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Nephron-Specific Deletion of Circadian Clock Gene Bmal1 Alters

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Nephron-Specific Deletion of Circadian Clock Gene Bmal1 Alters BASIC RESEARCH www.jasn.org Nephron-Specific Deletion of Circadian Clock Gene Bmal1 Alters the Plasma and Renal Metabolome and Impairs Drug Disposition † ‡ Svetlana Nikolaeva,* Camille Ansermet,* Gabriel Centeno,* Sylvain Pradervand, | †† Vincent Bize,* David Mordasini,*§ Hugues Henry,¶ Robert Koesters,** Marc Maillard, †† ‡‡ Olivier Bonny,* Natsuko Tokonami,* and Dmitri Firsov* *Department of Pharmacology and Toxicology and ‡Genomic Technologies Facility, University of Lausanne, Lausanne, Switzerland; †Institute of Evolutionary Physiology and Biochemistry, St. Petersburg, Russia; §Department of Nephrology, Hypertension and Clinical Pharmacology, Inselspital, Bern, Switzerland; |Department of Clinical Research, University of Bern, Bern, Switzerland; ¶Service of Biomedicine and ††Service of Nephrology, Department of Medicine, Lausanne University Hospital (CHUV), Lausanne, Switzerland; **Department of Nephrology, Tenon Hospital, Université Pierre et Marie Curie, Paris, France; and ‡‡Labeled Research Team (ERL) 8228–U1138 équipe 3, Centre de Recherche des Cordeliers, Paris, France 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 BASIC RESEARCH 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 (Bmal1lox/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 corre- lating 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 trans- porter 3 (SLC22a8). Collectively, these results indicate that the renal circadian clocks control a variety of meta- bolic/homeostatic processes at the intrarenal and systemic levels and are involved in drug disposition. J Am Soc Nephrol 27: 2997–3004, 2016. doi: 10.1681/ASN.2015091055 The circadian timing system is an essential physi- Received September 23, 2015. Accepted January 10, 2016. ologic regulatory mechanism that provides cells, S.N., C.A., G.C., and S.P. contributed equally to this work. tissues, organs, and finally, the whole organism with Published online ahead of print. Publication date available at an important functional advantage of anticipation www.jasn.org. of circadian changes in the environment that are imposed by the Earth’s rotation. The circadian tim- Correspondence: Dr. Natsuko Tokonami, The National Institute for Health and Medical Research (INSERM)/Université Pierre et Marie ing system is organized in a hierarchical manner, in Curie (UPMC) Paris 6/National Center for Scientific Research (CNRS), that the central oscillator located in the suprachias- Centre de Recherche des Cordeliers Génomique, Physiologie et matic nucleus of hypothalamus coordinates subsid- Physiopathologie Rénales, Equipe 3 U1138, Labeled Research Team (ERL) 8228, 15 rue de l’Ecole de Médecine, 75270 Paris Cedex, France, iary oscillators located in peripheral tissues. On the or Dr. Dmitri Firsov, Department of Pharmacology and Toxicology, molecular level, both the central and peripheral University of Lausanne, 27 rue du Bugnon, 1005 Lausanne, Switzer- oscillators share a common molecular clock land.Email:[email protected] or dmitri.fi[email protected] mechanism on the basis of cell–autonomous and Copyright © 2016 by the American Society of Nephrology J Am Soc Nephrol 27: 2997–3004, 2016 ISSN : 1046-6673/2710-2997 2997 BASIC RESEARCH www.jasn.org self–sustained transcriptional/translational feedback RESULTS loops. This core clock mechanism controls circadian expression of so–called output genes that, in turn, im- Validation of the Bmal1lox/lox/Pax8-rtTA/LC1 pose cell–specific functional rhythms (reviewed in refs. (Conditional Knockout) Knockout Model 1and2). The conditional inactivation of the Arntl gene encoding Previous studies have shown that the circadian timing BMAL1 was induced by 2-week treatment with doxycycline system plays a major role in renal function. In mice, whole- (DOX; 2 mg/ml in drinking water) of 8-week-old Bmal1lox/lox/ body inactivation of different elements of the molecular clock Pax8-rtTA/LC1 mice (hereafter referred to as conditional leads to abnormal circadian patterns of urinary sodium and knockout [cKO] mice). In parallel, the same DOX treatment potassium excretion, loss of the circadian rhythmicity of was provided to their littermate controls (Bmal1lox/lox mice; plasma aldosterone levels, and significant changes in arterial hereafter referred to as control mice). The quantitative PCR BP (reviewed in refs. 3–5). In humans, growing evidence (qPCR) analysis revealed that Bmal1 mRNA expression was suggests a possible link between dysregulation of renal circa- significantly reduced in the kidneys of cKO mice (2 months dian rhythms and development of hypertension and accelera- after the end of DOX treatment) (Supplemental Figure 1). ted progression of CKD. The importance of the circadian clock Immunohistochemical staining for BMAL1 and CRE expres- system in the kidney has been recently shown on the molecular sion was performed on day 5 of the 2-week DOX treatment level. Zuber et al.6 and Nikolaeva et al.7 have shown that hun- period or 2 months after the end of DOX treatment. As shown dreds of transcripts in the microdissected distal convoluted in Supplemental Figure 2, at day 5 of DOX treatment, the CRE tubule/connecting tubule and the cortical collecting duct was ubiquitously expressed along the renal tubule but not in exhibit significant circadian oscillations in their expression glomeruli or blood vessels in cKO mice. The BMAL1 protein levels and that the whole-body inactivation of circadian tran- was ubiquitously expressed in all kidney cells in control mice, scriptional factor Clock leads to dramatic changes in the tran- but in cKO mice, all tubular cells became negative for BMAL1 scriptomes of the distal convoluted tubule/connecting tubule staining at day 5 of DOX treatment (Supplemental Figure 3). and the cortical collecting duct. Gumz and colleagues8–10 have The specific tubular inactivation of BMAL1 was maintained in shownthatthecircadianclockproteinPER1exertsdiverse cKO mice 2 months after the end of DOX treatment (Supple- effects on the renal handling of sodium. Zhang et al.11 have mental Figure 3). shown that, among 12 tested mouse tissues, the kidney ex- To assess the effect of BMAL1 deficiency on circadian hibits the second greatest number of circadian transcripts (the mechanisms per se, we performed qPCR analysis of expression greatest number was found in the liver). However, the role of levels of several genes involved in the core clock (Cry1, Per2, the renal circadian clocks in the generation of functional and and Nr1d1)andDbp, an output gene that is directly controlled molecular rhythms in the kidney as well as the development of by the circadian clock. As shown in Supplemental Figure 1, the kidney disease remains largely unknown. To our knowledge, BMAL1 deficiency results in a significant attenuation in cir- the only study that addressed this question is a recent paper by cadian oscillations of mRNA expression of Cry1, Per2, Nr1d1, Tokonami et al.,12 which showed that ablation of the circadian and Dbp. The expression level of Bmal2, a paralog of Bmal1 clock in renin–secreting granular cells results in several that is thought to be able to rescue the BMAL1 deficiency in abnormalities, including increased GFR, decreased plasma al- some tissues,13 was increased in kidneys of cKO mice, but the dosterone levels, and low BP. Here, we studied the role of absolute levels of Bmal2 expression remained significantly circadian clocks in the nephron by using conditional and lower than those of other tested core clock components (Sup- inducible inactivation of BMAL1 protein, an indispensable plemental Figure 1). Collectively, these results suggested that element of the molecular clock (Bmal1lox/lox/Pax8-rtTA/LC1 the activity of the circadian clock in renal tubular cells is sig- mice). nificantly impaired. The renal tubular cells are involved in a wide variety of PAX8 is a paired–box transcriptional factor crucial to the essential functions, including maintenance of homeostasis, organogenesis and development of the kidney, the thyroid disposition of xenobiotics, and synthesis of molecules that are gland, and the Müllerian system. Traykova-Brauch et al.14 released into the bloodstream (e.g., bicarbonate and arginine). used a partial Pax8 promoter to target the expression
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