Diurnal Differences in Mitochondrial Bioenergetics is Lost in Bmal1 Knockout Rats

Abstract

In the kidney, 95% of O2 consumed through mitochondrial respiration is used for Na+ transport. The circadian clock regulates Na+ transport with a necessary diurnal variation for optimal health. The circadian clock transcription factor BMAL1 is proposed to regulate mitochondrial bioenergetic function; however, this has not been extensively investigated in the kidney. Therefore, we tested the hypothesis that renal mitochondrial respiration may be impaired or altered in a novel Bmal1 clock gene knock‐out rat model and is associated with loss of diurnal control of Na+ excretion. Male and female global Bmal1+/+ and Bmal1‐/‐ rats at 12‐14 weeks of age maintained in a regular 12‐hour light/dark cycle was used to measure renal mitochondrial O2consumption. Outer renal medullary tissue was dissected and prepared for respiration measurements at either day (ZT2‐4) or night (ZT14‐16) periods to correspond with minimum and maximum whole‐body energy consumption as well as peak and trough BMAL1 protein expression, respectively (n = 10, Bmal1+/+; n = 22, Bmal1‐/‐). Respiration was assessed using permeabilized tissue in the Oroboros Oxygraph and analyzed using Data Lab 2 software (O2K, Oroboros Instruments GmbH). Mitochondrial gene expression analysis was assessed using digital drop PCR. Data from both sexes were combined for each genotype at the two time points and analyzed by two‐way ANOVA. Mitochondrial state 3 O2 consumption was significantly higher (main effect of genotype p = 0.0310) in Bmal1‐/‐ (118 ± 156 pmol/s*mg) compared to BMAL1+/+ rats (75 ± 15 pmol/s*mg; Tukey post‐hoc p = 0.0303). We also observed a significant increase in complex IV activity in Bmal1‐/‐ rats during the dark vs. light period (164 ± 40 vs. 306 ± 40 pmol/s*mg; p=0.0031, t‐test). Mitochondrial‐associated genes, optic atrophy 1 (Opal1) and mitofusion 1 (Mfn1) show as significant day‐night difference in mRNA expression in kidneys from BMAL1+/+ control rats that is lost in Bmal1‐/‐ rats consistent with disrupted mitochondrial fusion and fission processes. Our lab previously reported that male Bmal1‐/‐ rats do not have the typical night‐day difference in Na+ excretion, which is consistent with our new data showing an alteration in mitochondrial function in kidneys from Bmal1‐/‐ rats. Therefore, our findings demonstrate that BMAL1 may play a role in maintaining the coupling of mitochondrial respiration and ATP generation in the kidney and support the hypothesis that diurnal Na+handling is regulated in part by BMAL1‐dependent mitochondrial function.