Secretin and body fluid homeostasis Jessica Y.S. Chu, Carrie Y.Y. Cheng, Vien H.Y. Lee, Y.S. Chan, Billy K.C. Chow  Kidney International  Volume 79, Issue 3, Pages 280-287 (February 2011) DOI: 10.1038/ki.2010.397 Copyright © 2011 International Society of Nephrology Terms and Conditions

Figure 1 SCT and its receptor in the kidney. (a) Bright-field photomicrograph of mouse kidney sections labeled with rabbit anti-mouse SCTR antibody raised in our laboratory (1:250). The specificity of this antibody was tested using sections obtained from SCTR-/- mice.19 SCTR was shown to localize in almost every segment of the nephron. (b) Diagrammatic illustration of the functions of SCT in the renal collecting duct tubules. SCT might be capable of inducing water reabsorption in renal collecting duct principal cells by binding to SCTR, which is localized on the basolateral membranes of the cells.19 On binding to its receptor, SCT activates an AC-cAMP–PKA cascade, which could then lead to an increase in AQP2 expression, probably via the subsequent phosphorylation of CREB and the docking of this factor onto the CRE site of the AQP2 gene promoter. In addition, SCT also induces the trafficking of AQP2 from the IV to the apical membrane of the collecting duct. This could allow water influx into the principal cells via AQP2 and water outflux from the cells via AQP3 and AQP4, which are constitutively expressed on the basolateral membrane. It is therefore possible that SCT could enhance water reabsorption from the collecting tubules. AC, adenylyl cyclase; AQP, aquaporin; cAMP, cyclic adenosine monophosphate; CRE, cAMP responsive element; CREB, cAMP responsive element binding protein; GTP, guanosine triphosphate; IV, intracellular vesicles; PKA, protein kinase A; SCT, secretin; SCTR, SCT receptor. Kidney International 2011 79, 280-287DOI: (10.1038/ki.2010.397) Copyright © 2011 International Society of Nephrology Terms and Conditions

Figure 2 Cellular distribution of SCT and SCTR in the central osmoregulatory regions. (a) SCT and its receptor were colocalized with Vp in the hypothalamic SON and PVN. The antibodies employed were rabbit anti-SCT (1:250 dilution; Phoenix Pharmaceuticals, Burlingame, CA), rabbit anti-SCTR (1:200 dilution; Chu et al.19), goat anti-Vp (1:400 dilution; Santa Cruz Biotechnology, Santa Cruz, CA), Alexa Fluor 594 donkey anti-goat IgG (1:500 dilution; Molecular Probes, Invitrogen, Carlsbad, CA), and Alexa Fluor 488 chicken anti-rabbit IgG (1:500 dilution; Molecular Probes. (b) Photographs of SFO section stained with antiserum against SCT and SCTR. Both SCT and its receptor were shown to be highly expressed within the SFO. Controls (c) were performed by liquid-phase preabsorption of the antiserum with either 0.1mM rat secretin or synthetic mouse secretin receptor peptide (R-A-E-C-L-R-E-L-S-E-E-K-K, which is present in the mouse secretin receptor) for 3h at room temperature before incubating with the sections. Bar=6μm. Ab, antibody; IgG, immunoglobulin G; PVN, paraventricular; Rb, rabbit; paraventricular; SCT, secretin; SCTR, SCT receptor; SFO, subfornical organ; SON, supraoptic; Vp, vasopressin. Kidney International 2011 79, 280-287DOI: (10.1038/ki.2010.397) Copyright © 2011 International Society of Nephrology Terms and Conditions

Figure 3 Centrally expressed SCT is a dipsogenic agent. (a) ICV-SCT (30μM) into the lateral ventricular induced Fos-immunoreactivities in the SFO when compared with ICV injection of isotonic saline (ICV-PBS). (b) Centrally expressed SCT, but not circulating SCT, induces drinking behavior. (A) Centrally injected SCT dose-dependently increase water intake and urine output. Data points are the mean fold±s.e.m. of values obtained in 15–20 rats per group. *P<0.05 and **P<0.01 versus PBS-treatment group. (B) Peripheral injection of SCT through osmotic minipump was not capable of increasing water intake in rats compared with those injected with isotonic saline (IV-PBS). ANGII, a dipsogenic agent when intravenously injected into the rat, was used as an internal control. ANGII, angiotensin II; ICV, intracerebroventricular; IV, intracellular vesicles; PBS, phosphate-buffered saline; SCT, secretin. Kidney International 2011 79, 280-287DOI: (10.1038/ki.2010.397) Copyright © 2011 International Society of Nephrology Terms and Conditions

Figure 4 Secretin is possibly a mediator of ANGII action. (a) Centrally, but not peripherally, injected ANGII induced central SCT expression. Results are presented in mean fold changes±s.e.m. (n=5). *P<0.05 versus PBS-treatment group. (b) Centrally injected ANG triggers SCT release into peripheral circulation. Blood samples (150μl) were collected every 5min from the jugular vein from -5min before to 30min (5-min interval) after ICV infusion of 5μg ANG. The results are presented in mean fold changes±s.e.m. relative to PBS-infused basal level of SCT in the plasma (0.099±0.005ng/ml). *P<0.05 and **P<0.01. ANG; angiotensin; ICV, intracerebroventricular; IV, intracellular vesicles; PBS, phosphate-buffered saline; SCT, secretin. Kidney International 2011 79, 280-287DOI: (10.1038/ki.2010.397) Copyright © 2011 International Society of Nephrology Terms and Conditions

Figure 5 Diagrammatic illustration of the functions of SCT in SFO and hypothalamus. In the SFO, SCT-stimulated c-fos expression is believed to function via the binding of SCT onto its receptor, hence triggering the activation of AC that increases the intracellular cAMP level. Activated PKA could then induce the phosphorylation of CRE binding protein (CREB), and hence the transcription of c-fos immediate early gene. SCT also functions as a dipsogenic peptide, probably through its action on the renin–angiotensin system within this region. In the hypothalamic magnocellular neurons, SCT-induced increases in cAMP might lead, via the activation of PKA, CREB, and/or activating transcription factor proteins, to the stimulation of Fos/Jun family of transcription factors. Subsequent dimerization of the product Fos with Jun protein could allow their interaction with the CRE sequences in Vp promoter, hence upregulating the Vp gene expression. As cAMP was demonstrated to upregulate the Vp gene transcriptionally via CRE and post-transcriptionally via enhancing the polyadenylation of its mRNA, SCT-induced cAMP could probably function directly to stimulate the Vp gene expression through CREB phosphorylation as well as to enhance the stability of vasopressin mRNA in the cytoplasm by increasing the polyadenylate tail length. In the hypothalamo–pituitary axis, SCT induces the secretion of Vp within the hypothalamic nuclei as well as from the posterior pituitary into the circulation. SCT itself could also be liberated into the fenestrated capillaries within the pars nervosa, exerting its action in the kidney. AC, adenylyl cyclase; cAMP, cyclic adenosine monophosphate; CRE, cAMP responsive element; CREB, cAMP responsive element binding protein; PKA, protein kinase A; SCT, secretin; SCTR, SCT receptor; SFO, subfornical organ; SON, supraoptic; Vp, vasopressin. Kidney International 2011 79, 280-287DOI: (10.1038/ki.2010.397) Copyright © 2011 International Society of Nephrology Terms and Conditions