This thesis aims to investigate if the endothelial protein C receptor is expressed in the murine kidney. This was done by performing flow cytometry and Western blot analysis on cultivated murine kidney endothelial cells (mKECs) as well as SDS-PAGE and Western blot analysis on murine kidney tissue. Flow cytometry was also performed on cultivated ARPE19 and 4T1 cells for comparison. It was discovered that ≥95,5% of the mKECs, ≥93,6% of the ARPE19 cells and ≥60,9% of the 4T1 cells express the receptor according to the flow cytometry data. A dot blot was performed to validate the primary antibody used for detection of EPCR in Western blot and SDS-PAGE. According to the dot blot, the primary antibody can be visualised in the dilution range from 1:2000 to 1:10. The dot blot also showed that the secondary antibody binds specifically to the primary antibody. Yet, Western blot analysis did not detect the receptor neither in mKECs nor tissue lysate. This was likely due to the fact that the primary antibody used did not bind specifically to the receptor, and may not be applicable for this method. SDS-PAGE did not show any indication that the receptor was present in the kidney tissue. In conclusion, it was discovered that the EPCR was expressed in the murine kidneys endothelial cells through flow cytometry, but the presented methods for Western blot and SDS-PAGE could not confirm the expression of the receptor.

<p>We defined hypertension in pregnancy as a composite of gestational hypertension, preeclampsia and eclampsia. The etiology of hypertension in pregnancy remains controversial. The three chapters of this thesis explore the risk of hypertension in pregnancy from various kidney conditions. Chapter 1 introduces the reader to the thesis. Chapter 2 is a systematic review that studied the risk of developing hypertension in pregnant women with chronic kidney disease but not on dialysis. We found that women with chronic kidney disease had at least a twofold higher relative risk of developing hypertension during pregnancy compared with women having no chronic kidney disease. Chapter 3 is a retrospective study looking at the risk of developing gestational hypertension and preeclampsia in women who had symptomatic gastroenteritis after drinking water infected with <em>E. coli</em> O157:H7 during the Walkerton outbreak in May 2000. We conducted this study using linked datasets at the Institute of Evaluative Sciences (ICES) Toronto, Ontario. We observed that there was no increased risk of developing gestational hypertension or preeclampsia among the symptomatic women compared with women from the neighbouring towns who were asymptomatic or did not drink the water. Chapter 4 is a protocol of a prospective cohort study recruiting female kidney donors and healthy non-donors as the comparative group to study pregnancy outcomes in these individuals. This is a multicentre study involving 12 transplant centres throughout Canada. There are 59 participants in this study to date (Feb 28, 2013) of which seven have been pregnant so far. Data collection for this study is ongoing.</p> / Doctor of Philosophy (PhD)

Master of Science / Department of Clinical Sciences / Gregory F. Grauer / Chronic kidney disease (CKD) is a common cause of morbidity and mortality in cats. The purpose of this study was to investigate the effects of Chinese rhubarb (Rheum officinale) supplementation on the progression of feline CKD. Cats with stable IRIS stage II or III CKD and without certain comorbidities were included in the study. Cats were randomly divided into 3 treatment groups and administered Chinese rhubarb extract (Group 1, Rubenal®, Vetoquinol, Forth Worth, TX; 75 mg tablet by mouth every 12 h), benazepril as a positive control (Group 2, 0.5 mg/kg by mouth every 24 h), or both (Group 3). Cats were fed a commercial renal specific diet and enteric phosphate binder as appropriate. Body weight, laboratory data, and blood pressure were recorded every 3 months. Variables between groups at enrollment and within groups over visits were compared with ANOVA and repeated measures ANOVA, respectively. A treatment by visit interaction term was included in all repeated measures models. Significance was set at p ≤ 0.05. Except for body weight there was no significant differences between treatment groups at enrollment. There was no significant change in body weight, hematocrit (Hct), UPC, serum creatinine, or systemic blood pressure over time as compared to baseline within any group. There was no significant difference between groups over time in regards to change in body weight, Hct, UPC, serum creatinine, or systemic blood pressure. The treatment by time interaction was non-significant in all models. Based on easily measured clinical parameters, this study failed to detect a significant difference in cats administered a Chinese rhubarb supplement, benazepril, or both.

Call number: LD2668 .T4 1964 B57 / Master of Science

The adult kidney consists of hundreds of thousands of fine epithelial tubules as functional units called nephrons. Nephrons have U-shaped tubules: loops of Henle that descend from the cortex to the medulla. This radial arrangement is critical to maintain water homeostasis in the kidney. Although Henle’s loops are crucial to renal physiology, the cue(s) they uses to navigate to the medulla are not understood. In this thesis, I investigate how the loop of Henle elongates during mouse renal development and show that it is probably guided to the medulla by diffusible, heparin-binding molecules. I used immumohistochemistry (IHC) on cryosections of embryonic kidneys to study the natural anatomy of the Henle’s loop. I used a low-volume culture system to allow embryonic kidneys (both natural and tissue-engineered) to form loops of Henle ex vivo and manipulated their direction of growth. Time-lapse imaging of Lgr-5 EGFP embryonic kidneys demonstrated the movement of the apex of the loop which suggested the idea of guidance cue(s) acting on the loop of Henle. Cut-and-paste experiments showed that loops appeared to be attracted to maturing collecting duct. Co-culture with an exogenous tubule inducer suggested the embryonic spinal cord as another source to attract the loops. Using raTAL (rat thick ascending loop of Henle) and 6TA2 (embryonic collecting duct cells) cell lines, I designed and performed a cell migration assay to test whether raTAL was attracted to 6TA2 cells. raTAL cells were notably attracted to 6TA2 cells compared to other cell lines. raTAL cells were also attracted to 6TA2-conditioned medium, which indicated that raTAL cells were attracted by secreted molecule(s). To begin to characterise those secreted molecule(s), heparin-binding protein-coated beads were used in the cell migration system and showed that at least one critical guidance factor is heparin-binding. From this study, I found that the apex of the Henle’s loop does move and loops are attracted by secreted molecule(s) possibly from the collecting duct. Although target molecule(s) were unidentified, this study provides the first mechanistic information about the guidance of the loop of Henle. Moreover, this was the first study of guidance of epithelial tubule shafts (rather than tips) adding to our understanding of general tubule morphogenesis.

Ischaemia/Reperfusion Injury (IRI) is the most common cause of acute kidney injury- a devastating clinical problem lacking any specific treatments to promote renal recovery. Macrophages (Mφ) are pleiotropic cells of the innate immune system, with roles spanning host defence, cytotoxicity, clearance of apoptotic cells and promotion of tissue repair. Mφ are also known to be important mediators of renal injury in other experimental models of renal disease including transplantation, obstruction and glomerulonephritis. This work sought to examine the role of Mφ in mediating renal IRI. Conditional renal Mφ and monocyte depletion prior to experimental IRI was achieved by administering diphtheria toxin to the CD11b-DTR transgenic animal. This had no impact on either renal function or structural injury. In contrast liposomal clodronate mediated Mφ depletion provided functional and structural protection from injury. Administration of exogenous apoptotic cells also protected renal function if delivered 24h prior to IRI. Immunodeficient SCID mice exhibited a protected injury phenotype after IRI, however derived no additional protection from the administration of either liposomal clodronate or i.v. apoptotic cells. These findings suggest that the protective phenotype must involve either lymphocyte populations or circulating antibody. Preliminary work demonstrates that SCID mice lack IgM natural antibody which deposits in the kidney in the first 30 minutes after IRI. It was also demonstrated that apoptotic cells bind IgM natural antibody present within the circulation. The potential for the key antioxidant enzyme Heme oxygenase-1 (HO-1) to protect renal function was also examined in aged mice using hemearginate (HA) - a potent HO-1 inducer. Echoing epidemiological studies in humans aged mice had increased susceptibility to IRI, whilst failing to induce medullary HO-1. The main site of medullary HO-1 induction by HA was in medullary Mφ, and the protective phenotype was abolished by Mφ ablation, implicating Mφ as the key mediators of HA induced protection in renal IRI. Final studies employed adenoviral transduction to overexpress HO-1 within bone marrow derived Mφ, leading to a modified phenotype with increased IL- 10 and phagocytosis, and reduced TNFα and NO production. When these were introduced in vivo after IRI renal function was improved, potentially due to accelerated clearance of renal platelet deposition.

Cette thèse vise à démontrer l’importance de la PI 3-kinase dans la régulation hormonale de la réabsorption rénale du sodium. Ce contrôle extrêmement précis, notamment par l’aldostérone, s’effectue au niveau du néphron distal. Nous avons utilisé comme modèle l’épithélium de cellules A6, dérivées du tubule distal de Xenopus Laevis. Le transport unidirectionnel de sodium s’effectue en deux étapes: depuis, l’entrée à partir du milieu luminal par des canaux sodiques épithéliaux (ENaCs) insérés dans la membrane apicale, jusqu’à la sortie vers le liquide extracellulaire par des pompes Na+/K+-ATPases, situées dans la membrane basolatérale. L’insuline augmente ce transport de sodium et la PI 3-kinase semble assurer un rôle-clef. Nous avons étudié l’importance de chacun des 3-phosphoinositides produits par la PI 3-kinase, sur le transport du sodium en ajoutant au milieu cellulaire des formes «perméantes» de ces phospholipides. Parmi ceux-ci, le PIP3 et dans une moindre intensité le PI(3,4)P2 augmentent ce transport. En revanche, le PI3P, le PI(3,5)P2, ainsi que le PI(4,5)P2 n’ont pas d’effet sur lui. Nous avons démontré par la technique du Western blot que la 3-phosphatase PTEN est exprimée dans les cellules A6. Cette phosphatase déphosphoryle le PIP3 en PI(4,5)P2. Nous avons surexprimé PTEN dans les cellules A6. Ceci réduit l’augmentation du transport du sodium induite par l’insuline, ainsi que celle induite par addition de la forme «perméante» de PIP3. Nous avons ensuite vérifié si d’autres agents qui activent la PI 3-kinase, stimulent également le transport de sodium à travers cet épithélium. A cette fin, nous avons d’abord vérifié que l’EGF et le peroxyde d’hydrogène, connus pour stimuler la PI 3-kinase dans d’autres systèmes, activent également cette enzyme dans les cellules A6. Tous deux augmentent ce transport. L’importance de l’augmentation induite par H2O2 est comparable à celle de l’insuline, tandis que l’effet de l’EGF est plus transitoire. Un dosage d’activité de la PI 3-kinase, nous a permis de démontrer que l’intensité de l’activation de la PI 3-kinase est corrélée avec l’amplitude de l’augmentation du transport du sodium. Par comparaison avec l’effet de l’insuline et de l’H2O2, l’EGF augmente faiblement l’activité de la PI 3-kinase et induit une faible augmentation du transport. Nous avons également examiné si la voie des MAPK influence la stimulation du transport du sodium par ces différents agents. Cette voie ne semble pas impliquée dans l’effet de l’insuline ou du peroxyde d’hydrogène. Par contre, elle diminue la stimulation du transport de sodium par l’EGF. L’effet de l’EGF sur le transport semble résulter d’un compromis entre l’activation de la voie de la PI 3-kinase qui l’augmente et l’activation de la voie des MAPK qui le diminue. En conclusion, une augmentation de PIP3, soit par addition de PIP3 exogène, soit par augmentation endogène sous l’effet de l’insuline ou d’autres agents stimulant la PI 3-kinase, augmente le transport du sodium tandis qu’une diminution de PIP3 endogène (par surexpression de PTEN) le diminue. L’importance de l’activation de la PI 3-kinase est quantitativement corrélée avec l’importance de l’augmentation du transport du sodium. La PI 3-kinase est donc un médiateur-clef de la régulation rénale de ce transport.

Haloalkenes were shown to react with cysteine, N-acetyl cysteine, or glutathione to form halogenated vinylthio (HVT) or saturated conjugates. When HVT were administered iv to rabbits, active transport in the renal tubules was inhibited 50% at doses as low as 20 mg/kg within 1 hr after dosing. There was sloughing of the renal brush border membrane with the injury progressing to a specific renal tubular necrosis of the S₃ segment. In vitro studies with renal tubules found that the HVT produced a dose-response related inhibition of acid/base transport, with complete inhibition of transport occurring at 1 mM. The cysteine conjugate synthesized from trichloroethylene, DCVC, inhibited tubular active transport 60 min after in vivo dosing (20-100 mg/kg), 45 min after exposure in the isolated perfused kidney (0.01-1 mM) and 15 min after incubation with isolated tubules (0.01-1 mM). All HVT conjugates had a similar potency with regard to transport inhibition in isolated tubules, with complete inhibition occurring at 1 mM within 15 min for cysteine conjugates compared to 45-60 min for the N-acetyl cysteine or glutathione conjugates. These latter conjugates are thought to be bioactivated to the cysteine conjugate prior to transport inhibition. Inhibition of tubular (gamma)-glutamyl transpeptidase by 1 mM AT-125 or 20 mM serine/borate prevented the inhibition of acid/base transport by the glutathione conjugate. In addition, the sequential formation of glutamate, glycine and the vinyl cysteine conjugate after tubule incubation with the glutathione conjugate provides further evidence for this bioactivation. The cysteine conjugates are thought to be further metabolized in tubules to a toxic intermediate by a brush border localized enzyme, C-S lyase. The inhibitor effect of this intermediate on acid/base transport is reversed in the presence of, or subsequent addition of, 6 mM exogenous glutathione. These studies provide further evidence for the nephrotoxicity of HVT, and formation of the nephrotoxic cysteine conjugates from glutathione and cysteine conjugates. The formation of saturated conjugates from CTFE was also demonstrated. These saturated and/or unsaturated conjugates may be responsible for haloalkene-induced nephrotoxicity.

No description available.

Muscle wasting and increased proteolysis is a major problem in chronic kidney disease (CKD). Exercise is potentially beneficial, but has been under-investigated in pre-dialysis CKD and could theoretically worsen acidosis through exercise-induced lactic acid generation. We therefore investigated effects of 6 months walking exercise with and without additional alkali therapy. 40 patients were recruited (23 male and 17 female, median age 58, range 20-83, mean eGFR±SEM 25.7±1.2ml/min/1.73m2). 20 undertook walking exercise at a Borg Rating of Perceived Exertion Rate (RPE) of 12-14 for at least 30 minutes, 5 times a week. The other 20 continued with normal physical activity (non-exercising controls). In addition to standard oral bicarbonate therapy (STD), 10 patients in each group were randomised to receive additional bicarbonate (XS). Blood and vastus lateralis muscle biopsies were drawn at baseline, one and six months. 18 exercisers (including 8 in XS group) and 14 controls (6 in XS group) completed the 6 month study. Exercise tolerance increased after 1 and 6 months in the exercisers, but not the controls, accompanied by a reduced acute lactate response in the XS, but not the STD exercising group. After 6 months of exercise, 9 intramuscular free amino acids showed striking depletion in the STD, but not XS bicarbonate group. This suggests an inhibition of active amino acid transporters, possibly the SNAT2 transporters that are inhibited by acidosis. Studies with cultured myotubes identified glucocorticoid as a possible mediator of acid s inhibitory effect on SNAT2. The preservation of amino acid concentrations in the XS exercising group was accompanied by strong suppression of ubiquitin E3-ligases MuRF-1 and MAFbx which activate proteolysis through the ubiquitin-proteasome pathway. However, other anabolic indicators (Protein Kinase B activation and suppression of the 14kDa actin fragment) were unaffected in the exercising XS group. Possibly because of this, overall suppression of myofibrillar proteolysis (3-methyl histidine excretion) and increased lean body mass (DEXA) were not observed in the exercising patients. As XS alkali had no effect in non-exercisers, it is concluded that alkali effects in the exercisers arose by countering exercise-induced acidosis. Sulphuric acid produced from the catabolism of sulphur-containing amino acids ingested in the diet is the main contributor to the daily titratable acid load and hence acidosis in CKD. In these patients the amount of sulphate excreted in urine over 24h varied widely between individuals. This directly correlated with 3-methyl histidine excretion suggesting that sulphate excretion may be a better clinical indicator of acidotic patients at long-term risk of cachexia than conventional measures such as venous bicarbonate. Studies with cultured myotubes confirmed that skeletal muscle is a source of sulphuric acid and showed that production of this acid is partly suppressed by L-Glutamine a potential novel way to control acidosis in CKD.