Identification of potential mediators of oxidative damage and targets for novel treatments for chronic kidney disease

Abstract

Chronic kidney disease (CKD) is a common and serious problem causing a significant burden to healthcare systems and, most importantly, the affected individual. Despite this, few successful treatments exist for CKD. Understanding the pathophysiological features that are common to chronic kidney pathologies is vital to identify targets for treatment. Oxidative stress has been implicated in all chronic diseases, acting via highly conserved pathways of disease progression. However, evidence to date shows limited benefit of antioxidants as therapies for CKD patients, suggesting a need to better identify the pathways that are stimulated. This thesis focuses on the role of oxidative stress, the regulation of mitochondrial homeostasis, the action of antioxidant compounds, and the disruption of oxidant signalling networks in chronic kidney pathologies (reviewed in Chapter 1). The aims of this project were to: (1) to use an in vitro model of oxidative stress-induced kidney injury to determine how a failure in balance between oxidative stress and oxidant control causes the cellular characteristics of CKD, then to determine whether exogenous antioxidants can prevent and restore renal cell bioenergetics and reduce cell injury; (2) to use an in vivo model of oxidative stress-induced kidney injury with molecular and metabolic imaging to measure potential biomarkers identified in Aim 1, in association with changes in kidney structure and function, and determine whether treatment with an antioxidant therapy modulates kidney disease pathology; and (3) to explore the links between biomarkers of oxidative stress and clinical characteristics of CKD in patients to determine whether a lifestyle intervention in conjunction with standard nephrology care improve systemic oxidative stress and whether this influences kidney function. The renal specific in vitro, in vivo, and clinical models and the methods used are explained in Chapter 2. Chapter 3 reports the separate and cumulative effects of oxidative stress, mitochondrial dysfunction and cell senescence in promoting loss of renal cells in an in vitro model of kidney disease. The results demonstrate that oxidative stress and cell senescence cause mitochondrial destabilization and kidney tubular epithelial cell loss. This may contribute to the development of cellular and tissue atrophy seen in CKD. Chapter 4 further defines the role of mitochondrial alterations as a consequence of a specifically-impaired oxidant signalling regulatory mechanism, namely, peroxisome proliferator-activated receptor-gamma (PPARg)-regulated mitochondrial biogenesis. Oxidative stress promoted mitochondrial destabilisation in kidney proximal tubular epithelial cells, in association with increased PPARg Serine 112 phosphorylation. Despite their positive effects in other tissues, PPARg agonists failed to protect proximal tubular epithelial (PTE) cells, and appear to be detrimental to kidney PTE cell health when oxidative stress induces the cell damage.Chapter 5 examined mediators of oxidative stress in an in vivo model of acute to chronic kidney injury progression, in a spatial and temporal manner via intravital multiphoton microscopy (MPM) in the mouse kidney. Fluorescence lifetime imaging microscopy (FLIM) coupled MPM identified significant and dynamic metabolic substrate utilisation by kidney tubular cells promoting oxidative stress in progressive CKD. Mitochondrial dysfunction is persistent in structurally-normal tubules of the chronically-damaged kidney, potentially enhancing free radical production in a progressive manner. Chapter 6 examined the influence of antioxidants in reducing oxidative stress-induced kidney damage using in vitro and in vivo models. In vitro studies revealed that NAC was superior in reducing oxidative stress-induced PTE cell apoptosis, compared to Coenzyme Q10, alpha(a)-tocopherol or trolox. In vivo studies utilised 4-6 week old C57Bl6 mice on a 5% NAC diet that underwent bilateral ischaemia-reperfusion injury (20 min). Intravital MPM, histopathology, and protein analysis revealed that NAC protects against early kidney damage, but enhances the progression of chronic kidney pathology by eliminating redox-sensitive endogenous cytoprotective signalling. FLIM of cortex and medulla highlight mitochondrial dysfunction and the negative effects of NAC. This does not support general antioxidants for the treatment of CKD. Chapter 7 examined systemic biomarkers of oxidative stress in stage 3-4 CKD patients. 136 patients from the LANDMARK-III cohort underwent standard nephrology care with or without an exercise and lifestyle intervention. Clinical tests and oxidative stress biomarkers (plasma isoprostanes, glutathione peroxidase, total antioxidant capacity) were taken at baseline and at 12 months. No significant differences were identified over 12-months in systemic oxidative stress markers, or eGFR. The results highlight the biological variability of systemic oxidative stress biomarkers in CKD patients. In summary (Chapter 8), this thesis presents oxidative stress as a common and unifying concept of pathophysiological change in chronic kidney pathology at a cellular level. Oxidative stress actively promotes cell death and cell cycle arrest in PTE cells whilst enhancing the secretion of pro-fibrotic factors to increase tubulointerstitial fibrosis. Correcting this process requires are targeted approach, however is increasingly difficult given the complexity of the cellular, physiological, and systemic responses to oxidative stress.