soluble cx3cl1 chemokines  (R&D Systems)

Journal: Frontiers in Pharmacology

Article Title: CX3CL1 Worsens Cardiorenal Dysfunction and Serves as a Therapeutic Target of Canagliflozin for Cardiorenal Syndrome

doi: 10.3389/fphar.2022.848310

Figure Lengend Snippet: CX3CL1 expression in response to glucose stimulation. (A) Blood glucose levels at 3 and 42 days in streptozotocin (STZ)-induced DM mice. Insulin treatment was initiated at 28 days after STZ injection and persisted for 14 days. * p < 0.05, n = 8 in each group. (B) Blood glucose levels at 3 and 42 days in NOD mice. The NOD mice were assigned to three groups: normal glucose (NG) group (some mice didn’t develop into hyperglycemia), NOD group (with twice random blood glucose level>20 mM) and NOD&insulin group. Insulin treatment protocol was the same as panel (A) . * p < 0.05, n = 8 in each group. (C) Representative western blotting of CX3CL1 in the heart and kidney tissue of mice at various time points after STZ-injection. (D) Semi-quantitation of CX3CL1 expression in panel (C) . * p < 0.05, vs . the corresponding 0 day, n = 5 per group. (E) Immunohistochemical detection of CX3CL1 expression in heart and kidney tissues of normal or diabetic mice. Scale bar = 100 μm. (F) Cardiac CX3CL1 and CX3CR1 mRNA expression in NOD mice. (G) Renal gene expression of CX3CL1 and CX3CR1 in NOD mice. * p < 0.05, vs . NG, n = 6 per group. (H) CX3CL1 expression detected by western blotting in heart and kidney tissues from DM mice with or without insulin treatment for 14 days. (I) Semi-quantitation of CX3CL1 in panel (H) . * p < 0.05 vs . WT&Vehicle, n = 5 in each group. (J) mRNA expression of CX3CL1 in neonatal rat cardiomyocytes (NRCM) and fibroblasts (NRCF), two types of renal cell lines HBZY-1 and NRK-52E exposed to different concentrations of glucose medium for 3 days. * p < 0.05 vs . 5.5 mM group, n = 5 in each group. Experiments presented in panels (A) , (B) , (D) , (I) , and (J) were analyzed using one-way ANOVA followed by Bonferroni’s post hoc test. Experiments presented in panels (F) and (G) were analyzed using two-tailed unpaired t -test.

Article Snippet: Cultured NRCM and HBZY-1 were starved of serum for 12 h and then were exposed to recombinant soluble CX3CL1 (chemokine domain; 537-FT-025; R&D) at 200 ng/ml for 24 h in the presence or absence of 10 μM Y27632 (a ROCK inhibitor; Selleck) or 5 μg/ml of an anti-CX3CR1 antibody (GTX27200; Genetex).

Techniques: Expressing, Injection, Western Blot, Quantitation Assay, Immunohistochemical staining, Gene Expression, Two Tailed Test

Journal: Frontiers in Pharmacology

Article Title: CX3CL1 Worsens Cardiorenal Dysfunction and Serves as a Therapeutic Target of Canagliflozin for Cardiorenal Syndrome

doi: 10.3389/fphar.2022.848310

Figure Lengend Snippet: sCX3CL1 promotes mitochondrial-dependent apoptosis in cardiomyocytes and renal cells. (A) Western blotting to detect RhoA and GTP-bound RhoA protein in neonatal rat cardiomyocytes (NRCM) and HBZY-1 cells after 60 min of stimulation with 200 ng/ml of sCX3CL1 (soluble CX3CL1). (B) Western blotting to detect upregulation of ROCK1 and cleaved ROCK1 (c-ROCK) expression in response to stimulation of NRCM and HBZY-1 cells with sCX3CL1 (200 ng/ml) for 24 h. (C) Semi-quantitation of RhoA-GTP, ROCK and cleaved-ROCK expression. * p < 0.05 vs . control, n = 5 in each group. Representative images of (D) calcein fluorescence or (E) TMRE fluorescence (Mitochondrial membrane potential) in cells cultured with sCX3CL1 alone or co-treated with either a CX3CR1 neutralizing antibody or a Rho kinase inhibitor (Y-27632). Scale bar = 100 μm. Semi-quantitative analysis of calcein (F) or TMRE (G) fluorescence intensity ( * p < 0.05 vs . sCX3CL1), n = 5 in each group. (H) Western blots analysis of Bax, cytochrome C (Cyto C) in NRCM and HBZY-1. Semi-quantitation analysis of Bax and Cyto C in NRCM (I) and HBZY-1 cells (J) . * p < 0.05 vs . sCX3CL1, n = 5 in each group. (K) Subcellular localization of AIF (apoptosis inducing factor) was detected in NRCM and HBZY-1 cells after sCX3CL1 stimulation or co-treatment with either a CX3CR1 neutralizing antibody or Y-27632. Scale bar = 30 μm. Experiments presented in panel (C) was analyzed using two-tailed unpaired t -test and in panels (F) , (G) , (I) , and (J) were analyzed using one-way ANOVA followed by Bonferroni’s post hoc test.

Article Snippet: Cultured NRCM and HBZY-1 were starved of serum for 12 h and then were exposed to recombinant soluble CX3CL1 (chemokine domain; 537-FT-025; R&D) at 200 ng/ml for 24 h in the presence or absence of 10 μM Y27632 (a ROCK inhibitor; Selleck) or 5 μg/ml of an anti-CX3CR1 antibody (GTX27200; Genetex).

Techniques: Western Blot, Expressing, Quantitation Assay, Control, Fluorescence, Membrane, Cell Culture, Two Tailed Test

Journal: Frontiers in Pharmacology

Article Title: CX3CL1 Worsens Cardiorenal Dysfunction and Serves as a Therapeutic Target of Canagliflozin for Cardiorenal Syndrome

doi: 10.3389/fphar.2022.848310

Figure Lengend Snippet: Effect of CX3CL1/CX3CR1 inhibition on cardiorenal dysfunction in mice with diabetes-induced CRS. (A) Representative M-mode echocardiographic images of mice at 6 weeks after STZ injection. Scale bars, 2 mm (upper panels), horizontal bars represent 100 ms. (B) Left ventricular fractional shortening (LVFS). (C) Microalbuminuria amount for 24 h. (D) Serum creatinine levels. (E) Serum NGAL levels. For panels (B–E) , * p < 0.05 vs . WT&STZ group; † p < 0.05 vs . WT&STZ&Insulin group. For panel (B) , n = 6 in WT&Vehicle, WT&STZ&Inuslin, and CX3CR1 −/− &Vehicle group; n = 7 in WT&STZ and CX3CR1 −/− &STZ group; n = 5 in WT&STZ&anti-CX3CL1 group. n = 5 per group in panels (C–E) . (F) Representative M-mode echocardiographic images. Scale bars, 2 mm (upper panels), horizontal bars represent 100 ms. (G) LVFS. (H) Concentrations of serum BUN (urea nitrogen). (I) Serum creatinine levels. (J) Urine creatinine levels. For panel (G–J) , * p < 0.05 vs . NOD&Vehicle group. In panel (G) , n = 8 in NG&Vehicle; n = 6 in NOD&Vehicle, NOD&sh-scr, and NOD&sh-CX3CR1 group; n = 7 in NOD&Inuslin; n = 5 in NOD&Anti-CX3CL1. n = 6 per group in panel (H) ; n = 5 per group in panels (I–J) . Experiments presented in panels (B–E) and (G–J) were analyzed using one-way ANOVA followed by Bonferroni’s post hoc test.

Article Snippet: Cultured NRCM and HBZY-1 were starved of serum for 12 h and then were exposed to recombinant soluble CX3CL1 (chemokine domain; 537-FT-025; R&D) at 200 ng/ml for 24 h in the presence or absence of 10 μM Y27632 (a ROCK inhibitor; Selleck) or 5 μg/ml of an anti-CX3CR1 antibody (GTX27200; Genetex).

Techniques: Inhibition, Injection

Journal: Frontiers in Pharmacology

Article Title: CX3CL1 Worsens Cardiorenal Dysfunction and Serves as a Therapeutic Target of Canagliflozin for Cardiorenal Syndrome

doi: 10.3389/fphar.2022.848310

Figure Lengend Snippet: Inhibition of CX3CL1/CX3CR1 attenuated apoptosis in mice with STZ-induced diabetes. (A) Representative images of TUNEL staining in heart sections at 6 weeks after STZ-injection. (B) Percentage of TUNEL-positive nuclei in heart tissue. (C) Representative images of TUNEL staining in kidney sections at 6 weeks after STZ-injection. (D) Percentage of TUNEL-positive nuclei in kidney tissue. (E) Bax immunostaining (brown staining) in heart tissue. (F) Bax immunostaining in kidney tissue. (G) Representative electronic micrographs of the heart featuring sarcomeres and mitochondria. (H) Representative electronic micrographs of the kidney featuring tubular mitochondria. For panel (B) and (D) , * p < 0.05 vs . WT&STZ mice; † p < 0.05 vs . WT&STZ&Insulin mice, n = 5 in each group. Scale bar = 100 μm in panel (A) and (C) , Scale bar = 1 μm in panel (G) and (H) . Experiments presented in panels (B) and (D) were analyzed using one-way ANOVA followed by Bonferroni’s post hoc test.

Article Snippet: Cultured NRCM and HBZY-1 were starved of serum for 12 h and then were exposed to recombinant soluble CX3CL1 (chemokine domain; 537-FT-025; R&D) at 200 ng/ml for 24 h in the presence or absence of 10 μM Y27632 (a ROCK inhibitor; Selleck) or 5 μg/ml of an anti-CX3CR1 antibody (GTX27200; Genetex).

Techniques: Inhibition, TUNEL Assay, Staining, Injection, Immunostaining, Mouse Assay

Journal: Frontiers in Pharmacology

Article Title: CX3CL1 Worsens Cardiorenal Dysfunction and Serves as a Therapeutic Target of Canagliflozin for Cardiorenal Syndrome

doi: 10.3389/fphar.2022.848310

Figure Lengend Snippet: Inhibition of CX3CL1/CX3CR1 attenuated fibrosis in diabetes mice. (A) Myocardial interstitial fibrosis in STZ-induced DM mice. (B) Perivascular fibrosis of heart in STZ-induced DM mice. (C) Fibrotic score of the renal interstitial area in STZ-induced DM mice. (D) Fibrotic score of the glomeruli area in STZ-induced DM mice. (E) Myocardial interstitial fibrosis in NOD mice. (F) Perivascular fibrosis of heart in NOD mice. (G) Fibrotic score of the renal interstitial area in NOD mice. (H) Fibrotic score of the glomeruli area in NOD mice. (I) Representative images of immunostaining for DAPI (blue), α-SMA (green) and vimentin (red) in heart tissue. Scale bar = 100 μm. (J) Representative images of immunostaining for E-cadherin (red), fibronectin (red), vimentin (red), and α-SMA (green) in kidney tissue. Scale bar = 100 μm. For panels (A–D) , * p < 0.05 vs . WT&STZ group; † p < 0.05 vs . WT&STZ&Insulin mice; n = 5 per group. For panels (E–H) , * p < 0.05 vs . NOD&Vehicle group; n = 6 per group. Experiments presented in panels (A–H) were analyzed using one-way ANOVA followed by Bonferroni’s post hoc test.

Article Snippet: Cultured NRCM and HBZY-1 were starved of serum for 12 h and then were exposed to recombinant soluble CX3CL1 (chemokine domain; 537-FT-025; R&D) at 200 ng/ml for 24 h in the presence or absence of 10 μM Y27632 (a ROCK inhibitor; Selleck) or 5 μg/ml of an anti-CX3CR1 antibody (GTX27200; Genetex).

Techniques: Inhibition, Immunostaining

Journal: Frontiers in Pharmacology

Article Title: CX3CL1 Worsens Cardiorenal Dysfunction and Serves as a Therapeutic Target of Canagliflozin for Cardiorenal Syndrome

doi: 10.3389/fphar.2022.848310

Figure Lengend Snippet: Canagliflozin (Cana) improved cardiorenal dysfunction and repressed diabetic induced or high glucose induced CX3CL1 expression. (A) M-mode of echocardiography. Scale bars, 2 mm (upper panels), horizontal bars represent 100 ms. (B) Left ventricular fractional shortening (LVFS). (C) Serum creatinine concentration. (D) Renal neutrophil gelatinase-associated lipocalin (NGAL) content. (E) CX3CL1 expression detected by Western blotting in heart and kidney tissues. (F) Semi-quantitation of CX3CL1 expression in heart. (G) Semi-quantitation of CX3CL1 expression in kidney. * p < 0.05 vs . WT&STZ mice; n = 6 per group. (H–M) Western blotting of CX3CL1 levels in cultured cardiomyocytes (H9C2), glomerular mesangial cells (HBZY-1), neonatal rat cardiac fibroblasts (NRCF) and renal tubular epithelial cells (NRK-52E). The cultured cells were exposed for 5 days either to normal concentration of glucose (5 mM, NG) or high concentration of glucose (33.3 mM, HG) as well as to HG for 3 days followed by NG for 2 days (HN) with/without treatment of Cana. * p < 0.05 vs . HG; † p < 0.05 vs . HN; n = 5 per group. Experiments presented in panels (B–D) , (F–G) , (I–J) , and (L–M) were analyzed using one-way ANOVA followed by Bonferroni’s post hoc test.

Article Snippet: Cultured NRCM and HBZY-1 were starved of serum for 12 h and then were exposed to recombinant soluble CX3CL1 (chemokine domain; 537-FT-025; R&D) at 200 ng/ml for 24 h in the presence or absence of 10 μM Y27632 (a ROCK inhibitor; Selleck) or 5 μg/ml of an anti-CX3CR1 antibody (GTX27200; Genetex).

Techniques: Expressing, Concentration Assay, Western Blot, Quantitation Assay, Cell Culture

Journal: Frontiers in Pharmacology

Article Title: CX3CL1 Worsens Cardiorenal Dysfunction and Serves as a Therapeutic Target of Canagliflozin for Cardiorenal Syndrome

doi: 10.3389/fphar.2022.848310

Figure Lengend Snippet: Illustration of the molecular mechanisms by which CX3CL1 promotes cardiorenal dysfunction induced by diabetics. CX3CL1 expression is upregulated in cardiac and renal cells by a high glucose environment. Persistent high CX3CL1 expression accelerates the mitochondrial apoptotic pathway through activation of RhoA/ROCK1-Bax signaling. In addition, CX3CL1 regulates fibroblast and epithelial cell phenotypic trans-differentiation through activation of TGF-β/Smad signaling. CX3CL1 leads to the onset of cardiorenal dysfunction in diabetes-induced cardiorenal syndrome type 5 (CRS5) due to its proapoptotic and profibrotic effects, while SGLT2 inhibitor could improve CRS5 at least partially by repressing CX3CL1 expression.

Article Snippet: Cultured NRCM and HBZY-1 were starved of serum for 12 h and then were exposed to recombinant soluble CX3CL1 (chemokine domain; 537-FT-025; R&D) at 200 ng/ml for 24 h in the presence or absence of 10 μM Y27632 (a ROCK inhibitor; Selleck) or 5 μg/ml of an anti-CX3CR1 antibody (GTX27200; Genetex).

Techniques: Expressing, Activation Assay

Journal: JCI Insight

Article Title: The homozygous CX3CR1-M280 mutation impairs human monocyte survival

doi: 10.1172/jci.insight.95417

Figure Lengend Snippet: Percent of dead CD14+ monocytes elicited by serum starvation in the presence or absence of 100 nM of soluble or full-length CX3CL1 in CX3CR1-WT/WT monocytes. Monocytes cultured in serum-containing media were assayed as control. n = 6. ***P < 0.001; ****P < 0.0001. Statistical analysis was performed using 1-way ANOVA with Tukey’s multiple comparisons test. Data represent the mean ± SEM.

Article Snippet: Recombinant human soluble CX3CL1 (R&D Systems, catalog 362-CX) was then added at a final concentration of 100 nM for 3, 10, or 30 minutes.

Techniques: Cell Culture, Control

Journal: JCI Insight

Article Title: The homozygous CX3CR1-M280 mutation impairs human monocyte survival

doi: 10.1172/jci.insight.95417

Figure Lengend Snippet: (A) Representative FACS histograms of propidium iodine (PI) staining in CX3CR1-WT/WT (upper panels) and CX3CR1-M280/M280 (lower panels) CD14+ monocytes following serum starvation in the presence or absence of 100 nM of CX3CL1. (B) Percent of dead CD14+ monocytes elicited by serum starvation in the presence or absence of 100 nM of CX3CL1 in CX3CR1-WT/WT (left panel), CX3CR1-WT/M280 (middle panel), and CX3CR1-M280/M280 (right panel) cells. Shown are paired experimental results with or without CX3CL1. (C) The percent decrease in cell death conferred by CX3CL1 exposure in serum-starved CD14+ monocytes is greater in CX3CR1-WT/WT compared with CX3CR1-WT/M280 cells, while no CX3CL1-induced decrease in cell death is seen in CX3CR1-M280/M280 cells. n = 22 CX3CR1-WT/WT, 11 CX3CR1-WT/M280 and 6 CX3CR1-M280/M280. *P < 0.05; ***P < 0.001; ****P < 0.0001. Statistical analysis was performed using paired 2-tailed t tests (B) or 1-way ANOVA with Tukey’s multiple comparisons test (C). Quantitative data represent the mean ± SEM.

Article Snippet: Recombinant human soluble CX3CL1 (R&D Systems, catalog 362-CX) was then added at a final concentration of 100 nM for 3, 10, or 30 minutes.

Techniques: Staining

Journal: JCI Insight

Article Title: The homozygous CX3CR1-M280 mutation impairs human monocyte survival

doi: 10.1172/jci.insight.95417

Figure Lengend Snippet: Shown are percent of dead monocytes elicited by serum starvation in the presence or absence of 100 nM of CX3CL1 after exposure to 50 μM of the PI3K inhibitor LY294002 (A, n = 5) or 20 μM of the MEK inhibitor PD98059 (B, n = 6), or 100 nM of the PI3K inhibitor wortmannin (C, n = 6) or 20 μM of the MEK inhibitor U0126 (D, n = 5). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. Statistical analysis was performed using 1-way ANOVA with Tukey’s multiple comparisons test. Data represent the mean ± SEM.

Article Snippet: Recombinant human soluble CX3CL1 (R&D Systems, catalog 362-CX) was then added at a final concentration of 100 nM for 3, 10, or 30 minutes.

Techniques: