Age-dependent changes in the kidney are often debilitating, can be life-threatening and are a significant cause of increasing health costs worldwide. Excessive fibrosis, a general lack of regenerative ability and an increase in apoptosis in cells that determine healthy renal function work together to cause chronic kidney disease (CKD). This thesis reviewed the literature and then tested hypotheses developed from this review, to provide information on the molecules and mechanisms that determine the age-dependent changes of CKD. Results in this thesis provide a comprehensive analysis of the molecular, structural and functional changes of age-related CKD, with particular attention paid to the longevity gene p66Shc. The present studies were able to make use of established ageing rodent colonies of various phenotypes. In the first of the research Chapters, rat models of age-related CKD linked with obesity and hypertension were used. The research tested the hypothesis that each cause of age-related renal change (ageing, obesity or hypertension) would have differing underlying genetic modifications that could explain any differences in renal structure and function. In particular, alterations in oxidant handling and energy metabolism were investigated to identify markers for age-related CKD. Young (3 months) and old (20-24 months) spontaneously-hypertensive rats (SHR), normotensive Wistar- Kyoto (WKY) and Wistar rats (normotensive, with excess visceral and peri-renal fat in ageing) (N = 4 per group) were compared for renal functional and physiological parameters, fibrosis, inflammation and oxidative stress. All of the analyses indicated the old obese Wistars had the greatest renal injury, inflammation and markers of oxidative stress. In particular, % phosphop66/ p66Shc, considered an oxidant stress marker, was significantly increased in these animals (p<0.05). These results suggest that obesity and hypertension have differing oxidant handling and signalling pathways that act in the pathogenesis of age-related CKD, and that obesity alone may be a key causative mechanism of age-related CKD. x Oxidative stress is thought to be a major cause of age-related CKD. In chapter 5, the following hypotheses were tested: (1) that the added stress of ischemia-reperfusion (IR) injury on the ageing kidney would create an environment for increased injury; and (2) that this injury could be modulated by using a short-term anti-ageing strategies. Old (20-24 month) and young (3 month) WKY rats (females, N = 4 per group) were used to compare the effects of bilateral, 45-minute, IR injury with and without calorie restriction (a 40% reduction in food from baseline) or vitamin E (daily gavage of 1000IU) for 10 days prior to IR surgery and then for the length of recovery from IR (4 days). Histological, functional and molecular analyses were used. Old rats had significantly worsened renal injury compared with young rats with IR. Proteins involved in oxidative stress (HO-1, p66Shc and phospho-p66Shc), survival (PKB and phospho-PKB), apoptosis (Bax, Bcl-2), inflammation and fibrosis (NF-κB, tumour necrosis factor-α/TNFα, transforming growth factor-β/TGFβ) were differentially expressed according to age and development of IR injury. Vitamin E-supplemented animals showed minimal improvement and calorie restriction generally worsened the outcome in both young and old animals. Changes in protein expression support the notion that these short-term calorie-restricted animals were in a catabolic state, perhaps similar to protein energy wasting seen in some of the human dialysis population. In chapter 6 in vitro experimental models using primary human renal proximal tubular epithelial cells (PTECs) were utilised. Successive passaging in culture of the PTECs showed increasing markers of senescence and oxidative stress. The degree of senescence correlated with expression of the oxidative stress marker phospho-p66Shc and alterations in other key signalling molecules. Hydrogen peroxide (5mM for 1 hour) was used to simulate a burst of oxidative stress and the effects of leptin and resveratrol was examined. Histological and molecular analyses demonstrated some links with the previous in vivo results, for example the involvement of phospho-p66Shc in the development of cell senescence but generally the in vitro experiments did not replicate in vivo xi findings. The lack of complex, heterogeneous, cellular and growth factor/cytokine interactions of the in vivo environment are thought to be a factor in this disappointing result. The tendency for development of CKD differs in males and females in ageing humans. In chapter 7 characteristics of age-related CKD in old male and female rats were compared, summarising data on the WKY rats from Chapters 3 to 5. Minor differences between males and females in histology, function and protein expression are described, but these do not adequately reflect the findings of gender dimorphisms in development of CKD, reported from human and experimental in vivo studies. These experiments demonstrate some of the pathogenetic mechanisms of age-related CKD. The results indicate pathways or molecules that may be targeted in future therapies or may be used as biomarkers of early development of age-related CKD. In particular, the modification of p66Shc may one day be used to minimize renal damage and promote health in the elderly.
| Identifer | oai:union.ndltd.org:ADTP/279285 |
| Creators | Christine Percy |
| Source Sets | Australiasian Digital Theses Program |
| Detected Language | English |
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