Monocyte/Macrophage-Specific Loss of ARNTL Suppresses Chronic Kidney Disease-Associated Cardiac Impairment

Abstract

Defects in Aryl hydrocarbon receptor nuclear translocator-like 1 (ARNTL), a central component of the circadian clock mechanism, may promote or inhibit the induction of inflammation by monocytes/macrophages, with varying effects on different diseases. However, ARNTL's role in monocytes/macrophages under chronic kidney disease (CKD), which presents with systemic inflammation, is unclear. Here, we report that the expression of Arntl in monocytes promoted CKD-induced cardiac damage. The expression of G-protein-coupled receptor 68 (GPR68), which exacerbates CKD-induced cardiac disease, was regulated by ARNTL. Under CKD conditions, GPR68 expression was elevated via ARNTL, particularly in the presence of PU.1, a transcription factor specific to monocytes and macrophages. In CKD mouse models lacking monocyte-specific ARNTL, GPR68 expression in monocytes was reduced, leading to decreased cardiac damage and fibrosis despite no improvement in renal excretory capacity or renal fibrosis and increased angiotensin II production. The loss of ARNTL did not affect the expression of marker molecules, indicating the origin or differentiation of cardiac macrophages, but affected GPR68 expression only in cardiac macrophages derived from mature monocytes, highlighting the significance of the interplay between GPR68 and ARNTL in monocytes/macrophages and its influence on cardiac pathology. Understanding this complex relationship between circadian clock mechanisms and disease could help uncover novel therapeutic strategies.

Keywords: ARNTL; cardiac pathology GPR68; chronic kidney disease; circadian clock mechanism.

Figure 1

Effect of ARNTL on induction of GPR68 expression in RAW264.7 and mouse primary monocytes by 5/6Nx-derived serum. (A) mRNA of Arntl in RAW264.7 incubated with 10% serum from Sham and 5/6Nx mice for 24 h. (B) Transcriptional regulation of Gpr68 using serum prepared from Sham or 5/6Nx mice. Number of nucleotide residues indicates distance from transcription start site (+1). RAW264.7 cells were transfected with Gpr68 (-1734)-Luc, Gpr68 (-1512)-Luc, Gpr68 (-1261)-Luc, Gpr68 (-27)-Luc, or pGL4.18. Values are expressed as mean ± S.D. (n = 4). (C) Influence of CLOCK/ARNTL on transcriptional activity of mouse GPR68. RAW264.7 cells were transfected with Gpr68 (-1734)-Luc in presence or absence of CLOCK and ARNTL-expressing vectors. Relative luciferase activity of pGL4.18-transfected cells in absence of CLOCK/ARNTL was set at 1.0. (D) High-ARNTL-expressing RAW264.7 was created by introducing an ARNTL expression plasmid. ARNTL expression levels were measured using Western blotting. (E) Protein levels of GPR68 in RAW264.7-transfected pcDNA3.1 or ARNTL-expressing vectors. (F) Loss of Arntl caused by CRE-LOXP system resulted in loss of ARNTL protein in monocytes. Monocytes isolated from monocytic ARNTL +/+ mice or monocytic ARNTL −/− mice. (G) Expression of Gpr68 mRNA in primary cultured monocytes, which were isolated from monocytic ARNTL +/+ mice or monocytic ARNTL −/− mice. mRNA levels of Gpr68 were assessed after treatment with serum from Sham or 5/6Nx WT mice for 24 h. Values are expressed as mean ± S.D. (n = 4–6). *, p < 0.05, **, p < 0.01 indicates significant differences between two groups (two-way ANOVA with Tukey–Kramer post hoc tests or Student’s t-test).

Figure 2

The effect of monocyte/macrophage-specific transcription factor PU.1 on the induction of GPR68 expression. (A) The 5/6Nx-derived serum did not increase the transcriptional activity upstream of Gpr68 in NIH3T3. NIH3T3 was transfected with Gpr68 (-1734)-Luc or pGL4.18 and incubated with 10% serum from Sham and 5/6Nx mice for 24 h. (B) Transcription factors binding upstream of Gpr68 analyzed by previous transcriptome analyses. The blue waveform shows the sequenced tags in ChIP sequence analysis for each transcription factor. The numbers on the horizontal axis indicate the distance from the transcription start site (kbp). (C) The PU.1 protein in RAW264.7 incubated with 10% serum from Sham and 5/6Nx mice for 24 h. (D) High-PU.1-expressing NIH3T3 was created by introducing a PU.1 expression plasmid. PU.1 expression levels were measured using Western blotting. (E,F) The mRNA levels of Gpr68 (E) and Arntl (F) in NIH3T3-transfected pcDNA3.1 or PU.1-expressing vectors were measured after incubation with 10% serum from Sham and 5/6Nx mice for 24 h. (G) A schematic of mouse Gpr68. The numbers indicate the distance from the transcription start site (+1). Black rectangles, E-box. The arrow symbols indicate the location on the gene where the primer sets localize for the analysis of ChIP. (H) The binding of endogenous ARNTL to the Gpr68 upstream region in NIH3T3-transfected pcDNA3.1 or PU.1-expressing vectors. Values are expressed as the mean ± S.D. (n = 3–5). **, p < 0.01 indicates significant differences between the two groups (two-way ANOVA with Tukey–Kramer post hoc tests or Student’s t-test).

Figure 3

The effect of the loss of monocyte-specific ARNTL on the 5/6Nx-induced induction of GPR68 expression. (A) The binding of endogenous ARNTL or CLOCK to the Gpr68 upstream region in Ly6G⁻/CD11b⁺/Ly6C⁺ cells prepared from ARNTL +/+ or ARNTL −/− Sham and 5/6Nx mice in the blood. The primer sets used are shown in Figure 2G. (B) The expression of Gpr68 mRNA in Ly6G⁻/CD11b⁺/Ly6C⁺ cells prepared from ARNTL +/+ or ARNTL −/− Sham and 5/6Nx mice in the blood and spleen. (C,D) Flow cytometry analysis was performed to detect high-GPR68-expressing Ly6G⁻/CD11b⁺/Ly6C⁺ cells in the blood and spleen. The ratio of high-GPR68-expressing monocytes in the blood and spleen. For all panels, values are expressed as the mean ± S.D. (n = 5–7). **, p < 0.01, ** indicates significant differences between the two groups (two-way ANOVA with Tukey–Kramer post hoc tests).

Figure 4

The effect of the deficiency of monocyte-specific ARNTL on 5/6Nx-induced cardiac injury. (A) Serum BNP concentrations in ARNTL +/+ or ARNTL −/− Sham and 5/6Nx mice. Values are expressed as the mean ± S.D. (n = 6). (B,C) Mutations in Arntl in monocytes ameliorated CKD-induced cardiac fibrosis. Panel (B) shows Masson’s trichrome staining of tissue fibrosis in blue. Scale bars indicate 1 mm (upper panel) and 50 μm (lower panel). Panel (C) shows the quantification of the fibrosis area under light microscopy. Values are expressed as the mean ± S.D. (n = 5). (D) The total amount of collagen throughout the ventricle. Values were corrected for total protein mass. Values are expressed as the mean ± S.D. (n = 5–6). (E) Cardiac TIMP-1 protein levels in ARNTL +/+ or ARNTL −/− Sham and 5/6Nx mice. Values were corrected for total protein mass. Values are expressed as the mean ± S.D. (n = 5–6). (F) The mRNA levels of Tnf-α and Il-6 and fibrosis-related factors (Col1a1, Col1a2, Mmp1a, Timp-1, and αSma) in the cardiac ventricle of ARNTL +/+ or ARNTL −/− Sham and 5/6Nx mice. The mean value of the Sham-operated ARNTL +/+ group was set to 1.0. Values are expressed as the mean ± S.D. (n = 5). *, p < 0.05, **, p < 0.01 indicates significant differences between the two groups (two-way ANOVA with Tukey–Kramer post hoc tests).

Figure 5

The effect of the loss of monocyte-specific ARNTL on renal function in 5/6Nx mice. (A) Masson’s trichrome staining for the kidneys prepared from ARNTL +/+ or ARNTL −/− Sham and 5/6Nx mice. Scale bars indicate 50 μm. (B) The total amount of collagen throughout the kidney. Values were corrected for total protein mass. Values are expressed as the mean ± S.D. (n = 5–6). (C) Renal TIMP-1 protein levels in ARNTL +/+ or ARNTL −/− Sham and 5/6Nx mice. Values were corrected for total protein mass. Values are expressed as the mean ± S.D. (n = 5–6). (D) The mRNA levels of Tnf-α and Il-6 and fibrosis-related factors (Col1a1, Col1a2, Mmp1a, Timp-1, and αSma) in the kidney of ARNTL +/+ or ARNTL −/− Sham and 5/6Nx mice. The mean value of the Sham-operated ARNTL +/+ group was set to 1.0. Values are expressed as the mean ± S.D. (n = 5). (E–H) The serum concentrations of creatinine (E), urea nitrogen (F), angiotensin II (G), and aldosterone (H), in ARNTL +/+ or ARNTL −/− Sham and 5/6Nx mice. (I) The mRNA levels of Tgf-β in the kidney of ARNTL +/+ or ARNTL −/− Sham and 5/6Nx mice. The mean value of the Sham-operated ARNTL +/+ group was set as 1.0. (J) The serum concentrations of retinol in ARNTL +/+ or ARNTL −/− Sham and 5/6Nx mice. In all panels, values are expressed as the mean ± S.D. (n = 4–5). **, p < 0.01; *, p < 0.05 significant difference between the two groups (one-way or two-way ANOVA with Tukey–Kramer post hoc tests).

Figure 6

The effect of the loss of monocyte-specific ARNTL on the 5/6Nx-induced induction of GPR68 expression. (A,B) The mRNA levels of Vcam1 and Sele in the cardiac ventricle or kidney of ARNTL +/+ or ARNTL −/− Sham and 5/6Nx mice. The mean value of the Sham-operated ARNTL +/+ group was set to 1.0. (C,D) The number of cardiac or renal F4/80⁺/Ly6G⁻/CD11b⁺/Ly6C⁺ cells (C) and F4/80⁺/Ly6G⁻/CD11b⁺/Ly6C⁻ cells (D) in each organ. The mean value of the Sham-operated ARNTL +/+ group in each organ was set as 1.0. (E) The mRNA levels of Gpr68 in the cardiac ventricle or kidney of ARNTL +/+ or ARNTL −/− Sham and 5/6Nx mice. The mean value of the Sham-operated ARNTL +/+ group was set to 1.0. (F) The expression of Gpr68 mRNA in cardiac F4/80⁺/Ly6G⁻/CD11b⁺/Ly6C⁺ and F4/80⁺/Ly6G⁻/CD11b⁺/Ly6C⁻ cells prepared from ARNTL +/+ or ARNTL −/− Sham and 5/6Nx mice ventricles. (G) The expression levels in the cardiac F4/80⁺/Ly6G⁻/CD11b⁺/Ly6C⁺ and F4/80⁺/Ly6G⁻/CD11b⁺/Ly6C⁻ cells of markers indicative of a subset of macrophages. Histograms showing the expression of each marker were obtained by flow cytometric analysis. For all panels, values are expressed as the mean ± S.D. (n = 4–7). *, p < 0.05, **, p < 0.01 indicates significant differences between the two groups (two-way ANOVA with Tukey–Kramer post hoc tests).