms lack of overlap with the distal tubule marker CAL (blue). The scale bar in a represents 10μm. Kidney International 2013 84, 501-508DOI: (10.1038/ki.2013.187) Copyright © 2013 International Society of Nephrology Terms and Conditions

Figure 2 Intrarenal ghrelin receptors (GRs) are enriched in collecting duct membranes from renal tissue in normal rats. Following collecting duct–specific immunoprecipitation (IP) with L1-CAM (an epitope specifically present on collecting duct cells and not distal tubule cells), membranes were probed for GR expression. As shown in the composite of representative western blots, GRs are enriched in collecting duct membranes (Gel 1). To ensure the collecting duct membrane specificity of the IP, aquaporin-2 (AQP-2), a known marker of collecting duct cells, was localized to the same fraction (positive control, Gel 4); however, distal tubule marker sodium chloride cotransporter (NCC) was not present in this fraction (negative control, Gel 2). To validate our ability to detect NCC altogether, we show NCC enrichment of total renal membranes (Gel 3). IB, immunoblot. Kidney International 2013 84, 501-508DOI: (10.1038/ki.2013.187) Copyright © 2013 International Society of Nephrology Terms and Conditions

Figure 3 Ghrelin-induced antinatriuresis is not observed in the presence of acute intrarenal epithelial sodium channel (ENaC) blockade. (a) Direct renal interstitial (RI) infusion of ghrelin () significantly reduces urine sodium excretion (UNaV) in normal rats. The ghrelin-induced antinatriuresis is abolished after the coinfusion of ghrelin+amiloride (), a specific blocker of the collecting duct ENaC, whereas the coinfusion of ghrelin+chlorothiazide (), a specific inhibitor of the distal tubule sodium chloride cotransporter (NCC), had no effect. Both RI infusions of amiloride () or chlorothiazide alone () induced natriuresis, ensuring adequate Na+ transporter blockade. RI infusion of 5% dextrose in water (D5W) () served as a time control and had no effect. (b) Mean arterial pressures (MAP) in response to conditions in a. RI D5W infusion (□), ghrelin infusion (■), ghrelin+amiloride (), ghrelin+chlorothiazide (), amiloride (), and chlorothiazide alone (). Results are reported as a percentage of corresponding baseline value. Data represent mean±s.e.; **P<0.01, ***P<0.001 from respective control period (two-tailed paired Student’s t-test). Analysis of variance: +P<0.05, ++P<0.01 from time control (RI D5W). Kidney International 2013 84, 501-508DOI: (10.1038/ki.2013.187) Copyright © 2013 International Society of Nephrology Terms and Conditions

Figure 4 Ghrelin-induced antinatriuresis is not observed in the presence of chronic, systemic epithelial sodium channel (ENaC) blockade. (a) Urine sodium excretion (UNaV) in response to renal interstitial (RI) infusion of 5% dextrose in water (D5W) () or ghrelin (), following 72h of systemic ENaC blockade with amiloride. (□) and (■) represent UNaV in response to RI D5W or ghrelin infusion in the absence of systemic amiloride (only systemic D5W treatment), respectively. (b) Mean arterial pressure (MAP) values in response to the conditions in a. Results are reported as a percentage of corresponding baseline value. Data represent mean±s.e.; **P<0.01, ***P<0.001 from respective control period (two-tailed paired Student’s t-test). Kidney International 2013 84, 501-508DOI: (10.1038/ki.2013.187) Copyright © 2013 International Society of Nephrology Terms and Conditions

Figure 5 Renal interstitial (RI) ghrelin infusion increases cortical collecting duct (CCD) apical membrane αENaC, without changes in total renal αENaC. Phosphorylated serum/glucocorticoid-regulated kinase-1 (pSGK1) also increases in response to ghrelin infusion, but not total or phosphorylated sodium chloride cotransporter (NCC). (a) CCD membrane-associated αENaC in response to RI infusion of 5% dextrose in water (D5W) (□) or ghrelin (■) after 1- or 3-h infusions. (b) Total renal αENaC protein expression in response to conditions in a. (c) Renal pSGK1/total SGK1 protein ratio in response to conditions in a. (d) Renal pT53-NCC/total NCC protein ratio in response to conditions in a. Blots in a were normalized to β-tubulin and blots in b, c, d were normalized to β-actin. Data represent mean±s.e.; **P<0.01, ***P<0.001 compared with control (two-tailed paired Student’s t-test). Kidney International 2013 84, 501-508DOI: (10.1038/ki.2013.187) Copyright © 2013 International Society of Nephrology Terms and Conditions

Figure 6 Renal interstitial (RI) ghrelin infusion results in increased microdialysate cAMP levels; these levels return to baseline in the presence of adenylyl cyclase (AC) inhibition with SQ-22536. RI cAMP levels in response to RI infusions of 5% dextrose in water (D5W) (□), ghrelin (■), or the coinfusion of ghrelin+AC inhibitor SQ-22536 (). Results are reported in pmol/ml and data represent mean±s.e.; **P<0.01 from respective control period (two-tailed paired Student’s t-test). Kidney International 2013 84, 501-508DOI: (10.1038/ki.2013.187) Copyright © 2013 International Society of Nephrology Terms and Conditions

Figure 7 Ghrelin-induced antinatriuresis is abolished in the presence of adenylyl cyclase (AC) inhibition with SQ-22536. (a) Urine Na+ excretion (UNaV) in response to renal interstitial (RI) infusions of 5% dextrose in water (D5W) (□), ghrelin (■), or the coinfusion of ghrelin+AC inhibitor SQ-22536 (). (b) Mean arterial pressure (MAP) in response to conditions in a. Results are reported as a percentage of corresponding baseline value. Data represent mean±s.e.; **P<0.01, ***P<0.001 from respective control period (two-tailed paired Student’s t-test). Kidney International 2013 84, 501-508DOI: (10.1038/ki.2013.187) Copyright © 2013 International Society of Nephrology Terms and Conditions

Figure 8 Sodium reabsorption. Diagram illustrating the mechanism by which ghrelin receptors (GRs) stimulate sodium reabsorption in the cortical collecting duct. AC, adenylyl cyclase; PKA, protein kinase A. Kidney International 2013 84, 501-508DOI: (10.1038/ki.2013.187) Copyright © 2013 International Society of Nephrology Terms and Conditions