PPARG agonist pioglitazone influences diurnal kidney medulla mRNA expression of core clock, inflammation-, and metabolism-related genes disrupted by reverse feeding in mice

Abstract

This study examined the influence of PPARG activation by pioglitazone (PG) on the mRNA of core clock, inflammation- and metabolism-related genes in the mouse kidney medulla as well as urinary sodium/potassium excretion rhythms disrupted by reverse feeding. Mice were assigned to daytime feeding and nighttime feeding groups. PG 20 mg/kg was administered at 7 am or 7 pm. On day 8 of the feeding intervention, mice were killed at noon and midnight. Kidney medulla expression of Arntl, Clock, Nr1d1, Cry1, Cry2, Per1, Per2, Nfe2l2, Pparg, and Scnn1g was determined by qRT PCR. We measured urinary K⁺ , Na⁺ , urine volume, food, and H₂ O intake. The reverse feeding uncoupled the peripheral clock gene rhythm in mouse kidney tissues. It was accompanied by a decreased expression of Nfe2l2 and Pparg as well as an increased expression of Rela and Scnn1g. These changes in gene expressions concurred with an increase in urinary Na⁺ , K⁺ , water excretion, microcirculation disorders, and cell loss, especially in distal tubules. PG induced the restoration of diurnal core clock gene expression as well as Nfe2l2, Pparg, Scnn1g mRNA, and decreased Rela expressions, stimulating Na⁺ reabsorption and inhibiting K⁺ excretion. PG intake at 7 pm was more effective than at 7 am.

Keywords: PPARG; clock genes; kidney; pioglitazone; reverse feeding.

FIGURE 1

Experimental flowchart. Sun () and moon () pictograms indicate the light and dark periods, respectively. Solid lines indicate feeding and fasting periods. Experimental schedule from day 0 to day 8: (a) mice entrained to a 12‐h light–dark cycle with ad libitum access to food and water. (b) mice in daytime feeding groups. (c) mice in nighttime feeding groups. (d) mice were killed on day 8 at noon (HALO 05) and midnight (HALO 17), and kidney specimens and serum samples were collected. Sacrification for histopathological analysis was performed at HALO 05. White arrows indicate the time of pioglitazone administration. HALO, hour after light onset.

FIGURE 2

Circadian changes in mouse clock gene mRNA transcription in kidney tissues. Expression of mRNA: (a) Per1; (b) Per2; (c) Cry1; (d) Cry2; (e) clock; (f) Arntl; (g) Nr1d1. Significant differences are shown by the horizontal line (p < 0.05). Y axis—Relative mRNA levels

FIGURE 3

Circadian changes of mRNA transcription of inflammation/metabolism‐related genes in mouse kidney tissues. Expression of mRNA: (a) Nfe2l2; (b) Pparg; (c) Rela; (d) Scnn1g. Significant differences are shown by the horizontal line (p < 0.05). Y axis—Relative mRNA levels.

FIGURE 4

Summary of the effect of feeding intervention and pioglitazone intake on mouse urine metabolism in rest or active periods: (a) urinary Na⁺ concentration; (b) urinary K⁺ concentration; (c) urine volume; (d) food intake; (e) H₂O intake. Mice had ad libitum access to water. In nighttime feeding groups, mice had access to food in dark period (7 pm–7 am) and were fasted in light (7 am–7 pm) period. In daytime feeding groups, mice had access to food in light period and were fasted in dark period. Urine volume, food, and H₂O intake were determined at 12‐h dark and light periods. Open and solid dots indicate the daytime and nighttime feeding, respectively. p‐value was calculated by two‐way anova followed by Bonferroni post hoc tests

FIGURE 5

Representative morphology (H&E) and comparisons of proportions of injured tubules of kidney: (a) the distal tubules (arrows) in control mice are mostly intact; (b) the majority of distal tubules are injured (arrows) in untreated mice; (c) decrease in injured distal tubules (arrows) in daytime feeding PG 7 am mice; (d) decrease in injured distal tubules (arrows) in daytime feeding PG 7 pm mice; (e) comparisons of proportions of total injured tubules; f, comparisons of proportions of the distal injured tubules. p‐value was calculated by chi‐square test.