Thesis (M.S.)--University of Wisconsin. School of Nursing, 1973. / eContent provider-neutral record in process. Description based on print version record.

Thesis (doctoral)--Lund University, 1997. / Added t.p. with thesis statement inserted.

Acute kidney injury (AKI), characterized by an abrupt loss of kidney function with retention of nitrogenous waste products, is common in the months to years following kidney transplantation and is associated with an increased risk of transplant failure (graft loss). Kidney transplant patients who experience graft loss and return to dialysis have an increased mortality risk and a lower quality of life. Research involving kidney transplant patients can prove challenging, as they are relatively small in number. To increase statistical power, researchers may utilize administrative databases. However, these databases are not designed primarily for research, and knowledge of their limitations is needed, as significant bias can occur. When using administrative databases to study AKI in kidney transplantation, the method used to define AKI should be carefully considered. The power of a study may be greatly increased if AKI can be accurately defined using administrative diagnostic codes because data on AKI will be universally available for all patients in the database. However, the methods by which diagnostic codes are assigned to a patient allow for error to be introduced. We confirmed that, when compared to the gold standard definition for AKI of a rise in serum creatinine, the diagnostic code for AKI has low sensitivity but high specificity in the kidney transplant population (the best performing coding algorithm had a sensitivity of 42.9% (95% CI 29.7, 56.8) and specificity of 89.3% (95% CI 86.2, 91.8) (Chapter 3). We therefore determined that for the study outlined in Chapter 4, defining AKI using diagnostic codes would significantly under-­capture AKI and misclassify patients. We decided to define AKI using only serum creatinine criteria even though this would limit our sample size (creatinine data was only available for a subset of patients in the administrative databases). In Chapter 4, we derived an index score to predict the risk of graft loss in kidney transplant patients following an admission to hospital with AKI. The index includes six readily available, objective clinical variables that increased the risk of graft loss: increasing age, increased severity of AKI (as defined by the AKIN staging system), failure to recover from AKI, lower baseline estimated glomerular filtration rate, increased time from kidney transplant to AKI admission, and deceased donor. The derived index requires validation in order to assess its utility in the clinical realm.

京都大学 / 0048 / 新制・論文博士 / 博士(医学) / 乙第13330号 / 論医博第2198号 / 新制||医||1044(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 長船 健二, 教授 羽賀 博典, 教授 山下 潤 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM

1. Little work has been done on the response of regular haemodialysis patients to dynamic exercise. A systematic study of exercise capacity and the underlying mechanisms is of particular importance because these patients are encouraged to return to as normal a way of life as possible. Accordingly, a select group of healthy young male patients and a group of older males have been studied during submaximal cycling. The young male patients were compared to a closely matched sedentary control group. The 17 subjects discussed represent the fittest of 40 patients tested. 2. In both groups there was decreased work capacity associated with disproportionate tachycardia, which was not obvious at rest. Blood pressure was measured with a sphygmomanometer. During exercise there was a striking rise in the systolic blood pressure in about half the patients from currently acceptable resting levels. This occurred in the absence of any clinical circulatory overload. Mild hyperventilation and disproportionate lactic acidosis was seen towards peak exercise, probably because, in spite of the decreased work capacity, the patients were much closer to their maximum performance. However, the limiting factors were clearly circulatory and not respiratory. 3. A number of the younger male patients were more intensively studied to determine why some remained relatively 'normotensive' during exercise while others developed systolic hypertension. Total blood volume, total body water and plasma renin activity were measured at rest. It was found that the 'normotensive' patients had normal body volumes and normal to high plasma renin activity, while the hypertensive subgroup had increased volumes and normal to low plasma renin activity. Thus, in these patients the blood pressure responses to exercise were largely volume dependent, albeit at a subclinical level. 4. Cardiac output was measured at rest and during exercise. All patients developed a variable hyperkinetic circulation during exercise which was not apparent at rest. The patients were all anaemic and (xi) their cardiac output response was very like that described in patients with anaemia unassociated with renal disease. However, some patients with striking anaemia developed a less hyperkinetic circulation than others who were not so anaemic. When the body volume and the blood pressure response on exercise were considered, those patients who were normovolaemic and 'normotensive' developed a hyperkinetic circulation on exercise appropriate to their degree of anaemia. Those with subclinical volume overload and a hypertensive response to exercise developed a much less striking hyperkinetic circulation, suggesting that the blood pressure and volume excess was depressing the anticipated cardiac output response to their underlying anaemia. 5. One patient with an arteriovenous shunt was studied twice, initially when hypervolaemic with a haemoglobin of 9,1gm/100 ml and again after ultrafiltration when he was normovolaemic but his haemoglobin had risen to 12,5 gm/100 ml. On the first occasion his cardiac output response was moderately hyperkinetic but he developed increasing hypertension with a high calculated total peripheral resistance. On the second occasion his cardiac output response fell within the normal range, his blood pressure was lower but not normal and his calculated total peripheral resistance was even higher than before. Thus, the blood pressure of these volume dependent patients is due to a high total peripheral resistance, but may not simply be on the basis of 'waterlogging' of the peripheral vasculature. Some other factor, such as structural thickening, must be considered. 6. It is suggested that the combination of tachycardia and hypertension which develop on mild exertion and which may not be obvious at rest, is the most potent cause of the increased cardiovascular mortality seen in dialysis patients. Simple exercise testing will reveal those with subclinical volume overload who are most at risk. It was striking that in the two groups tested those who developed striking hypertension on exercise were usually older, between 35 and 50 years. This accelerated aging of their vascular tree would correspond with recent data showing that dialysis mortality increases with age, and is about a decade earlier than in the general population. It is suggested that a more aggressive policy be adopted towards blood pressure fluctuations and that the resting blood pressure should be kept below 140/90 mm Hg at all times, if necessary by complementing ultrafiltration with drug therapy and/or bilateral nephrectomy at an early stage. 7. Thus simple exercise testing with blood pressure recordings not only serves as a yardstick of physical rehabilitation and long-term follow-up, but may also reveal or magnify abnormalities not obvious at rest.

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The proximal tubule of the renal cortical nephron is highly susceptible to intoxication by chemical agents. An in vitro system was developed to study directly the effects of nephrotoxic chemicals on this renal sub-organ fraction without the complication of extrarenal factors. Segments of proximal tubules were isolated by a mechanical method from the kidneys of young rabbits. Tubules obtained by this method retained biochemical, functional, and morphological features comparable to those existing in vivo. Preliminary acute susceptibility studies demonstrated that the isolated proximal tubule segments were sensitive to a variety of known nephrotoxic agents that target the proximal tubule. These agents include halogenated hydrocarbons, heavy metals, and a halogenated vinyl cysteine conjugate. Incubation conditions were optimized to maintain the viability of proximal tubule suspensions for up to four hours. Longer incubation times made it possible to establish a chronology of early tubule responses to chemical intoxication. Long term incubation of proximal tubule suspensions with two model nephrotoxins, cadmium chloride and S-(trans-1,2-dichlorovinyl)-L-cysteine, produced in vitro tubule response patterns similar to those reported in vivo for these agents. While not entirely representative of in vivo exposure conditions, suspensions of isolated proximal tubules are an easily obtained system that proved equally applicable as a screening technique for nephrotoxic compounds or as an in vitro system for delineating proximal tubule response to chemical insult.

11β-Hydroxysteroid Dehydrogenase Type 2 (11βHSD2) catalyses the inactivation of cortisol. In aldosterone target tissues co-expression of 11βHSD2 and mineralocorticoid receptors (MR) protects the receptor from activation by glucocorticoids. In the syndrome of Apparent Mineralocorticoid Excess, mutations in the HSD11B2 gene cause hypertension, which is thought to be driven by volume expansion secondary to sodium retention. 11βHSD2 mice are indeed hypertensive but paradoxically volume contracted, suggestive of a urine-concentrating defect. The current studies were designed to evaluate sodium and water homeostasis in 11βHSD2-/- mice. 11βHSD2-/- mice developed a severe and progressive polyuric-polydipsic phenotype. Despite basal polyuria, at <100 days 11βHSD2-/- mice had a functional concentration response when challenged with 24 hours water deprivation. At >180 days the exacerbated polyuria was associated with severe medullary injury in the null mice. Basal aquaporin 2 (AQP2) abundance was reduced in the 11βHSD2-/- mice at both <100 and >180 days. Moreover, vasopressin 2 receptor (V2R) stimulation failed to normalize the impaired response to water deprivation in >180 day null mice. Consequently, a renal origin to the polyuria was postulated. Indeed, mice in which 11βHSD2 had been selectively targeted in the brain had a normal water turnover. A key finding from these studies is that functional deletion of 11βHSD2 in the brain, specifically the nucleus of the solitary tract (NTS), resulted in an increased salt appetite. Moreover, the mice displayed a preference for 1.5% NaCl over water. Blockade of mineralocorticoid receptors (MR) significantly reduced NaCl intake. This is the first demonstration of an increased salt appetite in a model with normal renal function and in the absence of sodium depletion. These data implicate activation of MR on 11βHSD2 positive neurons in the NTS in the behavioural drive to consume sodium.

Exosomes are small lipid membrane bound vesicles formed as part of the endosomal pathway and released into the extracellular space following fusion of late endosomes with the plasma membrane. Exosomes have been shown to have a variety of biological roles and may represent a novel source of disease biomarkers. The objectives of this project were to develop a panel of techniques for identifying exosomes in human urine then establish an in vitro model to determine whether exosomes change with cellular activation. We then used the techniques developed with human urine to determine whether human cerebrospinal fluid (CSF) contains exosomes and applied a mass spectrometry based approach to characterise the exosomal proteome. We used western blot for three exosomal markers (tsg101, CD24 and flotillin- 1), isopycnic centrifugation on a sucrose density gradient and direct visualisation using transmission electron microscopy (TEM) to verify the presence of exosomes. Using GeLC-MS/MS, 88 proteins were identified in the urinary exosomes. Several of these proteins could be linked to diseases and specific sections of the nephron. A murine cortical collecting duct cell line was used to model exosome release into the urine. Firstly, exosome release was verified using the approach developed in the urine. Stimulation of the cells with desmopressin caused an increase in the presence of aquaporin 2 in the exosomes. This increase reflected a similar change in the cells and occurred over a similar time course. This supports the hypothesis that the exosomes reflect the state of the kidney cells. In contrast, stimulation with cisplatin did not alter the presence of Fetuin-A, a proposed biomarker of cisplatin-induced acute kidney injury, in exosomes and this was consistent with no change in Fetuin- A expression in the cells. The released exosomes may act as mediators of communication to other cells. Following incubation of mCCD cells with AQP2 containing exosomes AQP2 in the cell lysate was increased indicating interaction between the cells and exosomes and potentially internalisation. Exosomes have been shown to be released by neuronal cells in vitro. We identified exosomes in the CSF of humans using western blot for known exosomal markers, density determination and direct visualisation with TEM and Immuno-TEM using an antibody specific for the exosomal marker flotillin-1. Label-free quantitative mass spectrometry was used to compare multiple CSF samples. On a whole protein analysis 86% of the proteins identified varied by less than 2-fold in comparison to the average across samples. On a tryptic peptide analysis 75% of the peptides identified varied by less than 2-fold in comparison with the average across samples. We have demonstrated exosomes are present in urine, CSF and mCCD cell conditioned media. In the mCCD cell derived exosomes we have demonstrated that following stimulation the proteome of the exosomes changes and that this change reflects the change seen in the cells. For the urinary and CSF exosomes we have characterised their proteomes using GeLC-MS/MS. These findings are consistent with the hypothesis that exosomes are a rich source of information, including biomarkers, on their cells of origin.