LLC-PK1 CELLS MODEL 11-BETA-HYDROXYSTEROID DEHYDROGENASE TYPE-2 REGULATION OF GLUCOCORTICOID ACCESS TO RENAL MINERALOCORTICOID RECEPTORS

Mineralocorticoid receptors (MRs) are nonselective in vitro, binding corticosterone, cortisol, and aldosterone with similar affinity. In the distal nephron in vivo, MRs are selectively activated by aldosterone despite much higher glucocorticoid levels. This has been suggested to reflect the action of 11 beta-hydroxysteroid dehydrogenase (11 beta-HSD), which catalyzes rapid inactivation of corticosterone to 11-dehydrocorticosterone (cortisol to cortisone). However, cellular models of this effect have not been reported, and a recent study suggested that properties intrinsic to MR contribute to aldosterone selectivity. We have screened clonal mammalian cell lines for 11 beta-HSD activity. Pig kidney epithelial LLC-PK1 cells expressed by far the greatest 11 beta-HSD activity. In cell homogenates, this was NAD-dependent, with K-m for corticosterone of 34.4 nM and cortisol of 89.7 nM. Intact LLC-PK1 cells showed similar apparent K-m for corticosterone (13.9 nM) and cortisol (79.4 nM); only 11 beta-dehydrogenation was detected. These biochemical data indicate the expression of the type 2 isoform, 11 beta-HSD2. Using primers to conserved regions of 11 beta-HSD2, a reverse transcriptase-polymerase chain reaction product was obtained from LLC-PK1 cell RNA. Sequence analysis revealed close homology to previously cloned 11 beta-HSD2 cDNAs from several species. LLC-PK1 cell 11 beta-HSD activity was inhibited by carbenoxolone (IC50 similar to 10(-8) M) and high concentrations of estradiol or progesterone (10(-7) and 10(-6) M), but was induced at lower estradiol concentrations (10(-8) and 10(-9) M).

To examine whether the 11 beta-HSD2 activity in LLC-PK1 cells regulates corticosterone access to MR, cells were transfected with the corticosteroid-inducible mouse mammary tumor virus long terminal repeat-luciferase reporter construct. Cell transfection by a lipofection method did not alter 11 beta-HSD activity in LLC-PK1 cells. LLC-PK1 cells expressed low levels of MR (13.9 fmol/mg protein, dissociation constant (K-d) 0.3 X 10(-9) M for aldosterone) and glucocorticoid receptors (GR; 18.5 fmol/mg protein, K-d 0.3 x 10(-9) M for dexamethasone). Transfection with mouse mammary tumor virus long terminal repeat-luciferase reporter construct alone suggested that the endogenous levels of MR and GR were insufficient to affect transcription. However, cotransfection of LLC-PK1 cells with pRShMR, an MR expression plasmid, allowed at least 50-fold induction of luciferase with 10(-8) M aldosterone; the ED(50) 0.3 x 10(-9) M closely reflects the in vitro affinity of MR for aldosterone. Corticosterone only weakly induced luciferase (maximum of 6-fold induction). Carbenoxolone alone (10(-6) M) was without effect on luciferase expression but potentiated the action of corticosterone up to 5-fold. Thus, in LLC-PK1 cells, endogenous 11 beta-HSD appears capable of protecting MR from activation by corticosterone. These data support the hypothesis that 11 beta-HSD is a sufficient explanation for aldosterone-selective MR in the distal nephron.