Mapping cardiac remodeling in chronic kidney disease

Abstract

Patients with advanced chronic kidney disease (CKD) mostly die from sudden cardiac death and recurrent heart failure. The mechanisms of cardiac remodeling are largely unclear. To dissect molecular and cellular mechanisms of cardiac remodeling in CKD in an unbiased fashion, we performed left ventricular single-nuclear RNA sequencing in two mouse models of CKD. Our data showed a hypertrophic response trajectory of cardiomyocytes with stress signaling and metabolic changes driven by soluble uremia-related factors. We mapped fibroblast to myofibroblast differentiation in this process and identified notable changes in the cardiac vasculature, suggesting inflammation and dysfunction. An integrated analysis of cardiac cellular responses to uremic toxins pointed toward endothelin-1 and methylglyoxal being involved in capillary dysfunction and TNFα driving cardiomyocyte hypertrophy in CKD, which was validated in vitro and in vivo. TNFα inhibition in vivo ameliorated the cardiac phenotype in CKD. Thus, interventional approaches directed against uremic toxins, such as TNFα, hold promise to ameliorate cardiac remodeling in CKD.

Fig. 1.. Mapping cardiac remodeling in CKD.

(A) Mice underwent sham surgery, STNx, or IRI and were sacrificed at 70 or 147 days after surgery. (B) Serum urea, serum creatinine (S-creatinine), heart weight, normalized to tibia length, and ejection fraction at the time point of sacrifice in sham (SH), STNx, and IRI mice. (C) Representative trichrome-stained images of kidney and heart. Scale bars, 50 μm. (D) UMAP embedding of nuclei isolated from left ventricle of all mice. n = 33,039 nuclei from n = 12 mice. (E) Expression of marker genes per cluster (cardiomyocytes: Ryr2, Dmd, Myh6, Fhl2, and Ttn; endothelial cells: Vwf, Cgnl1, Smoc1, Bmx, Pecam1, Flt1, Adgrl1, and Prprb; fibroblasts: Pdgra, Egfr, Dcn, Gsn, Col3a1, and Col8a1; lymphatic endothelial cells: Flt4, Prox1, Lyve1, and Pdpn; lymphocytes: Btla, Cd2, Bcl11b, and Sell; macrophages: Mrc1, Lyz2, Cd86, Cd68, and Cd80; mesothelial cells: Muc16, Msln, and Upk3b; monocytes: Cd45, Csfr1r; RBC: Lock2, and Cd36; pericytes: Pdgfrb, Notch3, Mcam, and Rgs5; and VSMC: Myh11, Myl9, and Lmod). (F) PROGENGY pathway analysis in all cell types, STNx versus sham and IRI versus sham. (G) Predicted receptor-ligand interactions per group, sham, STNx, and IRI. *P < 0.05; **P < 0.001. CM, cardiomyocytes; EC, endothelial cells; Fib, fibroblasts; Lym, lymphocytes; LEC, lymphatic endothelial cells; Mac, macrophages; Mes, mesothelial cells; Mon, monocytes; Per, pericytes; RBC, red blood cells; VSMC, vascular smooth muscle cells.

Fig. 2.. Mechanisms of cardiomyocyte hypertrophy in CKD.

(A) Representative confocal images of WGA-stained left ventricular myocardium from mice after sham surgery, STNx, or IRI. Scale bars, 20 μm. (B) Feret diameter of cardiomyocytes from sham, STNx, and IRI mice. (C) Hypertrophy gene set enrichment in cardiomyocytes 1 to 4 from sham, STNx, and IRI mice (GSEA). (D) Diffusion map (DM) embedding of cardiomyocytes 1 to 4 from sham, STNx, and IRI mice color-coded per condition, (E) See (D) but color-coded per cardiomyocyte cluster. (F) Differentiation trajectory toward hypertrophic cardiomyocytes within DM from (D) to (E). (G) Hypertrophy gene set enrichment in DM of cardiomyocytes from (D) to (E). (H) Genes sorted along the pseudotime trajectory of cardiomyocyte hypertrophy in (F); the color represents z score of the log-normalized gene expression across pseudotime (0 to 100) cells. (I) GO terms sorted along the pseudotime trajectory of cardiomyocyte hypertrophy in (F); the color represents the −log₁₀ transformed P value of GO terms derived from highly expressed genes across pseudotime (one to five) cells. (J) AC16 cardiomyocyte cell line, treated with 5 or 10% uremic serum, compared to healthy serum; planimetric analysis of cross-sectional area per cardiomyocyte from fluorescence microscopic images, TMRM staining. (K) Hypertrophic gene set, expressed in AC16 cells after treatment with 5% healthy or 5% uremic serum. (L) PROGENY pathway analysis in AC16 cells after treatment with 5% healthy or 5% uremic serum. (M) Planimetric analysis of cross-sectional area per cardiomyocyte from fluorescence microscopic images, TMRM staining, cells either treated with healthy serum, uremic serum + vehicle, uremic serum + ruxolitinib, or uremic serum + etanercept. *P < 0.05; ^#P < 0.001.

Fig. 3.. Cellular source of cardiac fibrosis in CKD.

(A) ECM score in all cells of sham, STNx, and IRI mice. (B) ECM score, median in fibroblasts, pericytes, and VSMCs. (C) ECM score of fibroblasts, pericytes, and VSMCs by treatment group (GSEA). (D) Diffusion map embedding of all fibroblast clusters, gene expression of collagen 1a1 (Col1a1) and periostin (Postn), fibroblast to myofibroblast differentiation trajectory direction, and expression of selected genes along this differentiation trajectory. (E) Enriched genes along the trajectory. (F) GO terms along the fibroblast to myofibroblast differentiation trajectory. (G) Receptor-ligand interaction analysis from fibroblasts with all other cell types, STNx, and IRI versus sham. *P < 0.05; **P < 0.001.

Fig. 4.. Fate tracing and scRNA-seq of cardiac Gli1⁺ cells in CKD.

(A) Scheme of the experiment: Mice were tamoxifen pulsed (arrows) and subjected to IRI with contralateral nephrectomy at 14 days after the first tamoxifen dose. Mice were sacrificed at 126 days after IRI. (B) Representative trichrome-stained images and confocal images of the left ventricular myocardium from mice after IRI and sham surgery. Scale bars, 50 μm. (C) Serum urea at the time point of sacrifice. (D) Maximum developed pressure (Dp/dtmax) from Millar catheter before sacrifice. (E) Fluorescence-activated cell sorting gate Gli1 tdTomato–positive cells out of left ventricular cells. (F) UMAP embedding all sorted Gli1⁺/tdTomato⁺ cells from the left ventricle of all mouse groups. (G) Odds ratio of cell type distribution of all Gli1⁺ cell clusters, IRI versus sham. (H) Violin plots for expression of ECM using the ESM gene set. (I) PROGENY pathway analysis for all cell clusters. (J) Diffusion map embedding of Gli1⁺ cells. (K) Pseudotime trajectory analysis of fibroblast to myofibroblast differentiation. (L) Genes enriched along the pseudotime trajectory of fibroblast to myofibroblast differentiation. (M) GO terms enriched along the pseudotime trajectory of fibroblast to myofibroblast differentiation. sham. *P < 0.05; **P < 0.001. Myf, myofibroblasts.

Fig. 5.. Cardiac endothelial alterations in CKD.

(A) Representative confocal images of WGA and CD31 costaining of left ventricular myocardium from sham, STNx, and IRI mice. Scale bar, 20 μm. (B) Quantification of capillary length in heart sections from STNx, IRI, and sham mice. (C) UMAP embedding of all endothelial cells. (D) Expression of the angiogenic gene set in endothelial cell clusters per group (GSEA). *P < 0.05; **P < 0.001. (E) Top enriched GO terms in endothelial cell clusters, STNx versus sham. (F) Top enriched GO terms in endothelial cells, IRI versus sham. (G) Top KEGG pathways in endothelial cells, STNx versus sham. (H) Top KEGG pathways in endothelial cells 1 to 3, IRI versus sham. (I) RL interaction analysis of predicted interactions between endothelial cells 1 to 3 and all other cell types, STNx and IRI versus sham. (J) Sankey plot of endothelial cells 1 to 3 receiving signals from fibroblast 1, IRI versus sham.

Fig. 6.. Role of UTs in uremic cardiomyopathy.

(A) Gene set enrichment of genes associated to UTs on all cell types; STNx versus sham (IRI versus sham is shown in fig. S13). (B) HUVEC tube formation assay, treatment with 5 or 10% uremic serum versus healthy control serum, quantification of isolated branches and bright-field microscopic images. (C) HUVEC tube formation assay, treatment with endothelin 1 (Edn1) or endothelin 1 neutralizing antibody, quantification of total mesh area and bright-field microscopic images. Scale bars, 500 μm. Sham: ^#P < 0.001. (D) Expression of endothelin converting enzyme 1 (Ece1) in lymphatic endothelial cells and endothelial cells 1 to 3, per treatment group. (E) Size of AC16 cells after treatment with Edn1 or vehicle for 3 days. Scale bars, 25 μm.