Renal Mitochondrial Gene Expression is Dependent on Time of Day in Diet‐induced Obesity

Abstract

Obesity is an independent risk factor in the development of nephropathy and is associated with aberrations in renal energy metabolism. The circadian clock affects oxygen consumption and renal excretory rhythms in mice. However, the relationships between time of food intake, dietary fat content, and renal mitochondrial gene expression have not been explored. We hypothesized that feeding behaviors such as dietary fat content and circadian aspects such as the time of day and time of food intake would alter renal mitochondrial gene expression. Eight-week-old male C57Bl/6 mice were placed on a high (45%) fat or normal (10%) fat diet. After 20 weeks on their diets, the high fat diet group was further split into an ad libitum or reverse feeding schedule where food availability was restricted to the 12h lights on period. After 2 weeks of restricted feeding, mice were anesthetized with 4% isoflurane and euthanized in 4h intervals over a 24h period (ZT1, 5, 9, 13, 17, 21). Kidneys were removed and the cortex, outer and inner medulla were dissected and snap frozen in liquid nitrogen. Samples were stored at -80°C until used for analysis. We selected outer medulla samples from the ZT5 (lights on, inactive period) and ZT17 (lights off, active period) timepoints for analysis because these are associated with nadir and peak levels of energy metabolism in other tissues. Mitochondrial gene expression was measured in 84 genes by mouse-specific microarray analysis (RT2 Profiler PCR Array; Qiagen; Germantown, MD). Fold change/regulation was calculated using the delta-delta CT method. Mitochondria-specific genes within the outer medulla of diet-induced-obese mice display a pattern of expression that appears to be dependent upon the dietary fat content and time of day, but not the timing of food intake. When comparing the normal fat diet at the ZT5 and ZT17 timepoints, 24 genes were upregulated and 39 were downregulated consistent with a time-of-day difference in mitochondrial function. Comparing the high fat diet at the same timepoints yielded 18 upregulated genes and only one downregulated gene suggesting a loss of time-of-day mitochondrial function due to diet-induced obesity. When comparing the normal fat and high fat diet at ZT5, 23 genes were upregulated and 39 genes were downregulated. However, comparing the normal fat and high fat diets at ZT17 resulted in 43 upregulated genes and 20 downregulated genes, suggesting a differential effect of obesity at different times. When the high fat diet was restricted to the active period, 27 genes were differentially expressed between ZT5 and ZT17 indicating a minor improvement in circadian expression. Interestingly, many of the differentially expressed genes are involved in pathways related to membrane polarization and small molecule transport. Our results suggest that there indeed is an effect of time of day on the renal mitochondrial milieu, particularly in the context of diet induced obesity and specifically within the outer medulla. This concurs with previous observations that changes in oxygen consumption within peripheral tissues are under circadian regulation.