Urinary Extracellular Vesicle Analysis Reveals Early Signs of Kidney Inflammation and Damage in Single-Ventricle Pediatric Patients after Fontan Operation

Background: Extracellular vesicles present in urine (uEVs) are gaining considerable interest as biomarkers, to monitor and predict kidney physio-pathological state. Patients with single ventricle defects and hemodynamic stabilization by Fontan intervention may develop kidney dysfunction as one of the most prevalent extracardiac co-morbidity. Our study aimed to identify markers for monitoring and predicting kidney function in children with single ventricle heart defects who underwent Fontan surgery, as well to get physio-pathological insights on possible mechanisms of tissue damage and progression. Methods: We isolated uEVs from urine of 60 paediatric patients affected by single ventricle defects, and from 10 healthy subjects. We analysed uEVs for the expression of the reno-protective hormone Klotho, using super- resolution microscopy of single EVs and ELISA. Moreover, we analysed the level of marker of kidney regeneration, such as CD133 and CD24, and of inflammation using a bead-based cytofluorimetric multiplex analysis. Marker expression was correlated with demographical, clinical and surgical data of patients. Results: uEVs from children with single ventricle defects showed reduced levels of Klotho and CD133, compared with the one of healthy subject. In parallel, the levels of inflammatory markers (CD3, CD56, and HLA-DR) were significantly higher. Interestingly, levels of inflammatory markers correlated with age of patients and distance from surgery. Conclusion: This study demonstrates that EVs isolated from the urine of single ventricle patients. present distinct expression of biomarkers of fibrosis, regeneration and inflammation in respect to those of healthy subjects, suggesting the presence of early signs of kidney damage.Experimental scheme