Circulating Extracellular Vesicles in the Pathogenesis of Heart Failure in Patients with Chronic Kidney Disease

Background: Cardiovascular disease (CVD) causes more than 50% of deaths in patients with advanced chronic kidney disease (CKD). CVD degree correlates with CKD severity. Clinical studies suggest that kidney-derived factors contribute to the development of CVD in CKD, independently of co-morbidities like hypertension and hyperglycemia. However, to date, no kidney-specific humoral risk factor that triggers direct cardiotoxicity has yet been identified, primarily due to the paucity of studies in patients with reno-cardiac disease. Here, we investigate how, in CKD patients, circulating extracellular vesicles (EVs) facilitate pathological kidney-heart communication, thereby causing cardiotoxicity, impairing cardiac function and contributing to heart failure (HF) progression. Methods and results: EVs isolated from the plasma of patients with CKD (hCKD-EVs), but not healthy controls (hCtr-EVs), were cardiotoxic, significantly inducing apoptosis both in vitro and in vivo and impairing contractility of isolated adult rat primary CMs in vitro. Likewise, EVs isolated from both plasma and kidneys of adenine diet-induced CKD mice were also cardiotoxic. Pharmacologically depleting circulating EVs in CKD mice significantly recovered cardiac function and ameliorated HF, suggesting CKD-EVs play a causal role in HF pathogenesis. Small RNA sequencing and q-RT-PCR validation uncovered distinct miRNAs enriched in both human and mouse CKD-EVs, compared to Ctr-EVs. CKD-EV-miRNA mimics were cardiotoxic to human ventricular AC16 myocytes inducing apoptosis and to iPSC-derived cardiomyocytes, impairing contractility and downregulating contractile gene expression. Interestingly, levels of endogenous primary miRNAs corresponding to CKD-EV-miRNAs were significantly higher in CKD-kidney tissues, specifically in CD45-veCD31-ve renal cells, but not in CKD-livers, CKD-lungs or CKD-PBMCs from peripheral blood, a result that suggests that CKD-EV-miRNAs originate renally. Remarkably, CKD-EV-miRNA levels correlated with established markers of cardiac injury, uncovering the presence of sub-clinical heart disease and demonstrating heterogeneity in CKD patients not yet diagnosed with HF. Conclusion: Collectively, our human subject and mouse studies show that circulating CKD-EVs, carrying distinct renal-derived miRNAs, mediate the molecular crosstalk that contributes to the pathogenesis of HF in CKD. Consequently, CKD-EVs hold promise as diagnostic and prognostic biomarkers for early disease detection and as targets for novel therapeutic interventions in chronic reno-cardiac disease. Overall design: This study was approved by the institutional review boards of the Mount Sinai Hospital and University of Virginia (IRB# HSR20550). Peripheral blood was collected from adult patients with moderate to advanced chronic kidney disease (CKD) and healthy controls. CKD diagnosis followed KDIGO guidelines, and exclusion criteria included transplant history, active infection, malignancy, immunosuppressive therapy, pregnancy, bleeding disorders, and anemia. Plasma was isolated from blood collected in sodium citrate tubes and sequentially centrifuged to remove cells, platelets, and microvesicles. Extracellular vesicles (EVs) were isolated via ultracentrifugation (120,000 × g, 90 min), and total RNA was extracted using the miRNeasy micro kit (Qiagen). Small RNA libraries were prepared using the SMARTer smRNA-seq Kit (Takara Bio) and sequenced on an Illumina NovaSeq 6000 (2x150bp, paired-end). Raw reads were processed using Cutadapt to remove adapters and low-quality sequences, retaining reads 18–32 bp in length. Filtered reads were annotated against miRBase v22 for small RNA identification.