X WANG1,2, AJ KASSIANOS1,2,3,4, K KILDEY1,2, J POTRIQUET5, R WILKINSON1,2,3,4 and H HEALY1,2
1Conjoint Kidney Laboratory, Pathology Queensland, Brisbane, Queensland; 2Kidney Health Service, Royal Brisbane and Women’s Hospital, Brisbane, Queensland; 3Queensland University of Technology, Brisbane, Queensland; 4Medical School, University of Queensland, Brisbane, Queensland; 5QIMR Berghofer Medical Research Institute, Brisbane, Queensland
Aim: To characterise and compare apical and basolateral PTEC secreted exosomes under normal and diseased (inflammatory/hypoxic) conditions.
Background: PTEC are polarised epithelial cells and play a central role in kidney disease. Exosomes secreted from different sides of these cells may contain differential molecular cargo that protects or contributes to the disease process.
Methods: Transwell plates and inflammatory/hypoxic culture conditions were used to mimic the in vivo diseased environment. Exosomes were isolated separately from the apical and basolateral sides of PTEC. Exosomes were analysed for size/concentration (qNano) and exosomal CD9 and CD63 protein expression (Western blot). Protein and miRNA content were analysed by mass spectroscopy (MS) and sequencing respectively.
Results: Exosomes from normal and diseased culture conditions displayed a similar size (50-120nM) and morphology. Greater numbers of exosomes were secreted from the apical side compared to the basolateral side under all culture conditions. These apical exosomes expressed greater than fourfold more CD9 and CD63 protein compared to basolateral exosomes. In addition, apical exosomes from diseased conditions expressed twice as much CD9 and CD63 protein as normal condition apical exosomes. Protein and miRNA data from apical and basolateral secreted exosomes under normal, inflammatory and hypoxic conditions will be presented.
Conclusions: Collectively, our data indicate differential expression of exosome number and molecular cargo from apical versus basolateral secretion. These differences are exacerbated under inflammatory and hypoxic culture conditions that mimic early and late stage kidney disease. Further investigation of apical and basolateral exosome protein and miRNA cargo may identify novel targets with potential for biomarker and therapeutic development in renal disease.