Thromboxane receptor (TPr) activity is elevated in diabetes and contributes to complications of diabetic kidney disease (DKD). TPr blockade appears to have therapeutic potential. Several rodent models of DKD show attenuation of renal damage and proteinuria upon administration of the TPr antagonist, S18886. However, the cellular targets that underlie the injurious effects of TPr activation in DKD remain to be elucidated. A pilot study in our laboratory subjected a conditionally-immortalized mouse podocyte cell line to high glucose (25 mM D-glucose) and equibiaxial mechanical stretch (an in vitro simulator of increased glomerular capillary pressure associated with glomerular hyperfiltration in early diabetes). qRT-PCR revealed that exposure of podocytes to mechanical stretch (10% elongation) and high glucose for 6 hours yielded a 9-fold increase in TPr mRNA levels vs. controls (non-stretch, 5mM D-glucose + 25mM L-glucose) (p<0.05, n=5). We hypothesized that TPr expression and activity are increased in podocytes during the onset of DKD resulting in maladaptive effects on this key glomerular filtration barrier cell type. We showed that enhanced TPr signaling threatens podocytes viablility. Cultured podocytes treated with the TPr agonist, U-46619 (1 μM) for 24 hours are more vulnerable to apoptosis as quantified by Hoescht 33342 (20% cell death p<0.001, n=3) , TUNEL (30-fold increase, ns, n=3) and Annexin-V labeling (3-fold increase, p <0.001, n=3). To further support these in vitro findings, we developed a transgenic mouse with podocyte-specific overexpression of TPr. A construct consisting of a desensitization resistant mutant of the human TPr with both N- and C-terminal HA-epitope tags under the control of an 8.3 kb fragment of the immediate 5’ mouse NPHS1 promoter was cloned, isolated and injected into FVB/n oocytes that were implanted into pseudopregnant CD1 females. Founders were characterized for TPr transgene expression, and TPr transgene mRNA levels were detected by qRT-PCR. Our in vitro results suggest that increased TPr expression in podocytes of diabetic mice may contribute to filtration barrier damage and have important implications in the development and progression of DKD.

Cellular adaptation to conditions of stress is critical to the survival of all organisms. In mammalian cells, reduced oxygen availability, or hypoxia, triggers a myriad of alterations within molecular pathways aimed at ensuring sustained viability and functionality of vital organs. The master regulator of the hypoxic response is the heterodimeric HIF transcription factor, composed of an oxygen-labile HIFalpha subunit and a constitutively expressed and stable HIFbeta(ARNT) subunit. While experiments in mice have demonstrated the indispensability of HIF1alpha/HIF2alpha, unchecked hyperactivation of HIF is associated with pathological complications, such as the development of clear-cell renal cell carcinoma (ccRCC), the most common form of kidney cancer. In particular, overexpression of HIF2alpha has been intimately linked to ccRCC molecular pathogenesis. Here, we report that HIF2alpha overexpression leads to Akt-mediated hyperactivation of Hdm2, resulting in decreased activity of p53 and increased resistance to apoptosis. Significantly, we show that restoration of p53 activity via inhibition of Hdm2 reverses chemoresistance of otherwise refractory ccRCC cells. We also demonstrate that the picornavirus EMCV (Encephalomyocarditis virus) efficiently destroys ccRCC tumour cells in an NF-kappaB- and HIFalpha-dependent manner both in vitro and in a mouse xenograft model. This work provides pre-clinical evidence for the potential use of EMCV in an oncolytic virus-based approach to the treatment of advanced ccRCC. In addition, using a cell biology-based approach we reveal that inhibition of the DNA methyltransferase DNMT1 leads to enhanced HIF promoter-binding affinity and transactivation activity in a manner that is independent of changes in HIFalpha protein levels. This study uncovers a novel HIF regulatory mechanism in mammalian cells. In summary, the work presented here provides insight into the molecular mechanisms governing HIF activity and the effects of HIF on the molecular pathogenesis of ccRCC. Our findings have the potential to provide new therapeutic avenues for the treatment of kidney cancer.

Cellular adaptation to conditions of stress is critical to the survival of all organisms. In mammalian cells, reduced oxygen availability, or hypoxia, triggers a myriad of alterations within molecular pathways aimed at ensuring sustained viability and functionality of vital organs. The master regulator of the hypoxic response is the heterodimeric HIF transcription factor, composed of an oxygen-labile HIFalpha subunit and a constitutively expressed and stable HIFbeta(ARNT) subunit. While experiments in mice have demonstrated the indispensability of HIF1alpha/HIF2alpha, unchecked hyperactivation of HIF is associated with pathological complications, such as the development of clear-cell renal cell carcinoma (ccRCC), the most common form of kidney cancer. In particular, overexpression of HIF2alpha has been intimately linked to ccRCC molecular pathogenesis. Here, we report that HIF2alpha overexpression leads to Akt-mediated hyperactivation of Hdm2, resulting in decreased activity of p53 and increased resistance to apoptosis. Significantly, we show that restoration of p53 activity via inhibition of Hdm2 reverses chemoresistance of otherwise refractory ccRCC cells. We also demonstrate that the picornavirus EMCV (Encephalomyocarditis virus) efficiently destroys ccRCC tumour cells in an NF-kappaB- and HIFalpha-dependent manner both in vitro and in a mouse xenograft model. This work provides pre-clinical evidence for the potential use of EMCV in an oncolytic virus-based approach to the treatment of advanced ccRCC. In addition, using a cell biology-based approach we reveal that inhibition of the DNA methyltransferase DNMT1 leads to enhanced HIF promoter-binding affinity and transactivation activity in a manner that is independent of changes in HIFalpha protein levels. This study uncovers a novel HIF regulatory mechanism in mammalian cells. In summary, the work presented here provides insight into the molecular mechanisms governing HIF activity and the effects of HIF on the molecular pathogenesis of ccRCC. Our findings have the potential to provide new therapeutic avenues for the treatment of kidney cancer.

Typescript. / Thesis (Ph. D.)--University of Hawaii at Manoa, 1979. / Bibliography: leaves 86-97. / Microfiche. / xii, 97 leaves ill. 29 cm

Malnutrition is present in up to 48% of chronic kidney disease patients on the initiation of renal replacement therapy (dialysis)1. At this time, malnutrition is an independent and significant predictor of morbidity and mortality2. As a consequence of progressive deterioration in kidney function, symptoms of decreased appetite and reduced intake are common factors leading to the decline in nutritional status3. However, at present there is little evidence to inform nutrition assessment and intervention for pre-dialysis chronic kidney disease (CKD). The purpose of this study was to provide evidence for the nutritional management of CKD patients prior to dialysis with an aim to optimise nutritional status. To address this, an investigation comprising of two phases examining nutrition assessment and intervention in a sample of pre-dialysis Stage IV and V CKD patients was undertaken. Both phases of the study were conducted through Royal Brisbane and Women’s Hospital (RBWH) Department of Renal Medicine pre-dialysis clinic. Participants met the following criteria: adult (&gt18 years) Glomerular Filtration Rate (GFR) &lt30ml/min CKD, not previously seen by a dietitian for Stage IV CKD, absence of communication or intellectual impairment inhibiting their ability to undertake the intervention and an absence of malnutrition from a cause other than CKD. Phase I was a cross-sectional investigation into the performance of a range of tools assessing nutrition status, conducted at baseline of Phase II. Phase II was a randomisedcontrolled trial designed to determine if providing individual nutrition counselling with regular telephone follow-up resulted in improved body composition, nutritional status, dietary intake and quality of life, compared with standard care. A range of intermediate, clinical and patient-centred outcome measures were collected at baseline and twelve weeks. Body composition was measured by total body potassium counting (TBK), considered a gold-standard measure of body cell mass (BCM, the body’s functional metabolising tissue). Nutritional status was measured using Subjective Global Assessment (SGA) and a number of modified versions of SGA, 7-point SGA, Malnutrition Inflammation Score (MIS) and the scored Patient-Generated Subjective Global Assessment (PG-SGA). Dietary intake was measured using 3-day food records. Quality of life was measured by Kidney Disease Quality of Life Short Form version 1.3 (KDQOL-SFTM v1.3 © RAND University), combining the Short Form-36 (SF-36), with a kidney disease-specific module4. Statistical analysis was carried out using SPSS Version 13 (SPSS Inc, Chicago, IL, USA). Phase I analysis was based on descriptive and bi-variate statistics, including chi-square, t-test and ANOVA. For phase II, change variables (Week 12 – Week 0) were created for the outcome measures (BCM, SGA tools, dietary intake (energy and protein) and the 18 KDQOL-SFTM subscales). The assessment of change in outcome measures by treatment group was undertaken by ANCOVA, adjusting for baseline values. Further multivariate analysis (ANCOVA and MANCOVA models) were created for outcome variables when confounding variables were identified and adjusted for. In Phase I, 56 patients (Male n=34; age mean (±SD) 70.7 (±14.0); GFRMDRD 22.4 (±6.5) mL/min) underwent baseline assessment. In this population the prevalence of malnutrition was 19.6% (n=11, SGA B; no C ratings). Malnutrition was associated with lower body cell mass (mean BCM, 26.3 vs. 33.4 kg p=0.007), body weight (64.8 vs. 76.1 kg p=0.042), BMI (23.7 vs. 27.6 kg/m2 p=0.015) and greater weight loss over previous 6 months (-6.2 vs. -0.1 kg p=0.004). Body cell mass indexed for height (BCM-I kg/m3.5) had a relationship with MIS (r=-0.27 p=0.063) and scored PG-SGA (r=-0.27 p=0.060), but not with 7-point SGA (F(4) 2.24 p=0.080). PG-SGA best discriminated malnutrition based on a BCM-I cut-off of &lt5.25kg/ m3.5 of all the modified SGA tools. The scored PG-SGA including the global SGA rating is recommended for use in pre-dialysis CKD. In Phase II, 50 patients, (Male n=31 (62.0%); age 69.7 (±12.0) years; GFRMDRD 22.1 (±6.9) ml/min) completed the 12 week study period (intervention n=24; standard care n=26). At 12 weeks, there was a clinically significant improvement in all outcome measures in the intervention group. There was a 3.9% (95% CI, -1.0 to 8.7%) mean difference in change for Body Cell Mass between the treatment groups, represented by a significant decrease in the standard care group and maintenance in the intervention group. Nutritional status measured by SGA improved or was maintained (24/24) in the intervention group, however, decreased in 14% (4/26) of the standard care group. Energy intake significantly improved in the intervention group resulting in a mean difference in change of 17.7kJ/kg (8.2 to 27.2 kJ/kg). Quality of life improved significantly in 10 of the 18 sub-scales in the intervention group. Significant effect modification for gender was apparent for many of the outcome variables, with females responding most significantly to the intervention treatment. This study concluded that, overall, structured nutrition intervention limits the deterioration in nutritional status, improves dietary intake and quality of life in patients with CKD prior to the onset of renal replacement therapy. This thesis makes a significant contribution to the evidence base for nutritional management of pre-dialysis Stage IV CKD. The use of SGA for nutrition assessment and including PG-SGA to measure change is recommended for routine nutrition assessment of pre-dialysis CKD. The provision of individual nutrition counselling with regular follow-up, with a focus on promoting intake provides beneficial patient outcomes supporting optimal nutritional status in pre-dialysis CKD patients.

Thesis (MAppSc (Medical Laboratory Sc)) --University of South Australia, 1993

Thesis (MAppSc) -- University of South Australia, 1998

Doctor of Philosophy (PhD) / Healthcare services and health research aim to improve the physical and psychosocial well being of consumers, and to offer responsive services needed and valued by them. Research in chronic kidney disease (CKD) has predominantly focused on investigating biomedical aspects and evaluating technological or pharmacological treatment interventions to improve medical management. While research into assessing patients’ and caregivers’ quality of life, and symptom burden, is growing minimal attention has been given to gaining a broad and in-depth understanding about the experiences, psychosocial issues and needs of patients and their caregivers. These need to be considered when planning and delivering patient-centred care and health research across the whole trajectory of CKD. The studies that form the major part of this thesis explore the perspectives, needs and experiences of CKD patients and their caregivers, within a broad and multidimensional framework encompassing aspects of the nature of the health and illness experiences and consumer perspectives. In Chapter 2, to understand what is known about parental experiences of caring for a child with CKD, the relevant qualitative literature was systematically reviewed and synthesized. Three inter-related clusters were identified: intrapersonal, interpersonal and external experiences. In Chapter 3, to gain a more detailed and broader understanding of this topic, in-depth interviews were conducted with parents of 20 children with CKD and 4 major themes were identified: absorbing the clinical environment, medicalising parenting, disrupting family norms, and coping strategies and support structures. In Chapter 4, to assess the effectiveness of support interventions for caregivers of patients with CKD, a systematic review was conducted which identified only three eligible studies that assessed only the effect of educational material on caregiver knowledge, not other domains. In Chapter 5, to describe and compare the broad range and depth of experiences and perspectives from predialysis, dialysis and transplantation patients, data from patient focus groups were analysed. The 5 themes that emerged from this data were: personal meaning of CKD, managing and monitoring health, lifestyle consequences, family impact, and informal structures. In Chapter 6, the focus groups were also used to elicit research priorities and identify reasons that patients used to develop their research priorities. A patient focused research agenda was elicited for CKD and 5 reasons that patients used to develop their research priorities were identified: normalisation of life, altruism, economic efficiency, personal concerns and clinical outcomes. During the focus groups, participants repeatedly expressed frustration about the poor public profile, and lack of community-based information on CKD prevention. So in Chapter 7, to assess how Australian news media covered prevention and early detection of CKD, I analysed television and newspaper stories that referred to CKD prevention or early detection. Kidney disease in general, and particularly the prevention and early detection of CKD, received virtually no media attention. When mentioned, it was mainly in the context of transplantation and donor stories, and seldom prevention or early detection, which appears largely unnewsworthy in its current form. At best, CKD received peripheral mention as a secondary concern in diabetes and obesity news stories which focused on lifestyle solutions. In Chapter 8, to develop a checklist for explicit and comprehensive reporting of qualitative studies (in-depth interviews and focus groups), I performed a comprehensive search in relevant publications for existing checklists used to assess qualitative studies. Seventy-six items from 22 checklists were compiled into a comprehensive list. All items were grouped into three domains: 1) research team and reflexivity, 2) study design, and 3) data analysis and reporting. The overarching purpose of these studies was to gain a better understanding about the needs, experiences and perspectives of CKD patients and their caregivers. The findings describe the permanent, profound and pervasive impact of CKD on the lives of patients and caregivers across the whole illness trajectory. A more detailed and broader understanding about patient and caregiver perspectives, as presented in this thesis, can support a move towards advancing patient-centred healthcare and research in CKD.

Diss. (sammanfattning) Stockholm : Karol. inst., 2002. / Härtill 4 uppsatser.

Thesis (M.S.)--University of Missouri-Columbia, 2008. / The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Includes bibliographical references.