Chronic kidney disease mediates cardiac dysfunction associated with increased resident cardiac macrophages

Figures

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  Fig. 1. 5/6 nephrectomy results in chronic kidney disease. A Timeline of the 5/6 Nx procedure showing right kidney excision followed by left Nx at 2 weeks. B Plasma SDMA, BUN and creatinine (CREAT) levels in sham and nephrectomised (Nx) mice at 12 weeks. Each point represents individual mice (* P < 0.05, *** P < 0.001). C Representative images of kidney from sham or 5/6 Nx mice (scale bar = 1 mm) stained with Masson’s Trichrome (left). Collagen content as measured by colour deconvolution and CKD score (see materials and methods) (right, *** P < 0.001, **** P < 0.0001)

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  Fig. 2. Chronic kidney disease promotes cardiac remodelling. A Cardiac remodelling measured by echography in sham and 5/6 nephrectomy (Nx) mice showing heart weight (HW), left ventricular mass (LVM), ejection fraction, fractional shortening, and diastolic/systolic left ventricular posterior wall (LVPW) thickness, intraventricular septum (IVS) thickness, and left ventricular internal diameter (LVID). Parameters are normalised to tibia length. B Intracarotid mean, systolic (SBP) and diastolic (DBP) blood pressure expressed in mmHg for sham and 5/6 Nx mice (* P < 0.05, ** P < 0.01). C Plasma levels of cardiac troponin I in ng/mL for sham and 5/6 Nx mice. D Fold change from sham in expression of extracellular matrix and cardiac injury genes in hearts of 5/6 nephrectomised mice (12 weeks). Grey line represents no change from sham (* P < 0.05, ** P < 0.01). E Cardiomyocyte cell areas and geodesic diameters (length of the shortest path between two furthest points) in hearts of sham and 5/6 Nx mice and representative A488-WGA staining images (scale bar = 50 μm). Each point represents the mean of at least 300 to 3000 cardiomyocytes per animal (** P < 0.01)

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  Fig. 3. Cardiac macrophage numbers increase during chronic kidney disease. A F4/80+MHCIIhi and F4/80+MHCIIlo resident macrophage, Ly6C+MHCIIhi macrophage, F4/80+CCR2+MHCIIhi pro-inflammatory macrophage or Ly6C+ monocyte numbers per mg of heart tissue and proportion (stacked barplot) in sham and 5/6 nephrectomised (Nx) mice. (*** P < 0.001). B Representative parametric UMAP embedding for the visualisation of different leukocyte populations in sham (left) and 5/6 Nx (right) mice. C Number of CD68-positive macrophages per mm2 of cardiac tissue measured in whole heart slice and in the left ventricle (LV) and representative images showing CD68 MHCII co-staining (scale bar = 100 μm) in sham and 5/6 nephrectomised (Nx) mice (* P < 0.05). D Total blood monocyte (percentage of all leukocytes) and monocyte subset (non-classical and classical, as percentage of CD115+ monocytes) frequency over 12 weeks in sham and 5/6 Nx mice. Presented as percentage of all leukocytes (total monocytes) or Ly6C+ (classical monocytes) or Ly6C− (non-classical monocytes). Mean ± SD at each time point. n = 6. E) Proportion of Ki67-positive macrophages in CD68-positive macrophages measured in whole heart slice and…

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  Fig. 4. CCR2−/− mice do not show increased numbers of cardiac macrophages and cardiac dysfunction during chronic kidney disease. A F4/80+MHCIIhi and F4/80+MHCIIlo resident macrophage numbers per mg of heart tissue in vehicle and folate treated (wild-type) WT and CCR2−/− mice (* P < 0.05). B Ejection fraction (left) and fractional shortening (right) measured by echography in vehicle and folate treated WT and CCR2−/− mice (* P < 0.05). C Cardiac levels of troponin I in ng/mL in vehicle and folate treated WT and CCR2−/− mice. D Intracarotid mean, systolic (SBP) and diastolic (DBP) blood pressure expressed in mmHg in vehicle and folate treated WT and CCR2−/− mice. E Fold change from vehicle in expression of extracellular matrix and cardiac injury genes in folate treated WT and CCR2−/− mice. Grey line represents no change from control (* P < 0.05, ** P < 0.01). F Cardiomyocyte cell areas and geodesic diameters (length of the shortest path between two furthest points) in hearts of vehicle and folate treated WT and CCR2−/− mice. Each point represents the mean of at least 1000 to 3000 cardiomyocytes per animal (** P < 0.01, *** P < 0.001)

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  Fig. 5. CXCL10 blockade reduce cardiac macrophage numbers but did not protect from cardiac dysfunction. A Heatmap showing fold change in cardiac gene expression of chemokines in 5/6 Nx, folate-treated wildtype and folate-treated CCR2−/− mice (to their respective controls). B Heatmap showing fold change in plasma chemokine levels in 5/6 Nx, folate-treated wildtype and CCR2−/− mice (to their respective controls). C F4/80+ macrophage, lymphocyte, and neutrophil numbers per mg of heart tissue in vehicle-treated mice injected with PBS, or folate-treated mice injected with either anti-CXCL10 antibody or control IgG2a antibody(* P < 0.05, ** P < 0.01). D Representative images showing CD68 staining (scale bar = 50 μm) in vehicle-treated mice injected with PBS, or folate-treated mice injected with either anti-CXCL10 antibody or control IgG2a antibody (* P < 0.05). E) Number of CD68-positve macrophages per mm2 of cardiac tissue and proportion of Ki67-positive macrophages in CD68-positive macrophages (grey line) measured in whole heart slice in vehicle-treated mice injected with PBS, or folate-treated mice injected with either anti-CXCL10 antibody or control IgG2a antibody. F) Cardiomyocyte cell…

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  Fig. 6. Increase in plasma and cardiac cell expression and production of CXCL10 in human chronic kidney disease. A Heatmap showing mRNA expression fold change from healthy control in iPSC-derived cardiomyocytes (CM), human cardiac microvascular endothelial cells (EC) and primary ventricular fibroblasts (FB) treated with serum from healthy or CKD donors. Mean of two independent donors. B Heatmap showing protein secretion fold change from healthy control in supernatant of iPSC-CM, EC and FB treated with serum from CKD patients. Mean of two independent donors. C CKD patients or matched healthy controls were recruited and plasma levels of CXCL10 measured by ELISA. CKD patients were split into CKD stage 3, 4 or 5. Each individual donor was tested in triplicate. **** represents P < 0.0001 from healthy controls