| Popis: |
Podocytes are highly specialised epithelial cells, located on the urinary side of the glomerular filtration barrier. Disruptions to podocyte structure and function results in a disruption to the filtration process and proteinuria. Therefore, an understanding of factors that influence podocyte biology is desirable. Podocytes respond to insulin both in vitro and in vivo and the podocyte-specific knock-down of the insulin receptor results in a glomerular pathology with features characteristic of diabetic nephropathy. There is also evidence that podocyte insulin signalling is disrupted in models of insulin resistance and diabetes. The aim of this thesis was to investigate how both positive and negative modulators of insulin signalling specifically influence podocyte responses in vitro. Initial work was undertaken to characterise the responses of a recently developed mouse podocyte cell line. These cells express the insulin and insulin-like growth factor {IGF)-I receptors, and the phosphorylation of these proteins is evident following insulin stimulation. Downstream of receptor activation, increased phosphorylation of insulin receptor substrate (IRS) proteins and activation of phosphoinositide 3-kinase {PI3K)/Akt and MEK/ERK mitogen activated protein kinase (MAPK) cascades is observed. Furthermore, these cells activate glucose transport pathways following insulin stimulation. The effects of the anti-diabetic agent metformin and the selective chemical inhibition of protein tyrosine phosphatase 1B (PTP1B) were next examined, as potential enhancers of podocyte insulin signalling. Data herein demonstrates that these chemicals have direct effects on mouse podocytes in vitro and influence cellular glucose uptake. The modulation of podocyte signalling by diabetic factors, specifically the chronic exposure to high insulin, high glucose and inflammatory cytokines, was also investigated. Data reveals that the exposure of podocytes to a combination of these diabetic factors disrupts the activation of insulinstimulated signalling and glucose uptake. Individually, these factors exert their effects at different points within the signalling network. Quantitative peR arrays were also performed to identify changes at the mRNA level. Interestingly, the mRNA encoding the neurotransmitter Neuropeptide Y (NPY) was down regulated by 7-fold following cytokine exposure. Further investigation demonstrates that this peptide signals to both human and mouse podocytes in vitro stimulating the phosphorylation of Akt and ERK1/2, as well as influencing calcium signalling. Overall, work within this thesis demonstrates both the positive and negative modulation of podocyte insulin signalling in vitro and identifies a novel role for NPY in podocyte biology. |
