Proteomic Profiling of a Novel Immortalised Human Distal Convoluted Tubule Cell Line
- Bioengineering: Modeling, Diagnosis, Therapy
November 04, 2023 | Location: Exhibit Hall, Pennsylvania Convention Center
Abstract Time: 10:00 AM - 12:00 PM
- 400 Bioengineering
- Zhong, Chutong, University College London, London, United Kingdom
- Siew, Keith, University College London, London, United Kingdom
- Walsh, Stephen B., University College London, London, United Kingdom
Group or Team Name
- London Tubular Centre.
Tubulopathies are renal disorders with complex and inadequately understood pathophysiology, creating an urgent need for improved in vitro models to facilitate research. The development of a novel immortalised human distal convoluted tubule cell (hDCT) line represents a breakthrough in this field, offering a promising model for investigating the pathogenesis of tubular diseases. This research focuses on the proteomic characterisation of this cell line, a critical step towards understanding its utility for research into distal tubulopathies.
Four immortalised hDCT cell lines, primarily isolated from healthy volunteer’s urine, were gifted from Dr Kusaba and were cultured as previously described (Ikeda et al., 2020). Cells were treated in 8M urea and 0.1% SDS in the presence of phosphatase and protease inhibitors for protein extraction and cell lysates were analysed through Liquid Chromatography and tandem Mass Spectrometry (LCMS). Western blot analysis was performed for DCT-specific marker NCC (SLC12A3).
WNK1, SPAK, CAB39, CUL3, OxSR1 and PMCA1 (AT2B1) have been identified by LCMS from four hDCT cell lines at a high level, of which hDCT3 has an additionally abundant detection for WNK4 and KCC4 (SLC12A7). NCC was detected from all four hDCT cell lysates in the western blot.
The proteomic analysis of the novel immortalised hDCT cell line has unveiled the presence of key proteins of the DCT region. Notably, the identification of regulator proteins such as WNK1 and SPAK and ion channel corroborates the promising potential of this cell line as a representative model for studying DCT physiology. These findings serve as a preliminary validation of the hDCT cell line's suitability for tubular modeling, enriching our toolset for investigating renal disorders. In future research, we aim to focus on functional experiments for a more comprehensive characterisation of the cell line, solidifying its utility in unraveling the physiology of tubulopathies and potentially guiding targeted therapeutic developments.