Ms Xiangju Wang1,2, A/Prof Ray Wilkinson1,2,3, A/Prof Helen Healy1,2,3, Dr Andrew Kassianos1,2,3
1Conjoint Internal Medicine Laboratory, Pathology Queensland, Brisbane, Australia, 2Kidney Health Service, Royal Brisbane and Women’s Hospital, Brisbane, Australia, 3Faculty of Medicine, University of Queensland, Brisbane, Australia
Aim: To establish the role that sEV secreted by injured PTEC have in the ‘wave of death’ of CKD.
Background: In ~50% of people, CKD progresses despite removal/remediation of the initiating agent. Subcellular hypoxia in the mitochondrial-dense PTEC triggers multiple pathobiological pathways, including cell death and fibrosis characteristic of CKD. Amongst the signals generated by stressed PTEC are inside-out signals packaged into sEV, whose cargo are a communication pathway to neighbouring cells. In this work, we study the cell-cell communications of sEV in the ‘wave of death’ of CKD.
Methods: Ex vivo patient-derived PTEC were cultured under normal (21% O₂) and hypoxic (1% O₂) conditions on Transwell inserts to separate sEV secreted from apical versus basolateral surfaces. sEV were analysed for concentration (qNano) and cargo (miRNA sequencing). We profiled the injury in autologous PTEC co-cultured with harvested sEV using assays of DNA damage/cell death (flow cytometry, Western blot).
Results: Significantly increased numbers of sEV were secreted from the apical surface of hypoxic PTEC compared with normoxic PTEC. No significant differences in basolateral sEV numbers were observed between culture conditions. Biological pathway analysis of apical hypoxic sEV cargo found distinct miRNA signatures associated with processes of cellular injury/disease. Apical sEV produced by hypoxic PTEC selectively induced DNA damage (↑γ-H2AX), cellular necrosis (↑Annexin-V+/propidium iodide+ cells) and ferroptotic cell death (↓glutathione peroxidase-4, ↑4-hydroxynonenal) in autologous PTEC compared with apical normoxic sEV and basolateral sEV.
Conclusions: Our data establish PTEC-derived apical sEV as a key trigger of tubular death/loss in hypoxic CKD. This innovative concept of how tubular injury is propagated from the initiating insult into a ‘wave of death’ will have significant impact for CKD therapeutics.
Biography:
Dr Andrew Kassianos is a Senior Scientist at the Conjoint Kidney Research Laboratory, Queensland Health. Dr Kassianos has made significant contributions to understanding the cell-cell communication between discrete kidney cell and immune cell populations and the therapeutic potential of targeting this cross-talk in chronic kidney disease (CKD). His research is internationally recognised in the field of CKD pathobiology and his seminal work has translated studies from experimental animal models to the human system.