Volume 85, Issue 5, Pages 1058-1067 (May 2014) Anoctamin 1 induces calcium-activated chloride secretion and proliferation of renal cyst– forming epithelial cells  Bjoern Buchholz, Diana Faria, Gunnar Schley, Rainer Schreiber, Kai-Uwe Eckardt, Karl Kunzelmann  Kidney International  Volume 85, Issue 5, Pages 1058-1067 (May 2014) DOI: 10.1038/ki.2013.418 Copyright © 2014 International Society of Nephrology Terms and Conditions

Figure 1 Calcium-activated chloride secretion in principal-like MDCK cells (PLCs). (a) Original recordings of open-circuit Ussing chamber experiments. PLCs grown on permeable supports were stimulated with the P2Y2 receptor agonist UTP (100μmol/l) from the luminal or basolateral side of the monolayer. (b) Summary of the calculated equivalent short-circuit currents (Isc) induced by UTP in PLC compared with the nonstimulated monolayer (control, con), *paired t-test, P<0.05. (c) Original recording of an open-circuit Ussing chamber experiment. PLCs were treated with 1μmol/l of ionomycin (iono) to increase intracellular calcium. (d) Summary of the Isc induced by ionomycin in PLC compared with the nonstimulated monolayer (control, con) (number of experiments), *paired t-test, P<0.05. (e) Original recording of an open-circuit Ussing chamber experiment of PLCs stimulated with UTP in the presence of gluconate, which replaced extracellular chloride. (f) Summary of the Isc induced by UTP in PLCs compared with the nonstimulated monolayer (control, con) in the absence of chloride (number of experiments). (g) Summary of the Isc obtained from PLCs that, before UTP (100μmol/l) application, were preincubated with different inhibitors for 5min: without preincubation (control, con), clotrimazole (clotri; 10μmol/l), niflumic acid (NFA; 10μmol/l), 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS; 100μmol/l), and tannic acid (TA; 20μmol/l) (n=6–9 individual experiments), *analysis of variance (ANOVA), P<0.05. UTP, uridine triphosphate. Kidney International 2014 85, 1058-1067DOI: (10.1038/ki.2013.418) Copyright © 2014 International Society of Nephrology Terms and Conditions

Figure 2 Knockdown of ANO1 in principal-like MDCK cells (PLCs). (a) Original recording of an open-circuit Ussing chamber experiment using PLCs transfected transiently with siRNA directed against ANO1 (si-ANO1) or a scrambled sequence (Scramble). Cells were stimulated with UTP (100μmol/l, luminal). (b) Summary of the Isc induced by luminal UTP compared with nonstimulated monolayer (control, con) in PLCs transfected with siRNA directed against ANO1 (si-ANO1), ANO6 (si-ANO6), or a scrambled sequence (scrbld), *paired t-test, #unpaired t-test, P<0.05. (c) Summary of the Isc induced by basolateral stimulation with UTP compared with the non-stimulated monolayer (control, con) in PLCs described in b, *paired t-test, #unpaired t-test, P<0.05. (d) Summary of the Isc induced by ionomycin (1μmol/l) compared with the nonstimulated monolayer (control, con) in PLCs transfected with si-ANO1, si-ANO6 or scrambled, *paired t-test, #unpaired t-test, P<0.05. (e) Mean Isc values obtained from PLCs transfected with si-ANO1, si-ANO6, or scrambled and that were pre-incubated with different inhibitors for 5min before UTP (100μmol/l) application: without preincubation (control, con), clotrimazole (clotri; 10μmol/l), niflumic acid (NFA; 10μmol/l), 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS; 100μmol/l), and tannic acid (TA; 20μmol/l). (n=6–9 individual experiments), *paired t-test, #unpaired t-test, P<0.05. UTP, uridine triphosphate. Kidney International 2014 85, 1058-1067DOI: (10.1038/ki.2013.418) Copyright © 2014 International Society of Nephrology Terms and Conditions

Figure 3 Expression of ANO1 in cyst-lining epithelia. (a–c) Principal-like MDCK cell (PLC) cysts obtained from four individual experiments incubated with either control medium (Ctrl) or medium supplemented with 10μmol/l forskolin (Fsk) were stained for ANO1 (green). (a) ANO1 fluorescence intensities at the basal membrane, the center, and the apical membrane of control-treated cysts in comparison with forskolin-treated cysts, *unpaired t-test, P<0.05. Arrows in c indicate staining of ANO1 in the apical membrane. (b, c) Representative photos of PLC cysts in the absence (b) and presence of 10μmol/l forskolin (c) stained for ANO1. Scale bars: 20μm. (d, e) Metanephric kidneys E12.5–14 were cultured ex vivo in the presence of forskolin, leading to tubular dilation and cyst expansion, and then they were stained for ANO1 (green) and 4,6-diamidino-2-phenylindole (DAPI; blue) showing numerous cysts stained positive for ANO1 (#) with a few cysts stained negative for ANO1 (* in e). (f, g) Representative stainings of human autosomal dominant polycystic kidney disease (ADPKD) tissue for ANO1 (green) and DAPI (blue) showing many cysts stained positive for ANO1 (#) and a few cysts stained negative for ANO1 (* in f). lu indicates the lumen of each cyst; the dotted line in f represents the border of three cysts sticking together. a.u., arbitrary units. Kidney International 2014 85, 1058-1067DOI: (10.1038/ki.2013.418) Copyright © 2014 International Society of Nephrology Terms and Conditions

Figure 4 Inhibition of ANO1 in the principal-like MDCK cell (PLC) cyst model. (a) In the presence of 10μmol/l forskolin, PLC cysts were treated with 0, 10, and 20μmol/l tannic acid (TA). At day 5, cyst sizes relative to untreated cysts (set to 100%) and cyst numbers from four individual experiments comprising 1656–2048 cysts per condition are summarized. Right: representative photos of cysts in the presence of 0 (control), 10, and 20μmol/l TA at day 5. Scale bars: 50μm. (b) According to a, PLC cysts were treated with 0, 2.5, and 10μmol/l AO1. Cyst sizes relative to untreated cysts (set to 100%) and cyst numbers from three individual experiments comprising 335–701 cysts per condition are summarized. Right: representative photos of cysts in the presence of 0 (control), 2.5, and 10μmol/l AO1 at day 5. Scale bars: 50μm. *Wilcoxon signed-rank test, P<0.05. Kidney International 2014 85, 1058-1067DOI: (10.1038/ki.2013.418) Copyright © 2014 International Society of Nephrology Terms and Conditions

Figure 5 Inhibition of ANO1 in the embryonic kidney cyst model. (a) Embryonic kidneys E12.5–E13.5 were cultured on filters. After 24h, right kidneys were stimulated with 10μmol/l forskolin (Ctrl), and left kidneys were treated with 10μmol/l forskolin+10μmol/l tannic acid (TA). At day 5, cystic indices were examined (each group n=11). Right: representative photos of whole-mount embryonic kidneys at day 5. (b) Embryonic kidneys E12.0–E13.5 were cultured on filters. After 24h, right kidneys were stimulated with 10μmol/l forskolin (Ctrl), and left kidneys were treated with 10μmol/l forskolin+10μmol/l AO1. At day 5, cystic indices were examined (each group n=8). Right: representative photos of whole-mount embryonic kidneys at day 5. (c) Embryonic kidneys from B (n=5 incubated with forskolin, and n=5 incubated with forskolin+AO1) were stained for proliferating cell nuclear antigen (PCNA). Number of PCNA-positive cells normalized to the whole cell number was determined. Right: representative photos of sections from embryonic kidneys stained for PCNA. *Paired t-test, P<0.05. TA, tannic acid. Kidney International 2014 85, 1058-1067DOI: (10.1038/ki.2013.418) Copyright © 2014 International Society of Nephrology Terms and Conditions

Figure 6 Knockdown of ANO1 in the embryonic kidney cyst model. (a) Embryonic kidneys E12.5–E13.0 were cultured on filters. After 24h, right kidneys were stimulated with 10μmol/l forskolin and 10μmol/l morpholinos directed against a mismatched sequence (Ctrl MO), and left kidneys were treated with 10μmol/l forskolin+10μmol/l morpholinos directed against ANO1 (ANO1 MO). At day 5, cystic indices were examined (each group n=19). (b) Representative photos of whole-mount embryonic kidneys at day 5. (c) Representative photos of embryonic kidney sections treated with either Ctrl MO (left) or ANO1 MO (right) stained for ANO1 (green) and 4,6-diamidino-2-phenylindole (DAPI; blue). (d) Embryonic kidneys from A (n=5 Ctrl MO, and n=5 ANO1 MO) were stained for proliferating cell nuclear antigen (PCNA) and PCNA-positive cells normalized to the whole cell number were determined. (e) Representative photos of sections from embryonic kidneys stained for PCNA. *Paired t-test, P<0.05. Kidney International 2014 85, 1058-1067DOI: (10.1038/ki.2013.418) Copyright © 2014 International Society of Nephrology Terms and Conditions