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Primary murine cardiac endothelial cells exhibit Lcn2 up-regulation under uremic conditions.

ABSTRACT: Cardiovascular diseases, including uremic cardiomyopathy, remain the leading cause of mortality in patients with chronic kidney disease (CKD). Myocardial capillary rarefaction and microvascular dysfunction were proposed as potential mechanisms of uremic cardiomyopathy, but the understanding was hampered by the lack of in vitro models and primary myocardial endothelial cells (ECs). Methods & Results: We developed an in vitro model for studying uremic endothelial dysfunction using primary murine cardiac endothelial cells (pmCECs). We describe the isolation and culture protocol for pmCECs in parallel to the isolation of primary cardiomyocytes . We validated the cell lineage of pmCECs using flow cytometry (CD146⁺, CD31⁺) and isolating cells from transgenic mice with EC-specific reporter (Cdh5-Cre::tdTomato). pmCEC exhibited similar endothelial responses as Human umbilical vein endothelial cells (HUVECs) to hypoxia, including Hif1-α upregulation and enhanced angiogenesis. Exposure to hemodialysate from CKD patients significantly impaired pmCEC proliferation and angiogenic capacity. Bulk RNA sequencing revealed enrichment of oxidative stress pathways, including the up-regulation of lipocalin-2, a key component of these pathways. Increased lipocalin-2 expression in myocardial ECs was confirmed in vivo in Col4a3⁻/⁻ mice, a model of progressive CKD. In the Alport mice, lipocalin-2 correlated with diastolic dysfunction, reduced ejection fraction, and increased cardiac injury markers (pro-ANP, BNP). Conclusion: Primary murine cardiac ECs represent a useful in vitro model, facilitating studies of uremic cardiomyopathy and identification of potential molecular mediators, such as endothelial-derived lipocalin-2.

ORGANISM(S): Mus musculus

PROVIDER: GSE305397 | GEO | 2026/07/01

REPOSITORIES: GEO

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Project description:Introduction: Cardiovascular pathology is the main cause of death in patients with chronic kidney disease (CKD). CKD is associated with the accumulation of uremic toxins in the bloodstream due to their difficult elimination by renal substitution therapy. Indoxyl sulfate (IS) is one of the most abundant uremic toxins found in the blood of patients with CKD. We conducted an in vitro study to assess the mechanisms underlying IS-induced endothelial dysfunction that could lead to cardiovascular diseases. We studied not only endothelial cells but also their extracellular vesicles (EVs) owing to their capacity to act as messengers that transmit signals through their cargo. Methods: EVs were characterized using nanoparticle tracking analysis, transmission electron microscopy, and flow cytometry, and their tetraspanin expression was evaluated to confirm their identity as EVs. Subsequently, mass spectrophotometry of the protein lysates of cells and EVs was performed, followed by Gene Set Enrichment Analysis to identify the principal altered pathways. Additionally, qPCR and proliferation analyses were performed to confirm proteomic results. Results: Proteomic analysis of endothelial cells revealed that IS causes an increase in proteins related to adipogenesis, inflammation, and xenobiotic metabolism and a decrease in proliferation via mTOR. Inflammation due to TNF signaling via NFкB was confirmed by an increase in the expression of the target genes IL-6, MCP-1, and ICAM-1. Concerning EVs, extracellular matrix elements were reduced, and myogenesis genes were downregulated in response to UV irradiation and inflammation. Fatty acid metabolism also seemingly increases, which, along with adipogenesis and inflammation observed in cells, could be related to atherosclerosis. Conclusions: Endothelial cells treated with IS had more proteins related to adipogenesis, inflammation, and xenobiotic metabolism and fewer associated with proliferation. Furthermore, EVs from cells treated with IS may mediate endothelial dysfunction, since they present fewer extracellular matrix elements, myogenesis and inflammatory factors, and proteins downregulated in response to UV radiation.

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Primary murine cardiac endothelial cells exhibit Lcn2 up-regulation under uremic conditions.

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