Implementation of glomerular cell coculture and microfluidics to investigate basement membrane assembly

  • Nikki-Maria Koudis

Student thesis: Phd

Abstract

The glomerular basement membrane (GBM) is a complex, condensed network of extracellular matrix (ECM), and together with podocytes and glomerular endothelial cells it forms the glomerular filtration barrier (GFB). The GBM is essential for maintaining glomerular integrity and has a key role in size selective glomerular filtration. Current research heavily relies on in vivo model systems to investigate GBM components and to dissect mechanisms of basement membrane assembly and regulation. Yet these systems are costly and have ethical implications. In recent years, advances have been made in the development of in vitro models of the glomerular capillary wall. These cellbased approaches have not yet addressed how the basement membrane assembly is affected by exposure to flow. In this thesis, I developed and characterised a novel GFB in vitro, using cell sheet engineering and microfluidics to apply flow, and demonstrated its function as a permeability barrier. In addition, I determined the protein composition of GFB coculture matrix under static and flow conditions using mass spectrometry-based proteomics. I found that the structure of collagen IV was changed under flow, from diffuse to more complex network-like assembly, with increased fibre thickness, and percentage of high density matrix. In addition, an altered matrix protein composition was observed under flow conditions. Matrix proteins were identified specific to flow conditions and with an altered abundance. Together, this indicated a distinct cellular and matrix response to shear stress. Many matrix proteins specific to the GBM were detected in the GFB in vitro. This demonstrated the utility of the model for studying the GBM in vitro. In addition, I pioneered a protocol for the endogenous tagging of GBM genes, including type IV collagen, in immortalised podocytes using CRISPR-Cas9. Tagging of matrix proteins will provide a platform for real-time analysis of the GBM network assembly, turnover, and regulation. My findings will provide tools to study the GBM and identify protein candidates that are important regulators of changes in blood flow. Importantly, this could reduce the need for in vivo models and provide simple and less-costly approaches to study basement membrane components.
Date of Award31 Dec 2021
Original languageEnglish
Awarding Institution
  • The University of Manchester
SupervisorBrian Derby (Supervisor) & Rachel Lennon (Supervisor)

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