Figure 4
From: Antisense oligonucleotide development for the selective modulation of CYP3A5 in renal disease
Salt-Induced CYP3A5*3 Protein Expression, Enzyme Activity and Mineralocorticoid Receptor Signaling in HEK293 Cells. (a) Confluent HEK293 cells were exposed for 48 h to (A) control media or media supplemented with (B) 10 mM KCl, (C) 50 mM KCl, (D) 100 mM KCl, or (E) 100 mM NaCl. Two protein bands attributed to CYP3A5 were detected via western blot, including the full-length reference protein (CYP3A5WT) at 56 kilodaltons (kDa) and a truncated 30 kDa splice variant form (CYP3A5sv). The housekeeping gene GAPDH, detected at 37 kDa, was used to normalize sample loading. (b) Total CYP3A5 protein and the ratio of CYP3A5WT to CYP3A5SV protein normalized to GAPDH expression was determined using densiometric analysis in ImageJ. (c) CYP3A enzyme activity was monitored in HEK293 cells under normal conditions and with elevated KCl (12 mM; 48 h.) and PMOs (1 µM; 48 h.), as measured fluorometrically by BFC metabolism (see “Methods”). KCl supplementation significantly induced enzyme activity (p < 0.01; t-test) in HEK293 cells (KCl) compared to control cells (CON). The G4 disrupting PMO (G4) and the CYP3A5 start-site inhibitor (AUG) PMO both significantly prevented KCl induction of BFC metabolism (p < 0.01 compared to KCl treated control) and were not different from control cells. The scrambled (SCR) PMO control did not inhibit KCl induction of CYP3A activity (ANOVA p value < 0.01. Post test p < 0.01 = **, and p < 0.005 = ***). (d) LC MS/MS analysis of HEK293 cell media demonstrated KCl enrichment (12 mM, 24 h) rapidly stimulated the conversion of cortisol to 6β-hydroxycortisol. An ~ sixfold change in the metabolic ratio was observed for HEK293 cells grown in normal media (1.93 × 10−3, solid black) compared to 12 mM KCL-supplemented media (1.19 × 10−2, solid gray). (e) Conditioned media from HEK293 cells (+ /- KCl (12 mM); 48 h.) were added to a luciferase-based cell reporter system for mineralocorticoid receptor (MR) transactivation (IB00501-32; Indigo Biosciences). MR activity was significantly elevated > two-fold (p < 0.01; t-test) in HEK293 media treated with 12 mM KCl. The ligand-specific sensitivity of the MR activity assay was validated using a standard curve for aldosterone (1 nM sensitivity = ~ 100,000 RLU).
