Fig. 3: Knockdown and overexpression of Bmal1 increases and decreases NRF1 expression in HK2 cells.

HK2 cells were transfected (n = 3) with siBMAL1 for 72 h and used to measure protein (A, n = 3) and mRNA (B, n = 5–6). Mean ± SD, multiple unpaired t-test. Representative of two experiments. HK2 cells transduced with adenoviruses expressing green fluorescent protein (Adv-CONTROL) or BMAL1 (Adv-BMAL1) were used to detect proteins (C, n = 3) and mRNA levels (D, n = 5–6). Mean ± SD, unpaired two-tailed t-test. HK2 cells transduced with lentiviruses for expression of shBMAL1 (E) or adenoviruses for expression of human BMAL1 (F) were transfected with pGL3 plasmid for expression of luciferase under control of the NRF1 promoter and used to measure NRF1 promoter activity. Mean ± SD, n = 5–6, unpaired two-tailed t-test. WT RPTECs were transfected with siBmal1 or siControl for 72 h and used to measure mRNA levels (n = 4–5) (G, top), transcription factors (G, bottom), and proteins (H, representative of n = 5–6). Mean ± SD, multiple unpaired t-tests. I WT RPTECs transduced with Adv-Bmal1 or Adv-Control for 72 h were used to measure Nrf1 and Sglt2 mRNA. Mean ± SD, n = 5–6, multiple unpaired t-tests. BBMVs from the renal cortex in female (J) and male (K) mice were used to measure Nrf1 and Sglt2 proteins (n = 3). BBMVs prepared from chow-fed female (L) and male (M) Bmal1^+/+ and Bmal1^−/− mice at 12:00 h or 24:00 h were incubated with [¹⁴C]-αMG (0.5 µCi/mL) for the indicated times. Mean ± SD, n = 4. *p < 0.05, **p < 0.01, ***p < 0.001; two-way repeated measures ANOVA. Precision-cut kidney slices (PCKs) from female (N, n = 3) or male (O, n = 4) Bmal1^+/+ and Bmal1^−/− mice were cultured for 2 h and incubated with [¹⁴C]-αMG (0.5 µCi/mL) for 2 h. Mean ± SD, n = 3–4/group, unpaired two-tailed t-test. *p < 0.05, **p < 0.01 and ***p < 0.001.