CD46 knock-out using CRISPR/Cas9 editing of hTERT immortalized human cells modulates complement activation

PLoS One. 2019 Apr 8;14(4):e0214514. doi: 10.1371/journal.pone.0214514. eCollection 2019.

Abstract

The kidney is especially sensitive to diseases associated with overactivation of the complement system. While most of these diseases affect kidney glomeruli and the microvasculature, there is also evidence for tubulointerstitial deposition of complement factors. Complement inactivating factors on cell membranes comprise CD55, CD59 and CD46, which is also termed membrane cofactor protein (MCP). CD46 has been described as localized to glomeruli, but especially also to proximal tubular epithelial cells (RPTECs). However, human cell culture models to assess CD46 function on RPTECs are still missing. Therefore, we here performed gene editing of RPTEC/TERT1 cells generating a monoclonal CD46-/- cell line that did not show changes of the primary cell like characteristics. In addition, factor I and CD46-mediated cleavage of C4b into soluble C4c and membrane deposited C4d was clearly reduced in the knock-out cell line as compared to the maternal cells. Thus, human CD46-/- proximal tubular epithelial cells will be of interest to dissect the roles of the epithelium and the kidney in various complement activation mediated tubulointerstitial pathologies or in studying CD46 mediated uropathogenic internalization of bacteria. In addition, this gives proof-of-principle, that telomerized cells can be used in the generation of knock-out, knock-in or any kind of reporter cell lines without losing the primary cell characteristics of the maternal cells.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • CRISPR-Cas Systems*
  • Cell Line
  • Complement Activation*
  • Complement C4 / chemistry
  • Complement C4b / chemistry
  • Epithelial Cells / cytology*
  • Gene Editing
  • Gene Knockout Techniques*
  • Humans
  • Kidney Tubules / cytology
  • Membrane Cofactor Protein / genetics*
  • Telomerase / metabolism*
  • Telomere / ultrastructure
  • gamma-Glutamyltransferase / metabolism

Substances

  • CD46 protein, human
  • Complement C4
  • Membrane Cofactor Protein
  • Complement C4b
  • gamma-Glutamyltransferase
  • TERT protein, human
  • Telomerase

Grant support

This work was supported by the Houska award to RGV, as well as the Christian Doppler Society. The financial support by the Austrian Federal Ministry of Economy, Family and Youth, the National Foundation for Research, Technology and Development is also gratefully acknowledged. Evercyte GmbH and Horizon Genomic GmbH provided support in the form of salaries for authors RGV, MWi, and TF, as well as TB and DL. None of the funders had any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the ‘author contributions’.