Volume 79, Issue 8, Pages 843-852 (April 2011) Vasopressin-dependent coupling between sodium transport and water flow in a mouse cortical collecting duct cell line  Hans-Peter Gaeggeler, Yann Guillod, Dominique Loffing-Cueni, Johannes Loffing, Bernard C. Rossier  Kidney International  Volume 79, Issue 8, Pages 843-852 (April 2011) DOI: 10.1038/ki.2010.486 Copyright © 2011 International Society of Nephrology Terms and Conditions

Figure 1 Time course of vasopressin (AVP)-dependent expression of aquaporin 2 (AQP2) protein. mCCD cells were grown on plastic petri dishes in serum- and hormone-free culture media and then diluent (lanes 1–6) or AVP (10nm) (lanes 7–12) were added for time points indicated. Total protein extracts were analyzed by western blotting as indicated in the Materials and Methods. Kidney International 2011 79, 843-852DOI: (10.1038/ki.2010.486) Copyright © 2011 International Society of Nephrology Terms and Conditions

Figure 2 Expression of aquaporin (AQP)2, AQP3, and AQP4 in mCCD cells grown on filter cups in serum- and hormone-free culture medium in the presence of diluent (control), and AVP for 6 or 24h. Immunofluorescence was performed as described in the Materials and Methods. Kidney International 2011 79, 843-852DOI: (10.1038/ki.2010.486) Copyright © 2011 International Society of Nephrology Terms and Conditions

Figure 3 Vasopressin (AVP)-induced sodium transport measured by short-circuit current (Isc) method. The osmotic gradient was 1:2 (160mOsm apical and 320mOsm basolateral). (a) Time course. At t0, AVP 1pM (closed circles), 10pM (closed squares), or 100pM (closed lozenges) were added to the basolateral medium of test dishes and diluent to controls (open circles). At t4h, amiloride (10μm, arrow) was added to the apical membrane of all dishes (n=3). (b) Dose dependence was established by computing the responses observed in a at 10, 60, and 120min. Kidney International 2011 79, 843-852DOI: (10.1038/ki.2010.486) Copyright © 2011 International Society of Nephrology Terms and Conditions

Figure 4 Vasopressin (AVP)-induced water flow. (a) Time course. At t0, 10nm AVP was added to the basolateral milieu and diluent to controls. The osmotic gradient was 1:2 (160mOsm apical and 320mOsm basolateral). At any given time, apical fluid loss (open circles) and basolateral fluid gain (closed circles) were measured by gravimetry as described in the Materials and Methods. The AVP-induced differences over controls are shown (n=3). (b) Dose dependence established by computing the responses observed at 1, 10, and 100pM and at 1nm (n=3). Kidney International 2011 79, 843-852DOI: (10.1038/ki.2010.486) Copyright © 2011 International Society of Nephrology Terms and Conditions

Figure 5 Vasopressin (AVP)-induced water flow: dependence on osmotic gradients. Apical medium was diluted with culture medium 1:1 (n=7), 1:2 (n=3), 1:5 (n=4), and 1:10 (n=1). Each experiment was performed in triplicate dishes. Cells were incubated for 6h in the presence or absence of 1nm AVP and water flow measured as in Figure 4 Kidney International 2011 79, 843-852DOI: (10.1038/ki.2010.486) Copyright © 2011 International Society of Nephrology Terms and Conditions

Figure 6 Vasopressin (AVP)-induced sodium transport measured by the short-circuit current (Isc) method under isoosmotic condition 1:1 (320mOsm apical and 320mOsm basolateral). Cells were incubated for 24h in the presence of diluent (Control; open circles) or amiloride (10μm apical; Amil; closed circles) or AVP (10nM; AVP; open squares) or AVP+amiloride (AVP+Amil; closed squares). Independent experiments (n=3) were performed in triplicate. (a) Time course. Data are shown as difference of ΔIsc Isc (t)–Isc (t0). Mean Isc value at t0 and t1h are shown in Table 2. The dashed line indicates the level at which Isc is reversed. (b) Integration of Isc over 24h computed as described in Materials and Methods. **p<0.01; ***p<0.001. NS, not significant. Kidney International 2011 79, 843-852DOI: (10.1038/ki.2010.486) Copyright © 2011 International Society of Nephrology Terms and Conditions

Figure 7 Net flux of chloride, potassium, and sodium under isoosmotic condition across the monolayer after 24h of incubation in the presence of diluent (control; open bar), amiloride 10mmol/l (Am; light gray bar), vasopressin 10nm (AVP; gray bar), or vasopresssin+amiloride (AVP+Am; closed bar). Independent experiments (n=3) were performed, each in triplicate. Net fluxes were calculated as described in Supplementary Table S1 online. Kidney International 2011 79, 843-852DOI: (10.1038/ki.2010.486) Copyright © 2011 International Society of Nephrology Terms and Conditions

Figure 8 Effects of vasopressin on net water flow compared to net flux of positive charges across the epithelium. Vasopressin (AVP)-induced net water flow (a) and net flux of positive charges (b) from apical to basolateral under isoosmotic condition as described in Figure 6. Cells were incubated for 24h in the presence of diluent (control) or amiloride (10μm apical; Amil) or AVP (10nM; AVP) or AVP (10nm) +amiloride (10μm) (AVP+Amil). *p<0.05; **p<0.01; ***p<0.001. NS, not significant. Kidney International 2011 79, 843-852DOI: (10.1038/ki.2010.486) Copyright © 2011 International Society of Nephrology Terms and Conditions