Abstract: FR-PO397
Phenotypic Characterisation of Novel Immortalised Human Distal Convoluted Tubule Cells
Session Information
- Genetics, Development, Regeneration
November 04, 2022 | Location: Exhibit Hall, Orange County Convention Center‚ West Building
Abstract Time: 10:00 AM - 12:00 PM
Category: Development‚ Stem Cells‚ and Regenerative Medicine
- 500 Development‚ Stem Cells‚ and Regenerative Medicine
Authors
- Zhong, Chutong, University College London, London, London, United Kingdom
- Siew, Keith, University College London, London, London, United Kingdom
- Walsh, Stephen B., University College London, London, London, United Kingdom
Background
The distal convoluted tubule (DCT) is responsible for fine-tuning the final excretion of sodium, potassium, calcium and magnesium in the urine. The study of rare monogenic diseases (namely Gordon and Gitelman syndromes) have highlighted the physiological importance of the DCT in blood pressure control. However, the most commonly used cellular models of this segment are either not truly kidney cells (e.g. studies have shown HEK293 are more likely of neuronal lineage) or of murine origin, and thus there is need for advanced human DCT (hDCT) models. Recently, 4 immortalised hDCT models isolated from human urine hav been created by Dr Tetsuro Kusaba, but have not been biochemically or functionally characterized.
Methods
Western blot analysis on total cellular lysates by using NCC antibody detected three different states of NCC in all four hDCT cell lines. qPCR detected the presence of NCC mRNA at Cq 29.57-32.18 across all four samples (Human kidney positive control Cq 27, and HEK293 negative control Cq 45).
Results
Western blot analysis on total cellular lysates by using NCC antibody detected three different states of NCC in all four hDCT cell lines. qPCR detected the presence of NCC mRNA at Cq 29.57-32.18 across all four samples (Human kidney positive control Cq 27, and HEK293 negative control Cq 45).
Conclusion
The preliminary western blot and qPCR results confirmed NCC expression in all four hDCT cell lines and the position of bands agreed with previous studies (de Jong et al., 2003; DOI 10.1074/jbc.M303101200. Zhang et al., 2015. doi: 10.1093/hmg/ddv185.). Further experiments will be performed using other key markers expressed in DCT, including TRPM6 and parvalbumin, Calbindin, KS-WNK1, WNK4, human isoforms of SPAK and NCC. These cell lines could be the first validated hDCT cellular models which reflect the human DCT physiology and can be adapted to 3D cell culture system for functional experiments