Volume 58, Issue 4, Pages 1652-1663 (October 2000) Cyclosporine stimulates Na+-K+-Cl- cotransport activity in cultured mouse medullary thick ascending limb cells  Mai-Szu Wu, Chih-Wei Yang, Marcelle Bens, Kou-Cheng Peng, Hsiao-Mei Yu, Alain Vandewalle  Kidney International  Volume 58, Issue 4, Pages 1652-1663 (October 2000) DOI: 10.1046/j.1523-1755.2000.00326.x Copyright © 2000 International Society of Nephrology Terms and Conditions

Figure 1 Properties of cultured mouse thick ascending limb (TAL) cells. (A) Confluent TAL cells grown on Petri dishes formed layers of cuboid-shaped cells and formed small domes (magnification ×250). (B) Illustration of an ethidium bromide-stained 4% agarose gel showing the amplified products of expected size (406 bp) obtained with the mNKCC2 primers in microdissected medullary TAL (lane 1) and cultured TAL cells (7th passage, lane 2). As controls, no band was detected using non–reverse-transcribed RNA from cultured cells (lane 3) or by omitting cDNA (lane 4). The expression of β-actin was also shown as an internal control. Molecular weight standards (M) were the 1 kb ladder from GIBCO-BRL. (C) Time course of 86Rb+ influx performed on cultured TAL cells grown on Petri dishes and incubated without (▪) or with ouabain (•) and ouabain plus furosemide (▴). Each point is the mean ± SE from five separate experiments performed in duplicate. Kidney International 2000 58, 1652-1663DOI: (10.1046/j.1523-1755.2000.00326.x) Copyright © 2000 International Society of Nephrology Terms and Conditions

Figure 2 (A) An ethidium bromide-stained 4% agarose gel showing the amplified products of expected size (498 and 456 bp) obtained with the mROMK1 and mROMK2 primers in whole mouse kidney extract (lane 1) and cultured (10th passage) TAL cells (10th passage, lane 2). As controls, no band was detected using non–reverse-transcribed RNA from cultured cells (lane 3) or by omitting cDNA (lane 4). The expression of β-actin was also shown as an internal control. Molecular weight standards (M) were the 1 kb ladder from GIBCO-BRL. (B) Immunoblot of cultured mouse TAL cell homogenate (lanes 1 and 3) and membrane-enriched (lanes 2 and 4) fraction (50 μg) probed with the rat anti-ROMK antibody. A 40 kD band was detected in membrane-enriched (lane 2) and to a lesser extent in whole cell homogenate (lane 1). Almost no labeled band was detected by omitting the primary antibody in whole cell homogenate (lane 3) and membrane-enriched preparation (lane 4). The molecular weight markers are indicated on the left side of the figure. Kidney International 2000 58, 1652-1663DOI: (10.1046/j.1523-1755.2000.00326.x) Copyright © 2000 International Society of Nephrology Terms and Conditions

Figure 3 Effect of cyclosporine A (CsA), FK506, and rapamycin on 86Rb+ influx. The ouabain-sensitive (Os) and the ouabain-resistant furosemide-sensitive (Or-Fs) component of 86Rb+ influx were measured on sets of confluent TAL cells grown on Petri dishes and incubated without (control) or with 100 ng/mL CsA (CsA), 10-7 mol/L FK506 (FK) or 10-7 mol/L rapamycin (Rapa). Values are the mean ± SE of 10 separate experiments performed in duplicate. **P < 0.01; ***P < 0.001 vs. control values. Kidney International 2000 58, 1652-1663DOI: (10.1046/j.1523-1755.2000.00326.x) Copyright © 2000 International Society of Nephrology Terms and Conditions

Figure 4 Effects of CsA on the cell cAMP content. Cellular cAMP was measured in the absence (basal) or presence of 10-6 mol/L dDAVP on sets of cells incubated without (□) or with CsA (▪). Bars are the mean ± SE from eight separate experiments performed in triplicate. Kidney International 2000 58, 1652-1663DOI: (10.1046/j.1523-1755.2000.00326.x) Copyright © 2000 International Society of Nephrology Terms and Conditions

Figure 5 Kinetics of 36Cl- efflux from TAL cells grown on filters. Confluent cells were loaded with 36Cl- as described in the Methods section. After rinsing, fresh unlabeled PBS-CaCl2 medium was added to both apical and basal sides of the filters. The 1 mL interval sample-and-replace technique (Methods section) allowed measurement of the percentage of 36Cl- remaining in cells (A) and the rate constant of 36Cl- efflux from the apical side (B, upper panel) and basal side (B, lower panel) of the cell layers incubated in the absence (○, □) or presence of 100 ng/mL CsA (•, ▪) or CsA plus 10-4 mol/L NPPB added to the basal side of the cells (▴, □). Values are the mean ± SE from eight to 10 separate experiments. Kidney International 2000 58, 1652-1663DOI: (10.1046/j.1523-1755.2000.00326.x) Copyright © 2000 International Society of Nephrology Terms and Conditions

Figure 6 Kinetics of 86Rb+ efflux from TAL cells grown on Petri dishes. Confluent cells were loaded with 86Rb+ as described in the Methods section. After rinsing, fresh unlabeled PBS-CaCl2 medium was added to the dish, and the percentage of 86Rb+ remaining in cells (A) and the rate constant of 86Rb+ efflux (B) were measured on cells incubated in the absence (○, □) or presence of 100 ng/mL CsA (•, ▪) or CsA plus 10-4 mol/L Ba2+ (▴, □). Values are the mean ± SE from eight separate experiments. Kidney International 2000 58, 1652-1663DOI: (10.1046/j.1523-1755.2000.00326.x) Copyright © 2000 International Society of Nephrology Terms and Conditions

Figure 7 Effect of barium on the kinetics of 36Cl- efflux from TAL cells grown on filters. The percentage of 36Cl- remaining in cells (A) and the rate constant of 36Cl- efflux from the apical side (B, upper panel) and basal side (B, lower panel) were measured on confluent cells incubated in the absence (○, □) or presence of 100 ng/mL CsA (•, ▪) or CsA plus 10-4 mol/L Ba2+ added to the apical side of the cells (▴, □). Values are the mean ± SE from 10 separate experiments. Kidney International 2000 58, 1652-1663DOI: (10.1046/j.1523-1755.2000.00326.x) Copyright © 2000 International Society of Nephrology Terms and Conditions

Figure 8 Dual effects of CsA and barium on 86Rb+ influx. The ouabain-sensitive (Os) and the ouabain-resistant furosemide-sensitive (Or-Fs) component of 86Rb+ influx were measured on sets of confluent TAL cells grown on Petri dishes and incubated without and with 10-4 mol/L Ba2+ (Ba2+), 100 ng/mL CsA, or CsA plus Ba2+. Values are the mean ± SE of 10 separate experiments performed in duplicate. **P < 0.01; ***P < 0.001 vs. control values. Kidney International 2000 58, 1652-1663DOI: (10.1046/j.1523-1755.2000.00326.x) Copyright © 2000 International Society of Nephrology Terms and Conditions