Urinary Extracellular Vesicle Analysis Reveals Early Signs of Kidney Inflammation and Damage in Single Ventricle Paediatric Patients After Fontan Operation

Cristina Grange,1 Luca Deorsola,2,3 Beatrice Degiovanni,2,3 Dario Tomanin,4 Diego Prudente,1 Licia Peruzzi,2,5 Carlo Pace Napoleone,2,3 Benedetta Bussolati1,4 1Department of Medical Sciences, University of Torino, Torino, Italy; 2Città della Salute e della Scienza Hospital, Torino, Italy; 3Pediatric and Congenital Cardiac Surgery, Regina Margherita Children’s Hospital, Torino, Italy; 4Department of Molecular Biotechnology and Health Sciences, University of Torino, Torino, Italy; 5Pediatric Nephrology Dialysis and Transplant Unit, Regina Margherita Children’s Hospital, Torino, ItalyCorrespondence: Benedetta Bussolati, Department of Medical Sciences, University of Torino, via Nizza 52, Torino, 10126, Italy, Tel +39011-6706453, Email [email protected]: 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 characterize uEVs in children with single ventricle heart defects who underwent Fontan surgery, focusing on markers for monitoring and predicting kidney function, 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 to assess the presence of the reno-protective hormone Klotho, using super resolution microscopy of single uEVs and ELISA. Moreover, we analysed the levels of markers of kidney regeneration, such as CD133 and CD24, and of inflammation using a bead-based cytofluorimetric multiplex analysis. The markers’ levels were correlated with patients’ demographical, clinical and surgical data.Results: uEVs from children with single ventricle defects showed reduced levels of Klotho and CD133, compared with the ones of healthy subjects. 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 single ventricle patients, who underwent Fontan’s surgery, present altered levels of uEV biomarkers related to regeneration, inflammation and fibrosis, suggesting the presence of early signs of kidney damage and inflammation, compatible with the complexity of the pathology. Keywords: urine, exosomes, congenital heart defects, kidney disease, CD133, Klotho