27‐Hydroxycholesterol regulates human SLC22A12 gene expression through estrogen receptor action

Figures

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  FIGURE 1. Phylogenetic trees of uric acid reabsorption transporters. A, Schema of uric acid transporters in kidney proximal tubular cells. B and C, Phylogenetic trees of the uric acid transporters (B) amino acid sequences and (C) gene promoter sequences. Three reabsorption transporters are indicated in color. D, A diagram of the gene promoter regions. White boxes show potential ERE sequences, and three grey boxes of SLC22A12, #1‐#3, show the ERE sequences having ERE scores more than 0.8. E, A bar plot showing the ERE values of uric acid transporter gene promoter regions

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  FIGURE 2. SLC22A12 gene promoter activities. A, Schematic representation of a human URAT1/SLC22A12 gene promoter‐luciferase reporter construct. ERE#1, #2, and #3 represent the same site shown in Figure 1D. B‐E, HepG2 cells with stable expression of SLC22A12 gene promoter‐driven luciferase proteins were treated with E1 (B) or E2 (C) at the indicated concentration for 24 hours. D, The effect of 27HC on the SLC22A12 gene promoter activity in HepG2 cells. E, The effect of 3β‐hydroxy‐5‐cholestenoic acid on the URAT1/SLC22A12 promoter activity in HepG2 cells. The results were analyzed using one‐way ANOVA followed by Dunnett's tests. ** P < .01 vs. control (ctrl). NS, nonsignificant. All experiments in triplicate were performed independently three times

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  FIGURE 3. Involvement of ER and EREs in the SLC22A12 gene promoter activity. A, HepG2 cells with stable expression of SLC22A12 gene promoter‐regulated luciferase proteins were treated with ICI 182,780. The results were analyzed using one‐way ANOVA followed by Dunnett's multiple comparison tests. * P < .05, *** P < .001, **** P < .0001 vs. ctrl. B, HepG2 cells were treated with 27HC in the absence or presence of 100 µM ICI 182,780 for 24 hours. The results were analyzed using two‐way ANOVA followed by Sidak's multiple comparisons tests. * P < .05 vs. vehicle control for 27HC. Open column; vehicle control, gray column; 27HC‐treated. C, Consensus ERE sequence, potential ERE sequences on the SLC22A12 gene promoter, and sequence logo of the three EREs. The largest letter (TGAC) shows the conserved sequence among the sequences. D, The effect of 27HC on ERE mutant gene promoter activity. The two HepG2 cell lines that stably express intact or ERE mutant gene promoter‐driven luciferase proteins were treated with 10 µM 27HC for 24 hours. The results were analyzed using two‐way ANOVA followed by Sidak's multiple comparisons tests. * P < .05 compared with nontreatment of 27HC. Open column; vehicle…

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  FIGURE 4. Immunohistochemical analysis of kidneys from Cyp7b1 +/+ and Cyp7b1 −/− mice. A, Immunohistochemical analysis using an anti‐Urat1 antibody. Upper panel: Cyp7b1 +/+ (WT), lower panel: Cyp7b1 −/− (KO). Scale bar showed 200 µm. B, Image analysis on the signal intensities of Urat1 immunostaining in male and female PT cells. Open column; vehicle control, gray column; 27HC‐treated. The results were analyzed using a two‐tailed student's t test. **P < .01 vs. WT. Open column; WT mice, gray column; KO mice

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  FIGURE 5. SLC22A12 expression in human kidney organoids. A, A bar plot showing ERE values of human SLC22A12 and mouse Slc22a12 gene promoter regions. B, The experimental scheme of human kidney organoid culture with 27HC treatments. C, The effect of 27HC on SLC22A12 mRNA expression in the human kidney organoids. The experiment was performed in six independent experiments. *P < .05 vs. ctrl by two‐tailed student's t test

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  FIGURE 6. Graphical abstract. 27HC binds to ER and induces URAT1/SLC22A12 expression. This results in an increase of URAT1 in the apical membrane, and more uric acid is transported into the cells, leading to increased serum uric acid levels. The red arrow demonstrates the upregulation of SLC22A12 transcription. Blue arrows show the movement of uric acid. (A) basal and (B) metabolically dysfunctional condition