Circadian clock genes directly regulate expression of the Na ⁺/H⁺ exchanger NHE3 in the kidney

Background. Daily rhythms in mammalian physiology are generated by a transcription/translation feedback loop orchestrated by a set of clock genes. However, little is known about the molecular cascade from the clock gene oscillators to cellular function. Methods. The mRNA expression profiles of NHE3 and clock genes were examined in mice and rat kidneys. First, luciferase assays followed by a site directed mutagenesis of an E-box sequence were used to assess the CLOCK:BMAL1-transactivated NHE3 promoter activity. A direct binding of CLOCK:BMAL1 heterodimers to an E-box sequences of NHE3 promoter was confirmed by electrophoretic mobility shift assay (EMSA). Results. We present evidence that renal tubular NHE3, the Na⁺/H⁺ exchanger critical for systemic electrolyte and acid-base homeostasis, is a clock-controlled gene regulated directly by CLOCK:BMAL1 heterodimers in kidneys. NHE3 mRNA level in rat kidney displayed circadian kinetics, and this circadian expression was severely blunted in homozygous CRY1/2 double-deficient mice, suggesting that the transcriptional machinery of peripheral clocks in renal tubular cells directly regulates the circadian expression of NHE3. By analyzing the 5′ upstream region of the NHE3 gene, we found an E box critical for the transcription of NHE3 via the CLOCK:BMAL1-driven circadian oscillator. The circadian expression of NHE3 mRNA was reflected by oscillating protein levels in the proximal tubules of the rat kidney. Conclusion. NHE3 should represent an output gene of the peripheral oscillators in kidney, which is regulated directly by CLOCK:BMAL1 heterodimers.