Molecular clock is involved in predictive circadian adjustment of renal function

Abstract

Renal excretion of water and major electrolytes exhibits a significant circadian rhythm. This functional periodicity is believed to result, at least in part, from circadian changes in secretion/reabsorption capacities of the distal nephron and collecting ducts. Here, we studied the molecular mechanisms underlying circadian rhythms in the distal nephron segments, i.e., distal convoluted tubule (DCT) and connecting tubule (CNT) and the cortical collecting duct (CCD). Temporal expression analysis performed on microdissected mouse DCT/CNT or CCD revealed a marked circadian rhythmicity in the expression of a large number of genes crucially involved in various homeostatic functions of the kidney. This analysis also revealed that both DCT/CNT and CCD possess an intrinsic circadian timing system characterized by robust oscillations in the expression of circadian core clock genes (clock, bma11, npas2, per, cry, nr1d1) and clock-controlled Par bZip transcriptional factors dbp, hlf, and tef. The clock knockout mice or mice devoid of dbp/hlf/tef (triple knockout) exhibit significant changes in renal expression of several key regulators of water or sodium balance (vasopressin V2 receptor, aquaporin-2, aquaporin-4, alphaENaC). Functionally, the loss of clock leads to a complex phenotype characterized by partial diabetes insipidus, dysregulation of sodium excretion rhythms, and a significant decrease in blood pressure. Collectively, this study uncovers a major role of molecular clock in renal function.

Fig. 1.

(A) Temporal distribution of acrophases of all differentially expressed transcripts (fold change > 1.8). The fold change was calculated between the expression level of a transcript at each individual Zeitgeber time point and the mean value of expression determined from all six Zeitgeber time points. (B) Temporal distribution of acrophases of differentially expressed solute carriers (Slc, fold change > 1.8), see also Table S5. (C) Temporal distribution of acrophases of differentially expressed enzymes involved in phase I and phase II reactions of metabolism of xenobiotics and other lipophilic compounds (fold change > 1.8), see also Table S5. The analysis was performed on the following phase I/phase II enzymes: CYP450 enzymes, flavin containing monooxygenases, alcohol denydrogenase, epoxide hydrolases, aldehyde dehydrogenases, carboxylesterases, glutathione-S-transferases, N-acetyltransferases, sulfotransfearses, UDP glucoronosyltransferases, NADPH dehydrogenases, glycin-N-acetlytransferase, aldehyde oxidases, aldo-keto reductases, acyl-CoA-synthetases.

Fig. 2.

Temporal distribution of acrophases of transcripts that meet the circadian criteria (fitting to a cosine curve with a period of 24-h, adjusted P value < 0.1, amplitude > 1.8). See also Table S6 and Table S7.

Fig. 3.

Temporal expression profiles of circadian transcripts. (A) Transcripts that meet the circadian criteria in both DCT/CNT and CCD. The red and blue squares show the expression values (arbitrary units of microarray hybridization data) of DCT/CNT and CCD circadian transcripts, respectively. The red and blue solid lines show fitting of the microarray data to the cosine function. The black squares show the qPCR expression values (ZT0 = 100%) of these transcripts in the whole kidney RNA samples. The black dashed line shows fitting of qPCR data to the cosinor function. The qPCR was performed on pools of RNA composed of equivalent amounts of RNA prepared from five animals at each ZT time point. The qPCR data are expressed as arbitrary units normalized for β-actin expression. (B) Transcripts that meet the circadian criteria only in DCT/CNT. (C) Transcripts that meet the circadian criteria only in CCD. Abbreviations used are: V1aR, vasopressin receptor Type 1a; V2R, vasopressin receptor Type 2; Usp2, ubiquitin specific protease 2; Gilz (Tsc22d3), glucocorticoid-induced leucine zipper; Tfrc, transferrin receptor; Mapre2, microtubule-associated protein; Ptges, prostaglandin E synthase; Slc6a9, glycine transporter; Slc6a6, taurine transporter; aqp2, aquaporin 2; aqp4, aquaporin 4.

Fig. 4.

(A) Temporal expression pattern of Gilz, Usp2, adenylyl kinase 4 (Ak4), and Mapre2 RNA in whole kidneys of wild-type (solid line) or dbp/hlf/tef triple knockouts (dashed line). Data are expressed as the arbitrary units of qPCR amplification normalized for β-actin expression. The qPCR was performed on pools of RNA composed of equivalent amounts of RNA prepared from six animals at each ZT time point. (B) Expression V2R, V1aR, Aqp-2, Aqp-4, Tfrc, Usp2, Gilz, Dbp, and Mapre2 transcripts at ZT2 and ZT12 in whole kidneys of wild-type (white bars) or clock(−/−) triple knockouts (gray bars). Data are means ± SEM of qPCR amplification values that were obtained from seven mice. Statistical significance was calculated using unpaired t-test. *, P = 0.05; **P < 0.05; ***, P < 0.001.

Fig. 5.

Diurnal profile of blood pressure in wild-type and clock(−/−) mice. The systolic, diastolic, and mean blood pressures are indicates as black, gray, and black dashed lines, respectively. Error bars show SEM (n = 5).