Bone marrow (BM)-derived mesenchymal stromal cells (MSCs) can be induced to express cardiac-specific markers by embryonic cardiomyocytes in vitro. To determine whether this phenomenon occurs in vivo, we have developed a cell transplantation system using zebrafish embryonic recipients. We were unable to isolate expandable zebrafish kidney stromal (ZKS) cells from the kidney, the human BM equivalent; hence, we analyzed the established ZKS1 cell line. We found that ZKS1 expresses stromal genes, but also expresses hematopoietic genes not normally expressed by MSCs. Furthermore, we were unable to differentiate ZKS1 cells into adipocytes, osteoblasts or cardiomyocytes in vitro. We created a transgenic ZKS1(CMV:eGFP) cell line which, after transplantation into zebrafish blastulae, was observed within the host heart, among other tissues. Finally, pT2/S2tnnt2-GM2 and pT2/S2tnnt2-DsRed transposons were generated to mark ZKS1 cardiac differentiation. The zebrafish model established here will be useful for studying the molecular mechanisms of exogenous MSC cardiac differentiation in vivo.

Using Ontario health administrative data, we identified 12,143 adults with chronic kidney disease (CKD) who received outpatient nephrology care prior to start of renal replacement therapy (RRT) in order to study the effect of care-related factors in predicting late start of predialysis care (PDC, defined as first outpatient nephrology visit <6 months prior to RRT start) and to explore covariates which further quantify the PDC received. Lack of an usual provider of primary care (OR 0.76; 95%CI 0.66, 0.87) predicted late start of PDC. In addition to late start of PDC, number of nephrology visits (OR 0.97 per visit; 95% CI 0.96, 0.98), and having seen a nephrologist in only 1 or 2 of the 6 months prior to RRT start (OR 1.33; 95%CI 1.18, 1.51), were also independent predictors of one-year mortality, suggesting that other measures of PDC are needed to better characterize the care received.

Loss of the renal microvasculature is a common feature of almost all forms of chronic kidney disease (CKD). Here we explored the role of the angiogenic chemokine stromal cell-derived factor-1-alpha (SDF-1) and its cognate receptor CXCR4 in experimental and human CKD. CXCR4 was present on endothelial cells and podocytes, while SDF-1 was detectable on podocytes, arteriolar smooth muscle cells, interstitial fibroblasts and occasional endothelial cells. CXCR4 mRNA was elevated in the kidneys of rats with CKD and chronic antagonism of CXCR4 accelerated renal decline and capillary loss. Acute SDF-1 infusion activated glomerular endothelial nitric oxide synthase (eNOS) in vivo, while functional response to SDF-1 was impaired in glomerular endothelial cells derived from eNOS-/- mice. Finally, CXCR4 mRNA was also found to be increased in biopsies of patients with secondary focal segmental glomerulosclerosis. These observations indicate that local eNOS-dependent SDF-1/CXCR4 signaling exerts a compensatory reno-protective effect in the setting of CKD.

I conducted an integrative field study on male cottonmouths (Agkistrodon piscivorus), a common pitviper of the southeastern United States, to investigate the evolution of contrasting mating patterns in North American pitvipers (bimodal and unimodal annual patterns) and resolve conflicting information about the pattern exhibited by the cottonmouth. I determined a unimodal late summer peak of testosterone (T) and a muted unimodal seasonal cycle of the sexual segment of the kidney (a secondary sexual characteristic), both of which were correlated with the single peak of spermatogenesis in late summer. I also conducted a study to determine diel and seasonal variation of corticosterone (CORT), the effect of captive handling on CORT, and the relationship between CORT and T after captive handling, and detected a significant elevation of CORT and a significant decrease of T after capture in male cottonmouths, as well as a significant negative correlation between CORT and T.

Chronic kidney disease (CKD) and its cardiac complications represent a large and growing problem in Canada. The progression of CKD is driven by the activation of several final common pathways of injury, including fibrosis and oxidative stress. If left unchecked, these inter-connected processes lead to progressive damage and subsequent organ dysfunction. Current clinical therapies, consisting of aggressive blood pressure control and blockade of the renin-angiotensin system, fail to arrest this progressive injury in a significant number of patients. Early outgrowth cells (EOCs) represent a novel bone marrow-derived cell population that have been recently described to have tissue protective activity. In this work, we examined the effects of intravascular EOC infusion in two independent models of CKD, demonstrating potent anti-fibrotic renoprotective effects in the subtotally nephrectomized (SNX) rat, a well-established model of non-diabetic progressive CKD, and anti-fibrotic and anti-oxidant effects in the db/db mouse, a commonly used model of type 2 diabetic nephropathy. In the SNX rat, which is characterized by impaired cardiac relaxation reminiscent of a common and high risk clinical CKD phenotype, EOC infusion was also associated with improved cardiac structure and function. In both cases, infused EOCs were not retained in significant numbers within the diseased kidney or heart, but rather localized to distant organs such as the liver, spleen, and bone marrow. We further demonstrated that EOCs release soluble factors with anti-oxidant and anti-fibrotic activity in vitro, and that a cell-free preparation of EOC-derived factors can mimic the reno- and cardiac protective effects of the cells themselves when infused into the SNX rat. Taken together, our results demonstrate the therapeutic potential of an EOC-based strategy for the treatment of CKD and its cardiac complications, and provide the preclinical rationale for the design of clinical trials of EOC-based therapies for this devastating disease.

Chronic kidney disease (CKD) and its cardiac complications represent a large and growing problem in Canada. The progression of CKD is driven by the activation of several final common pathways of injury, including fibrosis and oxidative stress. If left unchecked, these inter-connected processes lead to progressive damage and subsequent organ dysfunction. Current clinical therapies, consisting of aggressive blood pressure control and blockade of the renin-angiotensin system, fail to arrest this progressive injury in a significant number of patients. Early outgrowth cells (EOCs) represent a novel bone marrow-derived cell population that have been recently described to have tissue protective activity. In this work, we examined the effects of intravascular EOC infusion in two independent models of CKD, demonstrating potent anti-fibrotic renoprotective effects in the subtotally nephrectomized (SNX) rat, a well-established model of non-diabetic progressive CKD, and anti-fibrotic and anti-oxidant effects in the db/db mouse, a commonly used model of type 2 diabetic nephropathy. In the SNX rat, which is characterized by impaired cardiac relaxation reminiscent of a common and high risk clinical CKD phenotype, EOC infusion was also associated with improved cardiac structure and function. In both cases, infused EOCs were not retained in significant numbers within the diseased kidney or heart, but rather localized to distant organs such as the liver, spleen, and bone marrow. We further demonstrated that EOCs release soluble factors with anti-oxidant and anti-fibrotic activity in vitro, and that a cell-free preparation of EOC-derived factors can mimic the reno- and cardiac protective effects of the cells themselves when infused into the SNX rat. Taken together, our results demonstrate the therapeutic potential of an EOC-based strategy for the treatment of CKD and its cardiac complications, and provide the preclinical rationale for the design of clinical trials of EOC-based therapies for this devastating disease.

Relative changes are detectable in the blood of end-stage renal disease (ESRD) patients during hemodialysis (HD) treatment using optical spectroscopy. However, the potential impacts of several confounding factors that could affect the detection of these changes have not been evaluated. The objectives of this thesis were to: 1) investigate how the variations and/or changes in acid-base and oxygen parameters during HD treatment can affect the optical signature of whole blood of ESRD patients, 2) to investigate the effect of heparin on the optical properties of whole blood and its impact on our method. Blood samples were drawn from 23 ESRD patients at 5 time points during a 4 hour HD treatment and sent for blood gas and blood spectroscopy analyses. No significant correlations were found between the changes in the blood transmittance spectra and acid-base and oxygen parameters. This indicates that the perturbations in these parameters due to HD procedures do not confound the detection of changes in the blood transmittance spectra of ESRD patients during HD treatment. Additionally, the effect of heparin in modifying the optical properties of whole blood does not confound the detection of changes in the blood of ESRD patients due to HD treatment using whole blood-based optical spectroscopy. ANOVA revealed significant (P<0.05) measurable changes in the blood transmittance spectra of ESRD patients during HD treatment. Significant spectral differences (P<0.05) were found between ESRD patients. The lack of uniform spectral characteristics across patients is

Statement of the problem: The epithelial sodium channels (ENaC) play an important role in regulation of blood pressure (BP). Although the genes are identical in Dahl salt sensitive (S) and Dahl salt resistant (R) rats, expression of ENaC subunits is increased in kidneys of S rats on high salt diet. Intracerebroventricular (icv) infusion of ENaC blocker benzamil prevents Na+ induced hypertension. It was not known whether ENaC subunits are expressed in the brain and whether or not brain ENaC plays a role in regulation of [Na+] in CNS. Hypothesis: 1. Epithelial sodium channels are expressed in the brain. 2. Expression of ENaC is increased in the kidneys and brain of Dahl S rats on high salt diet. 3. ENaC in the brain contributes to regulation of [Na+] in the CSF and brain interstitium. Methods of investigation: We studied expression and distribution of the ENaC subunits and assessed the effects of icv infusion of Na+-rich aCSF in Wistar rats or high salt diet in Dahl S rats in different areas of the brain. Function of ENaC in the choroid plexus was evaluated by studying the effects of benzamil and ouabain on Na+ transport. Major findings: In Wistar rats, both mRNA and protein of all three ENaC subunits are expressed in brain epithelia and magnocellular neurons in the supraoptic (SON) and paraventricular (PVN) nucleus. ENaC abundance is higher on the apical versus basolateral membrane of choroid cells. Benzamil decreases Na+ influx into choroid cells by 20-30% and increases CSF [Na+] by ~8 mmol/L. Na+ rich aCSF increases apical membrane expression of βENaC in the choroid cells and of α and βENaC in basolateral membrane of ependymal cells, but has no effect on neuronal ENaC. Expression of ENaC is higher in choroid cells and SON of Dahl S versus R rats and the higher expression persists on a high salt diet. High salt attenuates the ouabain blockable efflux of Na+ from choroid cells and has no effect on CSF [Na+] in Dahl R rats. In contrast, high salt does not attenuate ouabain blockable efflux of 22Na+ and CSF [Na+] increases in Dahl S. Main Conclusion: ENaC in the brain contributes to Na+ transport into the choroid cells and appear to be involved in reabsorption of Na+ from the CSF. Aberrant regulation of Na+ transport and of Na+K+ATPase activity, might contribute to increases in CSF [Na+] in Dahl S rats on high-salt diet. ENaC in magnocellular neurons may contribute to enhanced secretion of mediators such as ‘ouabain’ leading to sympathetic hyperactivity in Dahl S rats.

Fluoxetine (FLX), the active ingredient of Prozac™, is a member of the selective serotonin reuptake inhibitor (SSRI) class of anti-depressant drugs and is present in aquatic environments worldwide. Previous studies reported that FLX is an endocrine disruptor in fish, bioconcentrating in tissues including the brain. Evidence implicates that serotonin influences the activity of the hypothalamo-pituitary-interrenal (HPI) stress axis, thus exposure to FLX may disrupt the teleost stress response. This study examined in vitro cortisol production in rainbow trout (Oncorhynchus mykiss) head kidney/interrenal cells exposed to FLX and 14C-pregnenolone metabolism in head kidney microsome preparations of FLX-exposed trout. Results indicated that cells exposed in vitro to increasing concentrations of FLX had lower cortisol production and cell viability (versus control) and microsomes isolated from trout exposed to 54 μg/L FLX had higher pregnenolone metabolism versus those of control and low FLX-exposed (0.54 μg/L) trout.

Cadmium is a highly toxic chemical and has been classified by the International Agency for Research on Cancer as a human carcinogen. Cadmium is abundant in the environment, at specific work places, and in food and water. Toxicological responses to cadmium exposure include respiratory diseases, neurological disorders and kidney damage. The present study examined the effects of cadmium on heat shock protein (HSP) accumulation in Xenopus laevis A6 kidney epithelial cells. HSPs are molecular chaperones involved in protein folding and translocation. In response to environmental stress these proteins bind to unfolded protein and inhibit their aggregation. Stress-inducible hsp gene transcription is mediated by the heat shock promoter element (HSE), which interacts with heat shock transcription factor (HSF). In the present study, hsp30 and hsp70 mRNA and protein were induced by heat shock, as determined by northern and western blot analysis. Exposure of A6 cells to cadmium chloride also induced the expression of hsp genes. For example, northern and western blot analysis revealed that exposure of A6 cells to cadmium chloride induced the accumulation of hsp30 and hsp70 mRNA and their respective proteins. Western blot analysis also revealed that A6 cells recovering from a cadmium chloride treatment retained relatively high levels of HSP30 and HSP70 protein accumulation over 24 h after the removal of the stress. Treatments combining a mild heat shock and cadmium chloride resulted in a synergistic increase in hsp30 and hsp70 gene expression at mRNA and protein levels. Further experiments in which two stressors were combined revealed that synergistic effects occurred with varying cadmium concentrations and different temperatures. Immunocytochemistry and confocal microscopy were used to confirm the results attained from western blot analysis. Further, this technique allowed the determination of intracellular localization of HSP30 in A6 cells and the examination of cellular morphology and cytoskeletal structure during cadmium chloride treatments. A 2 h heat shock at 33°C resulted in the accumulation of HSP30 in the cytoplasm, whereas a 2 h heat shock at 35°C resulted in some HSP30 accumulation in the peripheral region of the nucleus. This is in contrast to cells treated with cadmium chloride, where HSP30 accumulation was restricted to the cytoplasm. A 14 h 50 μM cadmium chloride treatment resulted in the accumulation of HSP30 in approximately 10% of cells. The proportion of cells displaying HSP30 accumulation increased to 80% and 95% in cells treated with 100 μM and 200 μM, respectively. HSP30 accumulation frequently occurred in large granular structures. High concentrations of cadmium chloride resulted in cell membrane ruffling at areas of cell-cell contact, as well as actin disorganization. This study characterized the pattern of hsp gene expression, accumulation and localization under various cadmium chloride conditions. These results suggest that hsp30 and hsp70 gene expression can be used as potential biomolecular markers for cadmium exposure.