Diabetic nephropathy is the leading cause of end-stage renal disease, yet the mechanisms responsible for hyperglycemia-induced kidney injury have not been fully elucidated. Activation of the renin-angiotensin system and NADPH oxidase-dependent generation of reactive oxygen species are important mediators of chronic kidney disease. I first studied the effect of ACE2, an important enzyme in the renin-angiotensin system, in diabetic kidney injury in the Akita mouse and related the effect to angiotensin peptide and NADPH oxidase. I then demonstrated the interaction between Angiotensin II, the main substrate, and angiotensin-(1-7), the main product of ACE2, respectively, on cell signaling in mesangial cells to better understand the in vitro effect of ACE2. Finally I studied the effect of deletion of p47phox, a regulatory subunit of the NADPH oxidase, on initiation and progression of diabetic nephropathy in the Akita mouse and mesangial cell.
Administration of human recombinant ACE2 decreased angiotensin II levels, increased angiotensin-(1-7) levels, normalized NADPH oxidase activity in the Akita mice, and ameliorated diabetes-induced kidney injury. In vitro, hrACE2 attenuated both high glucose and ANG II–induced oxidative stress and NADPH oxidase activity in mesangial cells.
Ang-(1–7)-induced ERK1/2 phosphorylation in mesangial cells in a mas receptor-cAMP-PKA-dependent manner. This effect of ang-(1-7) on ERK1/2 phosphorylation is not mediated by AT1R, AT2R, epidermal growth factor or NADPH oxidase. Pre-treatment with Ang-(1-7) attenuated Ang II-induced NADPH oxidase activity and ERK1/2 activation also in a cAMP-PKA-dependent manner.
Deletion of p47phox not only reduced diabetes-induced kidney injury but also reduced hyperglycemia by increasing pancreatic and circulating insulin concentrations. p47phox-/- mice exhibited improved glucose tolerance but modestly decreased insulin sensitivity. Deletion of p47phox attenuated high glucose-induced activation of NADPH oxidase and pro-fibrotic gene expression in mesangial cells. There was a positive correlation between p47phox and collagen Iα1 mRNA levels in renal biopsy samples from control subjects and subjects with diabetic nephropathy.
The data generated in this thesis strongly suggest a protective role of ACE2, via Ang-(1-7), and a deleterious role of p47phox in diabetic nephropathy. Future therapeutic strategies should include enhancing ACE2 activity in the kidney and inhibiting p47phox-dependent activation of NADPH oxidase in both the kidney and the pancreas.
| Identifer | oai:union.ndltd.org:TORONTO/oai:tspace.library.utoronto.ca:1807/34787 |
| Date | 17 December 2012 |
| Creators | Liu, George |
| Contributors | Scholey, James |
| Source Sets | University of Toronto |
| Language | en_ca |
| Detected Language | English |
| Type | Thesis |
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