Investigating Chronic Kidney Disease - The Role of Vinculin in the Regulation of the Glomerular Filtration Barrier

UoM administered thesis: Phd

  • Authors:
  • Franziska Lausecker

Abstract

Glomerular podocytes are highly specialised kidney epithelial cells that form an integral component of the glomerular filtration barrier. Intercellular junctions, cell-matrix adhesions and the actin cytoskeleton in podocytes are crucial for maintaining the integrity of this barrier; genetic mutations in many of these components result in glomerular dysfunction. Podocytes must adapt to mechanical forces accompanying filtration. The molecular mechanisms of how podocytes adapt to mechanical force is poorly understood. In this thesis, I hypothesised that the mechanosensing protein vinculin, which localises to cell-matrix adhesions and intercellular junctions, has a key role in podocyte adaptation to mechanical force. Firstly I focused on the contribution of cytoskeletal and adhesion components to podocyte barrier function. I used specific inhibitors and siRNA-mediated protein knockdown strategies. Analysis of barrier function using Electric cell-substrate impedance sensing (ECISTM) revealed essential roles for cell-matrix adhesions, cell-cell junctions and the actomyosin machinery. Cell-matrix adhesions demonstrated a particularly important role in the barrier function of podocytes compared to other epithelial cell lines. Secondly I investigated the specific role of the mechanosensing protein vinculin in maintaining the integrity of the glomerular filtration barrier. To study the role of vinculin in vivo, I characterised a podocyte-specific vinculin knockout mouse. These mice demonstrated glomerular dysfunction particularly upon the induction of injury and at advanced age. Ultrastructural analysis with serial block-face scanning electron microscopy revealed an increased length of podocyte foot processes in vivo. To study the relevance of my findings in glomerular diseases kidney biopsy samples from patients with focal segmental glomerulosclerosis, minimal change disease and membranous nephropathy were analysed. This revealed a reduction of vinculin specifically in podocytes. Finally I investigated podocyte barrier in vitro, using primary podocytes from the podocyte-specific vinculin knockout mouse and I validated impaired barrier function found in vivo. Furthermore vinculin knockout podocytes demonstrated smaller adhesions, impaired focal adhesion signaling as well as faster cell migration compared to wild-type podocytes. This suggests that vinculin deficient podocytes are less adhesive, which results in aberrant filtration upon the induction of glomerular injury. Overall my PhD studies highlight the roles of the actin cytoskeleton, cell-cell junctions and cell-matrix adhesions contributing towards the podocyte filtration barrier in specific ways and particularly emphasises the role of vinculin in the maintenance of the glomerular barrier in response to injury.

Details

Original languageEnglish
Awarding Institution
Supervisors/Advisors
Award date1 Aug 2018