Corticosteroid-Dependent Sodium Transport in a Novel Immortalized Mouse Collecting Duct Principal Cell Line

Morphology of confluent cultured mouse cortical collecting duct (CCD) cells. (A) Phase-contrast micrograph of confluent cells grown on a Petri dish (passage 25). The cells are all cuboid and form domes. (B) Cells contain cytokeratins K₈-K₁₈, as detected with a polyclonal antibody. (C) Tight junctions appear as fine, regular outlines around all cells when labeled with the anti-ZO-1 antibody. (D) The nuclei of all cells are immunoreactive for large T antigen. (E) Lateral and basal locations of Na⁺,K⁺-ATPase pumps is illustrated by confocal laser analysis of preparations labeled with a specific anti-α-Na⁺,K⁺-ATPase antibody. (F and G) Cells grown on filters form confluent monolayers of closely apposed cuboid cells (F), separated by tight junctions and desmosomes (G). Bars: A, 50 μm; B through E, 10 μm; F and G, 1 μm.

Dolichos biflorus agglutinin (DBA) binding to intact collecting ducts and cultured CCD cells. (A) DBA specifically binds to intercalated cells from intact CCD (arrows), whereas the adjacent pairs of principal cells are not stained (arrowhead). (B and C) Only few cultured mpkCCD_c14 cells (less than 5%) are positively stained with DBA, while most of the cells surrounding and forming domes are not stained. Bar, 10 μm.

ENaC mRNA and protein in cultured mouse CCD cells. (A) cDNA and non-reverse-transcribed RNA were submitted to reverse transcription (RT)-PCR. Each sample was amplified (28 cycles) with sets of primers specific for α- (lane 1), β- (lane 4), and γ- (lane 7) rENaC and hGAPDH. As control, no amplified products were detected when the mRNA was non-reverse-transcribed (lanes 2, 5, and 8) or when cDNA was omitted (lanes 3, 6, and 9). (B) Proteins were pulsed with [³⁵S]methionine and immunoprecipitated with specific anti-α, -β, and -γENaC sera (lanes 1) and the corresponding preimmune sera (lane 2).

Effects of ENaC blocking agents on I_sc. I_sc was measured on confluent mpkCCD_c14 cells before and after adding amiloride ([UNK], ○), benzamyl amiloride (▪, □), or ethylisopropyl-amiloride (▴, ▵) to the apical side (A) or basal side (B) of the filters for 10 min. Values, expressed as percentage of inhibition of I_sc measured before the addition of the blocking agents, are the mean ± SEM from six separate experiments.

Action of aldosterone on I_sc. (A) Representative traces of I_sc measured in the absence (○) or presence of 10^-10, 10^-7, or 10^-6 M aldosterone ([UNK]). (B) Dose-dependent response of I_sc after 2 h of incubation with ([UNK]) or without (○) various concentrations of aldosterone. Values are means ± SEM of four to seven experiments. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001 versus untreated cell values (○).

Effect of aldosterone on I_sc and R_T. (A) I_eq was measured on confluent mpkCCD_c14 cells incubated without (○) or with ([UNK]) 5 × 10^-7 M aldosterone, added (arrows) to both basal and apical sides of the filters for various times. (Inset) Bars represent the mean values ± SEM (n = 4) from the 24-h aldosterone-treated cells in the absence (-) or presence (+) of apical benzamyl amiloride (B.Am). (B) R_T was measured on the same sets of filters incubated without (○) or with ([UNK]) aldosterone. Values are means ± SEM from six to eight individual filters from eight passages (18th to 30th passages).

Effects of carbenoxolone (CBX) on corticosterone-stimulated I_eq and identification of 11β-HSD2 in cultured mouse CCD cells. (A) I_eq was measured on sets of confluent mpkCCD_c14 cells grown on filters in the absence (○) or presence ([UNK]) of 5 × 10^-7 M corticosterone and corticosterone plus 10^-6 M carbenoxolone (▴) for various times. Corticosterone and carbenoxolone were added (arrow) to the apical and basal side of filters. Values are means ± SEM from six to eight individual filters from eight passages (20th to 35th passages). (B) Samples of cDNA (250 ng) from mouse kidney and cultured mpkCCD_c14 cells were amplified by PCR for 32 cycles using primers from m11β-HSD2 exon 2 and 4 (lanes 1 to 3) or exon 3 and 5 (lanes 4 to 6). Amplified products of expected sizes (412 bp: lanes 1 and 2; 475 bp: lanes 4 and 5) were obtained in both mouse kidney (lanes 1 and 4) and cultured CCD cells (lanes 2 and 5). As control, no amplified products were detected by omitting cDNA (lanes 3 and 6). Fs, fragment sizes of DNA molecular weight markers. (C) The conversion, expressed in percentage of total radioactivity, of 2 nM corticosterone (CS) to 11-dehydrocorticosterone (11-DHCS) was measured on confluent mpkCCD_c14 cells incubated with 2 nM [³H]corticosterone for 4 h at 37°C in the absence (□) or presence ([UNK]) of 10^-6 M CBX. Values are means ± SEM of four experiments.

Mineralocorticoid and glucocorticoid receptors in mouse cultured CCD cells. (A) Samples of cDNA (250 ng) from rat and mouse kidneys and cultured mpkCCD_c14 cells were amplified by PCR for 34 cycles using sets of rMR primers. Amplified products of the expected size (380 bp) were obtained in rat kidney (lane 1), mouse kidney (lane 2), and cultured CCD cells (lane 3). An additional band of amplified products (not identified) was also detected in mouse kidney and cultured cells. (B) Digestion of the amplified products with HindIII yielded two bands of expected size (263 bp and 117 bp) in both mouse kidney (lane 1) and cultured CCD cells (lane 2). As control, no amplified products were detected by omitting cDNA (lane 3). Fs, fragment sizes of DNA molecular weight markers. (C and D) Scatchard plots of [³H]aldosterone ([UNK]) and [³H]dexamethasone (▴) binding to mpkCCD_c14 cytosol. The cytosol was incubated with 0.1 to 40 nM [³H]aldosterone or [³H]dexamethasone for 4 h at 4°C. Bound (B) and free (F) hormone fractions were separated by the dextrancharcoal method. The curve was simulated from the best interaction model, which was one class of specific sites and nonspecific binding. (E) Gradient sedimentation analysis of [³H]aldosterone-receptor complex in the cytosol of mpkCCD cells. Cytosol was incubated with 10^-8 M [³H]aldosterone for 4 h at 4°C in the absence ([UNK]) or presence (○) of a 100-fold excess unlabeled aldosterone. Sedimentation markers were aldolase (A, 7.9 S) and bovine serum albumin (BSA, 4.6 S). (F) The cytosol was also incubated with 10^-8 M [³H]aldosterone without (black bar) or with a 10-fold (hatched white bars) or 100-fold (hatched black bars) excess of unlabeled aldosterone, RU26752, or RU486.

Action of aldosterone on the amounts of ENaC subunit mRNA and protein in cultured mouse CCD cells. Confluent mpkCCD_c14 cells grown on filters were incubated without (time 0) or with 5 × 10^-7 M aldosterone for 2 to 24 h. (A, Top Panel) cDNA from cells were submitted to RT-PCR and amplified with sets of primers specific for α- (▪), β- ([UNK]), and γ- (▴) ENaC and GAPDH, used as internal standard. The graph provides the fractional changes in ENaC subunits over GAPDH mRNA ratios (arbitrary unit), which were normalized to 1 for the untreated cells. (A, Bottom Panel) Illustration of time-dependent increase in α-ENaC mRNA expression induced by aldosterone. (B, Top Panel) Rates of α- (▪), β- ([UNK]), and γ- (▴) ENaC protein syntheses assessed by immunoprecipitation studies. The graph shows the fractional changes in ENaC subunits, normalized to 1 for the values from untreated cells. (B, Bottom Panel) Illustration of the aldosterone-dependent increase in α-ENAC protein synthesis over time. Values are means ± SEM from four separate experiments.