Volume 78, Issue 2, Pages 160-169 (July 2010) Vasopressin induces phosphorylation of the thiazide-sensitive sodium chloride cotransporter in the distal convoluted tubule  Nis B. Pedersen, Marlene V. Hofmeister, Lena L. Rosenbaek, Jakob Nielsen, Robert A. Fenton  Kidney International  Volume 78, Issue 2, Pages 160-169 (July 2010) DOI: 10.1038/ki.2010.130 Copyright © 2010 International Society of Nephrology Terms and Conditions

Figure 1 Novel phosphospecific Na+–Cl− cotransporter (NCC) antibodies label the distal convoluted tubule (DCT) in the rat and mouse kidney. (a–c) Show anti-pT53-NCC in rat DCT, but not in the late thick ascending limb (TAL) at macula densa (MD) (a) nor in connecting tubule (CNT) (c). Similar labeling was found for the anti-pT58-NCC and the anti-pT53/pT58-NCC. Panels d–f document a similar labeling pattern in the mouse kidney for the anti-pT58-NCC. G, glomerulus. Kidney International 2010 78, 160-169DOI: (10.1038/ki.2010.130) Copyright © 2010 International Society of Nephrology Terms and Conditions

Figure 2 Specificity of phosphorylated Na+–Cl− cotransporter (pNCC) antibodies. The pNCC antibodies show distinct staining of distal convoluted tubule (DCT) segments (a, e, and i). Pre-absorption of each pNCC antibody with a synthetic phosphopeptide corresponding to the targeted phosphorylation site completely abolished labeling (c, h, and l). In contrast, pre-absorption with either a synthetic non-phosphorylated peptide (b, f, j, and k) corresponding to the same region or a non-targeted pNCC phosphopeptide (d and g) did not affect pNCC labeling. The weak labeling of cTAL with the anti-pT53/pT58-NCC antibody was not blocked with the T53/T58-phosphopeptide (l). G, glomerulus; MD, macula densa; TAL, thick ascending limb. Kidney International 2010 78, 160-169DOI: (10.1038/ki.2010.130) Copyright © 2010 International Society of Nephrology Terms and Conditions

Figure 3 Laser scanning confocal microscopy of phosphorylated Na+–Cl− cotransporter (pNCC) with tubule segment-specific markers. (a) Biotinylated anti-pT53-NCC shows perfect co-localization with anti-pT58-NCC, (b) biotinylated anti-pT53-NCC shows perfect co-localization with anti-pT53/pT58-NCC, (c) biotinylated anti-pT53-NCC co-localizes with total NCC, (d) anti-pT53-NCC does not co-localize with the TAL-specific marker Tamm-Horsfall protein (THP), (e) anti-pT53-NCC partially co-localizes with the DCT2 and connecting tubule (CNT)-specific marker Calbindin and (f) anti-pT53-NCC does not co-localize with the CNT and collecting duct (CD) marker aquaporin-2 (AQP-2). DCT, distal convoluted tubule; TAL, thick ascending limb. Scale-bar: 10μm for a–c and 20μm for d–f. Kidney International 2010 78, 160-169DOI: (10.1038/ki.2010.130) Copyright © 2010 International Society of Nephrology Terms and Conditions

Figure 4 Immunogold electron microscopy of normal rat kidney sections shows that phosphorylated Na+–Cl− cotransporter (pNCC) is present only in the apical plasma membrane. In kidney sections from Wistar rats, immunogold electron microscopy shows that total NCC (a and b) is detected in the apical plasma membrane (arrows) and subapical vesicles (arrowheads). In contrast, both pT53-NCC (c and d) and pT58-NCC (e and f) were localized to the apical plasma membrane alone (arrows). Gold particle diameter=10nm. Kidney International 2010 78, 160-169DOI: (10.1038/ki.2010.130) Copyright © 2010 International Society of Nephrology Terms and Conditions

Figure 5 Phosphorylation of Na+–Cl− cotransporter (NCC) increases with acute deamino-Cys-1, d-Arg-8 vasopressin (dDAVP) treatment in vivo. Immunoblotting with anti-pT53-NCC, anti-pT58-NCC, anti-pT53/pT58-NCC and total NCC antibodies on whole kidney homogenates from Brattleboro rats treated intravenously with either saline (-) or dDAVP (+) for 15 or 60min. Representative immunoblots for the 60min treatment are shown. Data are band densities relative to control (mean±s.e.), n=4. *P<0.05 from control, #P<0.01 between 15 and 60min dDAVP. AU, arbitrary units. Kidney International 2010 78, 160-169DOI: (10.1038/ki.2010.130) Copyright © 2010 International Society of Nephrology Terms and Conditions

Figure 6 Immunogold labeling of phosphorylated Na+–Cl− cotransporter (pNCC) in Brattleboro rat kidney sections after acute deamino-Cys-1, d-Arg-8 vasopressin (dDAVP) treatment. Brattleboro rats treated intravenously with either saline or dDAVP (60min) and kidney sections subjected to immunogold electron microscopy. Under control conditions (a and c), weak pNCC labeling was observed in the apical plasma membrane. After dDAVP treatment (b and d), pNCC increased only in the apical plasma membrane. No gold particles were observed inside the cell. Gold particle diameter=10nm. Kidney International 2010 78, 160-169DOI: (10.1038/ki.2010.130) Copyright © 2010 International Society of Nephrology Terms and Conditions

Figure 7 Immunogold labeling of total Na+–Cl− cotransporter (NCC) in Brattleboro rat kidney sections after acute deamino-Cys-1, d-Arg-8 vasopressin (dDAVP) treatment. Brattleboro rats treated intravenously with either saline or dDAVP (60min) and kidney sections subjected to immunogold electron microscopy. Under control conditions (a), total NCC labeling was observed in the apical plasma membrane (arrows) and also in intracellular vesicles (arrowheads). After dDAVP treatment (b), no significant difference in the number of gold particles associated with the apical plasma membrane was observed. Gold particle diameter=10nm. Kidney International 2010 78, 160-169DOI: (10.1038/ki.2010.130) Copyright © 2010 International Society of Nephrology Terms and Conditions

Figure 8 The acute and chronic effects of deamino-Cys-1, d-Arg-8 vasopressin (dDAVP) on increasing phosphorylated Na+–Cl− cotransporter (pNCC) can occur independently from the effects of angotensin II and/or aldosterone. Munich-Wistar rats were fed a high-NaCl diet and treated subcutaneously with candesartan, candesartan+dDAVP for 4 days, or candesartan plus acute dDAVP (60min). (a) Immunoblotting shows that both acute and chronic administration of dDAVP increased pT53-NCC, pT58-NCC, and pT53/pT58-NCC abundance compared with candesartan alone. The abundance of total NCC was unchanged. (b) Graphs show summarized data. Data are band densities relative to control (mean±s.e.), n=5. *P<0.05 from Candesartan group alone. AU, arbitrary units. Kidney International 2010 78, 160-169DOI: (10.1038/ki.2010.130) Copyright © 2010 International Society of Nephrology Terms and Conditions

Figure 9 Effect of acute deamino-Cys-1, d-Arg-8 vasopressin (dDAVP) treatment on the kinases SPAK/OSR1 in vivo. (a) Immunoblotting on whole kidney homogenates from Brattleboro rats treated intravenously with either saline (-) or dDAVP (+) for 60min with total SPAK/OSR1 or active phosphorylated SPAK/OSR1 (see Materials and Methods). (b) Graphs show summarized data. Data are band densities relative to total kinase abundance (mean±s.e.), n=4. *P<0.05 from control. AU, arbitrary units. Kidney International 2010 78, 160-169DOI: (10.1038/ki.2010.130) Copyright © 2010 International Society of Nephrology Terms and Conditions

Figure 10 pSPAK/OSR1 increase in rats fed a high-salt diet alongside AT1-receptor blockade following either acute or chronic deamino-Cys-1, d-Arg-8 vasopressin (dDAVP) treatment. (a) Immunoblotting with total SPAK/OSR1 or active, phosphorylated SPAK/OSR1 of kidney homogenates from Munich-Wistar rats treated subcutaneously with either candesartan, candesartan+dDAVP for 4 days, or candesartan plus acute dDAVP (60min). (b) Graphs show summarized data. Data are band densities relative to total kinase abundance (mean±s.e.), n=5. *P<0.05 from candesartan, #P<0.05 between chronic dDAVP and acute dDAVP. AU, arbitrary units. Kidney International 2010 78, 160-169DOI: (10.1038/ki.2010.130) Copyright © 2010 International Society of Nephrology Terms and Conditions

Figure 11 Effect of deamino-Cys-1, d-Arg-8 vasopressin (dDAVP) on intracellular Ca2+([Ca2+]i). Enzymatically isolated renal tubules were plated on coverslips, and a differential interference contrast image (inset) and enhanced green fluorescent protein fluorescence signal (not shown) were acquired. The cells were loaded with the intracellular Ca2+-sensitive dye fluo-4. Representative traces show the fluo-4 signal from 5 cells (yellow, red; late distal convoluted tubule (DCT)/connecting tubule and light blue, green, dark blue; early DCT), indicated in the differential interference contrast image. The experimental procedure of measuring [Ca2+]i was (1) baseline recording in HEPES-buffered salt solution; (2) addition of dDAVP (2 × 10–10M) to the superfusate, as indicated; and (3) clamp of [Ca2+]i to extracellular Ca2+ concentration ([Ca2+]e) by ionomycin (2 × 10–6M) in normal Ca2+ (1.8 × 10–3M) buffer and in a Ca2+-free buffer containing EGTA (1 × 10–3M). Data are normalized to initial fluo-4 signal. All segments of the DCT respond to dDAVP. G, glomerulus. Kidney International 2010 78, 160-169DOI: (10.1038/ki.2010.130) Copyright © 2010 International Society of Nephrology Terms and Conditions