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: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Lipocalin-2 protects against renal ischemia/reperfusion injury in mice through autophagy activation mediated by HIF1α and NF-κb crosstalk.

doi: 10.1016/j.biopha.2018.09.023

Figure Lengend Snippet: Fig. 1. Lcn2 deficiency exacerbated renal injury after ischemia/reperfusion. (A) Serum creatinine concentration (SCr) of different groups of mice after renal ischemia/reperfusion. (B) Blood urea nitrogen (BUN) levels of different groups of mice after renal ischemia/reperfusion. (C) Representative micrographs showing the morphology of kidneys from different groups of mice. Magnification: ×200. (D) Quantitative assessment of tubular damage. (E) Protein level of Lcn2 in the kidney after renal ischemia/reperfusion. *P < 0.05 vs. sham-operated wild-type mice, ** P < 0.05 vs. ischemia wild-type mice at the same experimental conditions (n=6).

Article Snippet: To determine the effect of Lcn2 on autophagy in ischemia/reperfusion-induced AKI, different concentrations of human Lcn2 recombinant protein (50, 100, 200, 400, and 1000 ng/mL, R&D systems, Minneapolis, MN) were added to the medium during hypoxia conditions.

Techniques: Concentration Assay

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Lipocalin-2 protects against renal ischemia/reperfusion injury in mice through autophagy activation mediated by HIF1α and NF-κb crosstalk.

doi: 10.1016/j.biopha.2018.09.023

Figure Lengend Snippet: Fig. 2. Lcn2 deficiency reduced autophagy and increased apoptosis in I/R mice. (A.) The TUNEL staining of different groups in the kidney of WT and Lcn2−/−mice 24 h after I/R. (B.) The protein levels of LC3I/LC3II, p62 and cleaved-caspase3 in the kidney of WT and Lcn2−/−mice 24 h after I/R. (C.) Quantitative analysis of protein expression of LC3II, p62 and cleaved-caspase3 in the kidney of WT and Lcn2−/−mice 24 h after I/R. *P < 0.05 vs Sham Vehicle, **P < 0.05 vs. ischemia wild-type mice at the same experimental conditions, n = 5 in each group.

Article Snippet: To determine the effect of Lcn2 on autophagy in ischemia/reperfusion-induced AKI, different concentrations of human Lcn2 recombinant protein (50, 100, 200, 400, and 1000 ng/mL, R&D systems, Minneapolis, MN) were added to the medium during hypoxia conditions.

Techniques: TUNEL Assay, Staining, Expressing

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Lipocalin-2 protects against renal ischemia/reperfusion injury in mice through autophagy activation mediated by HIF1α and NF-κb crosstalk.

doi: 10.1016/j.biopha.2018.09.023

Figure Lengend Snippet: Fig. 3. Activation of autophagy with enhanced Lcn2 expression attenuated apoptosis in HK-2 cells under hypoxia conditions. (A) The protein levels of Lcn2, LC3I/ LC3II, and Cleaved-caspase3 in HK-2 cells after different dose cobalt chloride treatment for 24 h. (B) Quantitative analysis of protein expression of LC3II and cleaved- caspase3 in HK-2 cells. (C) The protein levels of Lcn2, LC3I/LC3II, and Cleaved-caspase3 in HK-2 cells after hypoxia (1% O2) for 6 h followed with re-oxygenation for different time. (D) Cell viability of HK-2 cells suffered from hypoxia 6 h followed with re-oxygenation 6 h with or without 3-MA (5 mM before 1 h of H/R) treatment. (E) The protein levels of LC3I/II and Cleaved-caspase3 in HK-2 cells suffered from hypoxia 6 h followed with re-oxygenation 6 h with or without 3-MA (5 mM before 1 h of H/R) treatment. (F and G) Quantitative analysis of protein expression of LC3 II and cleaved-caspase3 in HK-2 cells. *P < 0.05 vs ctrl group, n=5 in each group.

Article Snippet: To determine the effect of Lcn2 on autophagy in ischemia/reperfusion-induced AKI, different concentrations of human Lcn2 recombinant protein (50, 100, 200, 400, and 1000 ng/mL, R&D systems, Minneapolis, MN) were added to the medium during hypoxia conditions.

Techniques: Activation Assay, Expressing

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Lipocalin-2 protects against renal ischemia/reperfusion injury in mice through autophagy activation mediated by HIF1α and NF-κb crosstalk.

doi: 10.1016/j.biopha.2018.09.023

Figure Lengend Snippet: Fig. 4. Lcn2 activated autophagy and attenuated apoptosis of HK-2 cells in an in vitro hypoxia model. (A.) The protein levels of LC3II/LC3I and cleaved-caspase3 in HK-2 cells treated with different dose Lcn2 during hypoxia condition. CoCl2 :300 uM for 6 h. (B. and C.) Quantitative analysis of protein expression of LC3II and cleaved-caspase3 in HK-2 cells. (D) The protein levels of Lcn2, p62, LC3II/LC3I and cleaved-caspase3 in HK-2 cells after transfecting with Lcn2-siRNA in normal conditions and H/R (hypoxia 6 h and reoxygenation 6 h) condition. (E.) Quantitative analysis of protein expression of Lcn2, p62, LC3II/LC3I and cleaved-caspase3. *P < .05 vs control group, **P < 0.05 vs. ischemia at the same experimental conditions, n = 5 in each group.

Article Snippet: To determine the effect of Lcn2 on autophagy in ischemia/reperfusion-induced AKI, different concentrations of human Lcn2 recombinant protein (50, 100, 200, 400, and 1000 ng/mL, R&D systems, Minneapolis, MN) were added to the medium during hypoxia conditions.

Techniques: In Vitro, Expressing, Control

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Lipocalin-2 protects against renal ischemia/reperfusion injury in mice through autophagy activation mediated by HIF1α and NF-κb crosstalk.

doi: 10.1016/j.biopha.2018.09.023

Figure Lengend Snippet: Fig. 5. Lcn2 negatively regulated NF-κb-p65 signaling pathway. (A) The protein levels of NF-κb pathway relative proteins in the kidney from different groups of mice. (B) Quantitative analysis of protein expression of NF-κb pathway relative proteins. (C) Representative western blot gel documents and summarized data showing the levels of NF-κb pathway relative proteins in HK-2 cells with or without Lcn2 (1000 ng/ml 6 h) treatment. *P < 0.05 vs control group, **P < 0.05 vs. ischemia at the same experimental conditions, n = 5 in each group.

Article Snippet: To determine the effect of Lcn2 on autophagy in ischemia/reperfusion-induced AKI, different concentrations of human Lcn2 recombinant protein (50, 100, 200, 400, and 1000 ng/mL, R&D systems, Minneapolis, MN) were added to the medium during hypoxia conditions.

Techniques: Expressing, Western Blot, Control

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Lipocalin-2 protects against renal ischemia/reperfusion injury in mice through autophagy activation mediated by HIF1α and NF-κb crosstalk.

doi: 10.1016/j.biopha.2018.09.023

Figure Lengend Snippet: Fig. 6. The protective effects of Lcn2 was mediated by HIF1α. (A and B) Representative western blot gel documents and summarized data showing the levels of HIF, Lcn2, LC3 I/II and cleaved-caspase3 in HK-2 cells after inhibiting HIF-1a with or without CoCl2 (300 μM 6 h) treatment. (C and D) Representative western blot gel documents showing the levels of p-p65, p-IKBa, LC3 I/II and cleaved-caspase3 in HK-2 cells with Lcn2 treatment under hypoxia conditions after reducing HIF1α. *P < 0.05 vs control group, **P < 0.05 vs. ischemia at the same experimental conditions, n = 5 in each group.

Article Snippet: To determine the effect of Lcn2 on autophagy in ischemia/reperfusion-induced AKI, different concentrations of human Lcn2 recombinant protein (50, 100, 200, 400, and 1000 ng/mL, R&D systems, Minneapolis, MN) were added to the medium during hypoxia conditions.

Techniques: Western Blot, Control

Journal: Developmental Neurobiology

Article Title: Zebrafish cerebrospinal fluid mediates cell survival through a retinoid signaling pathway

doi: 10.1002/dneu.22300

Figure Lengend Snippet: Loss of Rbp4 increases cell death. (A) Rbp4 sequence coverage from mass spectrometry (red). (B–E) Brightfield dorsal and lateral (B′–E′) view of control MO (B), control MO + p53 MO (C), rbp4 MO (D) and rbp4 MO + p53 MO (E) embryos. (F) Quantification of TUNEL after rbp4 MO and mRNA rescue. (G–J, L–O, R–S) Dorsal view of TUNEL (green) and propidium iodide (red). (G–J) TUNEL staining in control MO + p53 MO (G), control MO + rbp4 mRNA+ p53 MO (H), rbp4 MO+ p53 MO (I), rbp4 MO + rbp4 mRNA + p53 MO (J). (K) Method for RA rescue of rbp4 MO . (L–O) TUNEL staining in control MO + p53 MO + DMSO (L) control MO + p53 MO + RA (M), rbp4 MO+ p53 MO + DMSO (N), rbp4 MO + p53 MO + RA (O). (P) Quantification of TUNEL in RA rescue of rbp4 MO . (Q) Method of injection of Rbp4 inhibitor, A1120. (R‐S) TUNEL staining in DMSO (R) or A1120 (S) injected embryos (T) Quantification of TUNEL in A1120 or DMSO injected embryos. Data represented as mean ± SEM. F = forebrain, M = midbrain, H = hindbrain. Scale bars = 50 μm.

Article Snippet: Factors used include: E10.5 mouse CSF (frozen), physiological saline/artificial CSF (118 mM NaCl, 2 mM KCl, 10 mM MgCl 2 , 10 mM HEPES, 10 mM glucose), Caspase 3 Inhibitor I (Millipore; 500uM), Insulin like growth factor 2 recombinant human (IGF2, US Biologicals, 25ng/mL), Fibroblast growth factor 2 (FGF2, Sigma, 300 μg/mL), all‐trans RA (Sigma, 10 −8 M), all‐trans retinol (Sigma, 348 nM), recombinant human RBP4 (R&D systems, 2 ng/μL), A1120 (Sigma 30 × 10 −8 M), Citral (Sigma 250 μM), 4‐Diethylaminobenzaldehyde (DEAB, Sigma, 10 μM).

Techniques: Sequencing, Mass Spectrometry, Control, TUNEL Assay, Staining, Injection

Journal: Developmental Neurobiology

Article Title: Zebrafish cerebrospinal fluid mediates cell survival through a retinoid signaling pathway

doi: 10.1002/dneu.22300

Figure Lengend Snippet: RA synthesis is required for cell survival and Rbp4 can be transported from the yolk to the CSF. (A–C) Dorsal view of TUNEL (green) and propidium iodide (red) in embryos injected with DMSO (A), Citral (B) or DEAB (C). (D) Quantification of TUNEL after injection of inhibitors into wild‐type embryos every two hours from 22 to 36 hpf. (E) Experimental design for injection of human RBP4 protein into yolk of rbp4 morphants. (F) Quantification of TUNEL positive cells in rbp4 morphants after injection of RBP4 or Kaede protein. (G–L) Dorsal view of TUNEL (green) and propidium iodide (red) in control MO + p53 MO (G,I,K) or rbp4 MO+ p53 MO (H,J,L) embryos injected with DMSO (G,H), human RBP4‐His protein (I,J) or Kaede‐His protein (K,L). (M) Model of retinoid signaling pathway in the embryonic brain. Inhibitors used in red. (N) Model of Rbp4 + RA precursor transport. Rbp4 bound to retinol/RA precursors can be transported from the yolk to CSF (left, red arrows) where it is taken up from the CSF by a RA metabolizing cell, oxidized into RA and promotes cell survival (right). Data represented as mean ± SEM. F = forebrain, M = midbrain, UP = unpunctured, P = punctured, D = drained. Scale bars = 50 μm.

Article Snippet: Factors used include: E10.5 mouse CSF (frozen), physiological saline/artificial CSF (118 mM NaCl, 2 mM KCl, 10 mM MgCl 2 , 10 mM HEPES, 10 mM glucose), Caspase 3 Inhibitor I (Millipore; 500uM), Insulin like growth factor 2 recombinant human (IGF2, US Biologicals, 25ng/mL), Fibroblast growth factor 2 (FGF2, Sigma, 300 μg/mL), all‐trans RA (Sigma, 10 −8 M), all‐trans retinol (Sigma, 348 nM), recombinant human RBP4 (R&D systems, 2 ng/μL), A1120 (Sigma 30 × 10 −8 M), Citral (Sigma 250 μM), 4‐Diethylaminobenzaldehyde (DEAB, Sigma, 10 μM).

Techniques: TUNEL Assay, Injection, Control

Journal: Developmental Neurobiology

Article Title: Zebrafish cerebrospinal fluid mediates cell survival through a retinoid signaling pathway

doi: 10.1002/dneu.22300

Figure Lengend Snippet: Rbp4 is required for cell survival. (A–F) Dorsal view of TUNEL (green) and propidium iodide (red) in unpunctured (A), punctured (B) and drained (C) embryos injected with DMSO or drained embryos injected with RBP4 only (D), retinol (RE) only (E), or combination of retinol (RE) and Rbp4 (F). (G) Quantification of TUNEL after RBP4/retinol injection. Data represented as mean ± SEM. F = forebrain, M = midbrain, H = hindbrain. Scale bars = 50 μm.

Article Snippet: Factors used include: E10.5 mouse CSF (frozen), physiological saline/artificial CSF (118 mM NaCl, 2 mM KCl, 10 mM MgCl 2 , 10 mM HEPES, 10 mM glucose), Caspase 3 Inhibitor I (Millipore; 500uM), Insulin like growth factor 2 recombinant human (IGF2, US Biologicals, 25ng/mL), Fibroblast growth factor 2 (FGF2, Sigma, 300 μg/mL), all‐trans RA (Sigma, 10 −8 M), all‐trans retinol (Sigma, 348 nM), recombinant human RBP4 (R&D systems, 2 ng/μL), A1120 (Sigma 30 × 10 −8 M), Citral (Sigma 250 μM), 4‐Diethylaminobenzaldehyde (DEAB, Sigma, 10 μM).

Techniques: TUNEL Assay, Injection