Abstract
Introduction: Specialised cells of glomeruli, podocytes, possess a similar morphology and proteome to specialised cells of the brain, neurons. Clinical associations between neurodegenerative diseases (ND), diabetes, and chronic kidney disease are well documented. It was hypothesised that molecular mechanisms leading to cellular dysfunction in the two cell types are therefore similar. The aim of this PhD was to characterise in vitro changes in a known renally-expressed protein (tau) involved in ND in diabetic podocytopathy. Additionally, this PhD aimed to identify other ND-associated proteins that become differentially regulated in podocytes in diabetes and investigate the biological functions of such proteins in the podocyte.Methods: Conditionally immortalised human and mouse podocytes were cultured in physiologically high concentrations of insulin, glucose, interleukin-6 and tumour necrosis factor-α to recapitulate an in vivo diabetic environment. Expression and activation of tau was investigated via qPCR, western blotting, and immunofluorescence. Transcript-/proteomic data sets generated by our group were screened for genes involved in ND, in several renal cell lines in diabetic conditions. This identified upregulation of TANK binding kinase 1 (TBK1), a protein associated with Amyotrophic Lateral Sclerosis (ALS), in the podocyte in diabetes. Validation of TBK1 upregulation was attempted via qPCR, western blotting, and immunofluorescence. Generation of transgenic TBK1 podocytes was performed.
Results: Tau is phosphorylated in podocytes grown in diabetic conditions (the AT100 and S404 phosphosites). Many other ND-associated genes are differentially expressed within diabetic podocytes, namely TBK1, which shows upregulation of mRNA and protein in and human and mouse podocytes. Podocyte growth in diabetic conditions induced nuclear translocation of phosphorylated TBK1 and aggregation/association with PML-nuclear bodies and SUMO. Podocyte loss of TBK1 induced podocyte hypertrophy and actin cytoskeleton disorganisation.
Conclusion: ND-associated proteins are expressed in podocytes and become differentially regulated in response diabetic conditions. Additionally, TBK1 is involved in regulating podocyte cytoskeletal actin organisation.
| Date of Award | 3 Oct 2023 |
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| Original language | English |
| Awarding Institution |
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| Supervisor | Gavin I Welsh (Supervisor) & Richard J M Coward (Supervisor) |