Abstract: TH-OR40
Targeted Isolation of Urine-Derived Renal Tubular Cells for Personalised Medicine
Session Information
- Technology-Based Approaches in Nephrology
November 03, 2022 | Location: W308, Orange County Convention Center‚ West Building
Abstract Time: 04:48 PM - 04:57 PM
Category: Pathology and Lab Medicine
- 1700 Pathology and Lab Medicine
Authors
- Zhong, Chutong, University College London, London, London, United Kingdom
- Walsh, Stephen B., University College London, London, London, United Kingdom
- Siew, Keith, University College London, London, London, United Kingdom
Background
The kidney achieves homeostasis through the complex actions of the nephron; a heterogenous structure made up of 14+ segments. Rare monogenic tubular diseases are often characterised by impairment of specific tubular segments. Isolation and characterisation of patient-specific urine-derived renal tubular epithelial cells (uRTEC) from disease-relevant segments can assist diagnosis and treatment planning.
Methods
Urine samples collected from healthy volunteers and patients with genetic tubular disease were pelleted by centrifugation at 400 RCF for 10min at RT, and the cell pellet was washed with a 50:50 mixture of DMEM:F12 (supplemented with 10% FBS, 100U/ml penicillin, 100µg/ml streptomycin, 1X insulin-transferrin-selenium, 2.5µg/ml nicotinamide, 500µg/ml hydrocortisone). Targeted isolation and enrichment of segment-specific cells was performed using a magnetic beads conjugated to target-specific antibodies/lectins. Primary urine-derived cells were either fixed using 4% w/v formaldehyde-PBS for 15min at RT at 60% confluency, or lysed with TRI reagent at 90% confluency for RNA isolation. Cell types were validated by staining with fluorescently-tagged marker antibodies/lectins and qPCR of segment specific mRNAs.
Results
Primary urinary cells successfully cultured from patients’ urine samples with different morphologies presented and can be maintained to the third passage. As can be seen in Figure 1, cells from several patients stained positively with Dolichos Biflorus Agglutinin indicating the presence of distal convoluted tubule cells. These was validated by confirmation of expression of NCC.
Conclusion
uRTEC can be routinely isolated from patient’s urine, targeted for enrichment, and successfully subcultured for several passages. Future work, would aim to utilise these cells in 3D “Organ-on-a-Chip” systems, where we could potentially artificially reconstruct patient’s tubules from primary urinary cells and conduct individualised pharmacological experiments to optimize treatments, thereby bringing true personalised medicine to nephrology.