Chronic kidney disease (CKD) affects over 10% of the global population. Cardiovascular complications, including heart failure, vascular disease and sudden cardiac death correlate with CKD stages and cause most CKD-related deaths. While the accumulation of uremic toxins (uremia) is proposed to be a major driver of cardiovascular risk in CKD, the exact molecular and cellular cues remain elusive.
We recently showed that platelets regulate cardiac fibrosis and heart failure by controlling the activation of a profibrotic (SPP1+) macrophage subset via CXCL4. While platelet dysfunction in CKD results in both thromboembolic and hemorrhagic events, a potential role of uremia-induced platelet dysfunction in heart failure remains to be examined. We hypothesize that uremia alters thrombopoiesis, producing dysfunctional platelets with profibrotic and proinflammatory characteristics that drive heart failure in CKD.
To investigate this, we performed a transcriptome analysis of megakaryocytes and platelets in a murine model of time-resolved AKI-to-CKD transition. Kidney failure was induced in C57BL/6 wild-type mice by daily intraperitoneal injection of aristolochic acid for 5 days. Bone marrow cells were isolated and enriched for megakaryocytes on days 1, 4, 10, and 28 after the injections, followed by single-cell RNA sequencing (scRNAseq). In addition, we performed bulk RNA sequencing (bulkRNAseq) of murine platelets isolated from peripheral blood.
Bioinformatic analysis of 46,000 cells identified 13 hematopoietic clusters, including 29,900 cells of the megakaryocyte lineage. Using the algorithm Augur, differentiated megakaryocytes were ranked among the cell populations most affected by uremia. Subclustering and trajectory inference revealed 3 megakaryocytic clusters derived from a common progenitor population.
Linear regression analysis of differentially expressed genes in platelet (bulkRNAseq) and megakaryocyte (scRNAseq) data demonstrated that uremia-induced transcriptomic changes in megakaryocytes are transmitted to platelets. Gene set enrichment analysis showed enrichment of genes involved in immune cell migration and activation in megakaryocytes at early stages after uremia induction. At later timepoints, gene sets involved in platelet activation were enriched. Progeny analysis confirmed increased activity of profibrotic and proinflammatory signaling via TGFb and TNFa in uremia.
To validate potential driver genes in vitro, we established a coculture of megakaryocytes differentiated from induced pluripotent stem cells (iPSC) and cardiac organoids composed of iPSC-derived cardiomyocytes, fibroblasts and endothelial cells. The megakaryocytes express the platelet markers CD41 and CD42b as well as marker genes of mature megakaryocytes in our scRNAseq data. In a coculture, GapmeR-mediated knockdown of target genes in megakaryocytes is used to assess their effects on cardiac organoids. To model profibrotic and proinflammatory conditions of heart failure, cardiac organoids are exposed to Doxorubicin and TGFb.
In sum, uremia induces a proinflammatory and profibrotic activation of megakaryocytes that is transmitted to platelets. Alongside the in vitro validation of identified pathways in cocultures of genetically perturbed megakaryocytes and cardiac organoids, we will perform bulkRNAseq of platelets from CKD patients to further define the mechanisms of heart failure in CKD driven by altered platelets.
