Journal: Nature Communications

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

doi: 10.1038/s41467-021-23050-x

Figure Lengend Snippet: a Functional analysis of the genes using the KEGG database. Detailed data are presented in Supplementary Table . b , c The mRNA levels of Gpr68 in mouse primary cultured monocytes after treatment with TNFα, IL-6, angiotensin II, or retinol/RBP4 at the indicated concentrations for 24 h. d – f The protein levels of GPR68 and phosphorylated STAT5 (phos-STAT5), and the mRNA levels of Clock and Arntl in mouse primary cultured monocytes after treatment with 1 or 3 µM of retinol/RBP4 for 24 h. g Schematic representation of the upstream region of the mouse Gpr68 , Clock , and Arntl genes. The numbers indicate the distance from the transcription start site (+1). The circled letters (orange circles) indicate the location on the gene where each of the different primer sets localize for analysis of ChIP. h , i ChIP analysis of retinol/RBP4-induced changes in the binding amount of STAT5 protein to the upstream region of Clock and Arntl genes in mouse primary cultured monocytes or the collected monocytes prepared from Sham and 5/6Nx mice. The primer sets used qPCR are illustrated in g . j ChIP analysis of retinol/RBP4-induced changes in the binding amount of CLOCK and ARNTL protein to the upstream region of Gpr68 genes in mouse primary cultured monocytes. Using primer sets illustrated in Fig. . k Left panel shows the protein levels of STRA6 in monocytes transfected with siRNA against STRA6 (uncropped images are presented in Supplementary Fig. ). Right panels show STRA6-dependent phosphorylation of STAT5 and expression of GPR68, CLOCK, and ARNTL by serum from Sham and 5/6Nx mice in mouse primary cultured monocytes. For all panels, graphs show the mean ± SD of individual mice in independent experiments ( h , j n = 5; the others n = 4 for each group). Statistical significance was determined using one-way ANOVA with Dunnett’s post hoc tests ( c ), two-tailed Student’s t -tests ( d – f , h – j ), or two-way ANOVA with Tukey–Kramer post hoc tests ( k ). Numbers and P -values are shown in the graph. Source data are provided as a Source Data file.

Article Snippet: The calibration curve for calculating RBP4 concentrations was made by the quantified values of recombinant RBP4 protein levels (mouse; R&D systems, Inc., Minneapolis, MN, USA.

Techniques: Functional Assay, Cell Culture, Binding Assay, Transfection, Phospho-proteomics, Expressing, Two Tailed Test

Journal: Nature Communications

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

doi: 10.1038/s41467-021-23050-x

Figure Lengend Snippet: a Correlation between serum retinol and RBP4 levels in 5/6Nx mice fed with normal or vitamin A-free diet. b Dietary deficiency of vitamin A suppresses the upregulation of Gpr68 , Clock , and Arntl mRNA levels in circulating monocytes. The mean value of the Sham group was set as 1.0. c Double immunofluorescence labeling of GPR68 with F4/80 in the ventricle slices prepared from 5/6Nx mice fed with normal or Vitamin A-free diet. High-GPR68-expressing cells are double-labeled (yellow) with F4/80. The scale bar indicates 50 μm. d Serum BNP concentrations in Sham and 5/6Nx mice fed with normal or vitamin A-free diet. e Dietary deficiency of vitamin A ameliorates CKD-induced cardiac fibrosis. The Masson’s trichrome staining show tissue fibrosis in blue. Scale bars indicate 1 mm (upper panel) and 50 μm (lower panel). For all panels, graphs show the mean ± SD of individual mice in independent experiments. Statistical significance was determined using one-way ANOVA with Tukey–Kramer post hoc tests ( a , b , d ). Numbers and P -values are shown in each graph. Source data are provided as a Source Data file.

Article Snippet: The calibration curve for calculating RBP4 concentrations was made by the quantified values of recombinant RBP4 protein levels (mouse; R&D systems, Inc., Minneapolis, MN, USA.

Techniques: Immunofluorescence, Labeling, Expressing, Staining

Journal: Nature Communications

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

doi: 10.1038/s41467-021-23050-x

Figure Lengend Snippet: a Dose-dependent increase in the expression of GPR68 mRNA (left) and its protein (right) in human primary cultured monocytes by treatment with retinol/RBP4 for 24 h. b The phosphorylation state of STAT5 in human primary cultured monocytes after treatment with retinol/RBP4. c Schematic representation of the upstream region of the human GPR68 , CLOCK , and ARNTL genes. The circled letters (green circles) indicate the location on the gene where each of the different primer sets localize. d The binding of CLOCK, ARNTL, and STAT5 to regions, described in c , in human (healthy) primary cultured monocytes after treatment with retinol/RBP4 for 24 h. The primer sets used in qPCR are illustrated in c . e LPS-stimulated release of TNFα and IL-6 from human primary cultured monocytes after treatment with retinol/RBP4 for 24 h. f Serum retinol and RBP4 levels in CKD patients. The upper panel shows relationship between retinol and RBP4 levels. Lower panels show these levels stratified by serum creatinine levels (Scr). g , h Relationship retinol levels to mRNA expressions of GPR68 , IL-6 , TNFα , CLOCK , or ARNTL in human primary cultured monocytes incubated in media containing 20% serum collected from healthy subjects and CKD patients stratified by Scr. The release of TNFα and IL-6 from human primary cultured monocytes were assessed after incubation in the media containing 20% serum for 24 h. i Comparison of the values stratified by BNP concentrations. Left panel shows the expression levels of GPR68 mRNA in human primary cultured monocytes incubated same as in g . Right panel shows serum retinol levels. For panel f – i , each plot shows a value obtained using an individual human serum. For all panels, graphs show the mean ± SD in independent experiments ( a , b , d , e n = 5 for each group, f – i shown in each graph). Statistical significance was determined using one-way ANOVA with Dunnett’s post hoc tests ( a ), two-tailed Student’s t -tests ( a , b , d , g ), or one-way ANOVA with Tukey–Kramer post hoc tests ( e , i ). Numbers and P -values are shown in each graph.

Article Snippet: The calibration curve for calculating RBP4 concentrations was made by the quantified values of recombinant RBP4 protein levels (mouse; R&D systems, Inc., Minneapolis, MN, USA.

Techniques: Expressing, Cell Culture, Phospho-proteomics, Binding Assay, Incubation, Comparison, Two Tailed Test

Journal: Nature Communications

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

doi: 10.1038/s41467-021-23050-x

Figure Lengend Snippet: Serum levels of retinol and RBP4 were increased during chronic renal failure. The retinol-bound RBP4 activates JAK2/STAT5 signaling in circulating monocytes through STRA6 and induces the expression of GPR68 via activation of CLOCK/ARNTL. The high-GPR68-expressing monocytes infiltrate into the heart potential for producing inflammatory cytokines and their cardiac infiltration exacerbates inflammation and fibrosis.

Article Snippet: The calibration curve for calculating RBP4 concentrations was made by the quantified values of recombinant RBP4 protein levels (mouse; R&D systems, Inc., Minneapolis, MN, USA.

Techniques: Expressing, Activation Assay

Journal: Science Advances

Article Title: Atf3-mediated metabolic reprogramming in hepatic macrophage orchestrates metabolic dysfunction–associated steatohepatitis

doi: 10.1126/sciadv.ado3141

Figure Lengend Snippet: ( A and B ) Lv-Atf3 , Atf3 M−/− , or control mice were fed a WD. After 20 weeks, HSCs from the mice were isolated and relative mRNA levels of genes involved in fibrogenesis were determined by qRT-PCR ( n = 3 per group). ( C and D ) Hepatic macrophages from Atf3 fl/fl or Atf3 M−/− mice (fed a chow diet) were isolated, and total RNA was extracted for RNA-seq ( n = 3). Some biological processes were regulated by Atf3 (C). The heatmap shows that some cytokines were up-regulated or down-regulated by Atf3 (D). ( E ) mRNA levels. ( F ) Protein levels. ( G and H ) Atf3 fl/fl or Atf3 M−/− mice were fed a WD. After 12 weeks, liver macrophages were isolated and cultured for 24 hours. Then, the media were collected and cocultured with freshly isolated hepatocytes (G) or HSCs (H) in the presence of anti-Rbp4 (10 mg/ml). After 12 hours, relative mRNA levels of genes involved in fibrogenesis were determined. ( I and J ) Liver macrophages from C57BL/6J mice were isolated and then infected with Lv-Gfp or Lv-Atf3 for 24 hours. The mRNA or protein levels of Rbp4 were measured. ( K and L ) Lv-Gfp or Lv-Atf3 liver macrophages were cultured for 24 hours. The media were collected and cocultured with primary hepatocytes (K) or mouse HSCs (L) in the presence of recombinant mouse Rbp4 protein or vehicle (5 μg/ml). The mRNA levels of hepatocytes or HSCs were determined. ( M and N ) Primary hepatocytes or mouse HSCs were infected with lentiviruses expressing shRNA against scramble sequences (Lv-shScr) or Stra6 (Lv-shStra6) for 24 hours and then incubated with recombinant mouse Rbp4 protein or vehicle (5 μg/ml). The mRNA levels of hepatocytes (M) or HSCs (N) were measured. * P < 0.05; ** P < 0.01. NS, not significant.

Article Snippet: Recombinant mouse Rbp4 protein (catalog no. 3476-LC-050) and recombinant mouse FGF-21 protein (catalog no. 8409-FG-025/CF) were purchased from R&D Systems.

Techniques: Control, Isolation, Quantitative RT-PCR, RNA Sequencing, Cell Culture, Infection, Recombinant, Expressing, shRNA, Incubation

Journal: Science Advances

Article Title: Atf3-mediated metabolic reprogramming in hepatic macrophage orchestrates metabolic dysfunction–associated steatohepatitis

doi: 10.1126/sciadv.ado3141

Figure Lengend Snippet: ( A to H ) Hepatic macrophages from Rbp4 M−/− mice were isolated and then infected with Lv-Gfp or Lv-Atf3 for 24 hours. The macrophages were transplanted into C57BL/6J male mice and then fed a WD for 18 weeks ( n = 6 per group). (A) Hepatic macrophage Atf3 protein levels. (B) Body weight and liver weight. (C) Plasma ALT/AST. (D) Hepatic lipid levels. (E) Hepatic FFA levels. (F) Hepatic hydroxyproline levels. (G) Representative liver section images stained by Oil Red O or picrosirius red. (H) Hepatic mRNA levels. ( I ) C57BL/6J mice were fed chow diet or WD fed for 8 or 16 weeks. Hepatic macrophages were isolated, and Rbp4 protein levels were determined. ( J ) Plasma Rbp4 protein levels of Lv-Atf3 mice or Atf3 M−/− mice were measured. ( K to S ) Atf3 M−/− mice were fed a WD for 13 weeks, and the mice were intraperitoneally (i.p.) injected with 100 μg of anti-Rbp4 antibodies (per mouse) or vehicle once every 6 days for 30 days ( n = 6 per group). The experimental procedures are illustrated in (K). The levels of hepatic lipids (L), FFAs (M), hydroxyproline (N), and plasma ALT/AST were analyzed. (O) Representative liver section images stained by H&E or picrosirius red. (P) MASH score. (Q) Fibrotic area. (R) Plasma ALT/AST. (S) Hepatic mRNA levels. ( T ) Simplified model depicting the role of hepatic macrophage Atf3 in regulation of MASH in mice. * P < 0.05; ** P < 0.01.

Article Snippet: Recombinant mouse Rbp4 protein (catalog no. 3476-LC-050) and recombinant mouse FGF-21 protein (catalog no. 8409-FG-025/CF) were purchased from R&D Systems.

Techniques: Isolation, Infection, Clinical Proteomics, Staining, Injection