Alteration of circadian machinery in monocytes underlies chronic kidney disease-associated cardiac inflammation and fibrosis

Dysfunction of the circadian clock has been implicated in the pathogenesis of cardiovascular disease. The CLOCK protein is a core molecular component of the circadian oscillator, so that mice with a mutated Clock gene (Clk/Clk) exhibit abnormal rhythms in numerous physiological processes. However, here we report that chronic kidney disease (CKD)-induced cardiac inflammation and fibrosis are attenuated in Clk/Clk mice even though they have high blood pressure and increased serum angiotensin II levels. A search for the underlying cause of the attenuation of heart disorder in Clk/Clk mice with 5/6 nephrectomy (5/6Nx) led to identification of the monocytic expression of G protein-coupled receptor 68 (GPR68) as a risk factor of CKD-induced inflammation and fibrosis of heart. 5/6Nx induces the expression of GPR68 in circulating monocytes via altered CLOCK activation by increasing serum levels of retinol and its binding protein (RBP4). The high-GPR68-expressing monocytes have increased potential for producing inflammatory cytokines, and their cardiac infiltration under CKD conditions exacerbates inflammation and fibrosis of heart. Serum retinol and RBP4 levels in CKD patients are also sufficient to induce the expression of GPR68 in human monocytes. Our present study reveals an uncovered role of monocytic clock genes in CKD-induced heart failure.


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Sample size
No statistical method was used to predetermine sample sizes, but our sample sizes are similar to those reported in previous publications ( For WB, Can Get Signal Immunoreaction Enhancer Solution (Toyobo) was used for dilution of antibodies. For Immunofluorescence histochemical staining, Can Get Signal Immunostain Immunoreaction Enhancer Solution (Toyobo) was used for dilution of antibodies. For ChIP, antibodies were diluted in purified water with 16.7 mM Tris-HCl, 167 mM NaCl, 1.2 mM EDTA, 1.1 % Triton X-100, 10 % SDS. For FCM, antibodies were diluted in PBS with 10 % FBS and 10 mM EDTA.

Validation
All the antibodies used in this study were commercial antibodies. Validation of all antibodies was performed by the respective companies.
anti  None of the used cell lines is listed in ICLAC database.

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Laboratory animals
Clock mutant mice (C57BL/6J-ClockmlJt/J) were purchased from the Jackson Laboratory (Bar Harbor, ME, USA) and backcrossed to wild-type Jcl: ICR mice (Charles River Laboratory Japan, Inc.; Yokohama, Japan) for more than eight generations to improve breeding and offspring care. Male ICR mice were housed in a light-controlled room (lights on from ZT0 to ZT12) at 24 ± 1 °C and 60% ± 10% humidity, and the animals had free access to water and AIN-93G pelleted diet (positive control diet groups contained 4000 IU of vitamin A/kg) or a vitamin-A-free AIN-93G pelleted diet (Oriental Yeast Co., ltd.).