Nephron-Specific Deletion of Circadian Clock Gene Bmal1 Alters the Plasma and Renal Metabolome and Impairs Drug Disposition

Abstract

The circadian clock controls a wide variety of metabolic and homeostatic processes in a number of tissues, including the kidney. However, the role of the renal circadian clocks remains largely unknown. To address this question, we performed a combined functional, transcriptomic, and metabolomic analysis in mice with inducible conditional knockout (cKO) of BMAL1, which is critically involved in the circadian clock system, in renal tubular cells (Bmal1^lox/lox/Pax8-rtTA/LC1 mice). Induction of cKO in adult mice did not produce obvious abnormalities in renal sodium, potassium, or water handling. Deep sequencing of the renal transcriptome revealed significant changes in the expression of genes related to metabolic pathways and organic anion transport in cKO mice compared with control littermates. Furthermore, kidneys from cKO mice exhibited a significant decrease in the NAD⁺-to-NADH ratio, which reflects the oxidative phosphorylation-to-glycolysis ratio and/or the status of mitochondrial function. Metabolome profiling showed significant changes in plasma levels of amino acids, biogenic amines, acylcarnitines, and lipids. In-depth analysis of two selected pathways revealed a significant increase in plasma urea level correlating with increased renal Arginase II activity, hyperargininemia, and increased kidney arginine content as well as a significant increase in plasma creatinine concentration and a reduced capacity of the kidney to secrete anionic drugs (furosemide) paralleled by an approximate 80% decrease in the expression level of organic anion transporter 3 (SLC22a8). Collectively, these results indicate that the renal circadian clocks control a variety of metabolic/homeostatic processes at the intrarenal and systemic levels and are involved in drug disposition.

Keywords: arginase; circadian clock; kidney excretory rhythms; metabolome; organic anion transporter.

Figure 1.

Analysis of genes differentially expressed in kidneys of Control and cKO mice revealed enrichment of processes related to the cellular metabolism and organic anion transport. (A) Heat maps of normalized expression values of genes significantly affected in kidneys of cKO mice at ZT4, ZT16, or both time points (false discovery rate <5%). Expression values were mean centered, variance normalized, and subjected to hierarchical clustering (complete linkage) using Pearson correlation as the similarity metric. (B) Gene ontology analysis showing significantly enriched biologic processes (Bonferroni correction) among 552 genes differentially expressed in kidneys of control and cKO mice at ZT4 and ZT16. Significant biologic processes were summarized and classified with REVIGO. Ctrl, control.

Figure 2.

Altered renal metabolism in cKO mice. (A) Quantification of mitochondrial DNA (mtDNA) in kidneys of control and cKO mice (ZT8). Relative amounts of mtDNA and nuclear DNA were quantitated by qPCR of mitochondrially encoded NADH dehydrogenase 1 (mt-Nd1) and nuclear Ppia (cyclophilin) genes (mean±SD; n=6; t test). (B) NAD⁺-to-NADH ratios in the kidney (ZT12) and the liver (ZT12) of control and cKO mice (mean±SD; n=6; t test).

Figure 3.

Altered arginine production/degradation in cKO mice. (A) Western blot analysis of ARGII protein expression in the proximal straight tubules (PSTs) microdissected from kidneys of control and cKO mice (ZT8). Three mice per genotype were used for this analysis. (B) Arginase activity in the kidney (ZT0) and the liver (ZT0) of control and cKO mice (mean±SD; n=3; t test). (C) l-arginine (L-Arg) levels in kidney tissue of control and cKO mice (ZT8; mean±SD; n=6; t test). (D) nitric oxide synthase–specific l-citrulline (L-Citr) formation in kidney tissue of control and cKO mice (ZT8; mean±SD; n=6; t test). prot, Protein.

Figure 4.

Lower OAT3 mRNA and protein expression correlates with impaired natriuretic response to furosemide and decreased urinary excretion of furosemide in cKO mice. (A) qPCR analysis of Oat3 (Slc22a8) mRNA expression in kidneys of control (white circles) and cKO (gray circles) mice (mean±SD; n=6). (B) Western blotting with anti-OAT3 or anti-glyceraldehyde 3-phosphate dehydrogenase antibodies on protein extracts prepared from kidneys of control and cKO mice (n=5 mice in each group) at ZT16. (C) Rightward shift in the natriuretic response to furosemide in cKO mice (mean±SD; n=5). The half maximal inhibitory concentration (IC50) values were calculated using the GraphPad PRISM 6 software (GraphPad Software, La Jolla, CA) at ZT16. (D) Lower urinary excretion of furosemide in cKO mice. Furosemide was dosed in the urine collected for 10 minutes followed by the 0.3-μg/g body wt furosemide bolus (mean±SEM; n=8; t test). Body wt, body weight; Ctrl, control; iv, intravenous; UNaV, sodium excretion rate; v, vehicle.