Kidney disease remains a public health problem worldwide, where there is an increase in the number of incidence of Acute Kidney Injury (AKI) in hospitalized patients. The AKI is characterized by an abrupt decrease in renal function, resulting in the inability of the kidneys to perform itÂs basics functions. The Ischemia/Reperfusion (I/R) injury can be defined as all changes resulting from the deprivation and the re-establishment of the oxygen supply to tissues and organs. It is a complex process that results in the production of reactive oxygen species (ROS) and oxidative stress damage. The Brazilian Red Propolis is a complex mixture collected and produced by Apis mellifera bees, and seems to be promising in this context, attenuating the oxidative and nephrotoxic effect in the kidney. This experimental study aims to replicate and standardize a framework of AKI by Ischemia/reperfusion (I/R) in rats, and analyze the possible protective renal effects of Red Propolis Alcohol Extract (RPAE) at a dose of 150mg/kg on markers and pathological variables. Wistar rats, adult males, were divided into 4 groups, using an induction and treatment regimen. We evaluated biochemical parameters indicative of renal function (creatinine, urea, creatinine clearance) tubular function (FENa+ and FEK+), oxidative profile through MDA (malondialdehyde) and activity of antioxidant enzyme GSH, in addition to histological analysis and immunohistochemistry. The RPAE significantly altered almost all parameters investigated. The results demonstrated the protective effect of the extract in the dose used against the nephrotoxicity: decreased serum levels of creatinine (1.8  0.5 vs 2,7Â0,9) and urea (181.1  65.6 vs 274,3Â91,81), reversed the increase in FENa+ (0,58Â0,30 vs 1,03Â0,39) and FEK+ (64,74Â52,44 vs 134,4Â54,94), reversed the decrease of creatinine clearance (0,41Â0,14 vs 0,073Â0,048), decreased MDA levels (90,22Â20,82 vs 133,9Â23,36) and increased GSH (1784Â297,4 vs 1267Â229,5), decreased the rate of acute tubular necrosis (2,0Â0,7 vs 3,6Â0,5), increased the expression of eNOS (2,20,4 vs 0,60,5) and Heme-oxygenase (2,60,5 vs 1,40,5). Therefore, occurred protection of renal function, protection of the tubular damage and oxidative stress. These results describe for the first time the effect of Red Propolis on a model of AKI induced by I/R, suggesting the protective effect of the extract against this type of injury. / As doenÃas renais apresentam um problema de saÃde pÃblica mundial, aonde hà um aumento nos nÃmeros de incidÃncia de LesÃo Renal Aguda (LRA) em pacientes hospitalizados. A LRA caracteriza-se por uma reduÃÃo abrupta da funÃÃo renal, resultando na incapacidade dos rins em exercer suas funÃÃes bÃsicas. A lesÃo por isquemia/reperfusÃo (I/R) pode ser definida como as alteraÃÃes resultantes da privaÃÃo seguida do re-estabelecimento do fornecimento de oxigÃnio para tecidos e ÃrgÃos. à um processo complexo, que resulta na produÃÃo espÃcies reativas de oxigÃnio (EROs) e dano por estresse oxidativo. A PrÃpolis Vermelha brasileira à uma mistura complexa coletada e produzida pelas abelhas Apis mellifera, e parece ser promissor nesse contexto, atenuando assim o efeito oxidativo e nefrotÃxico no rim. Este estudo experimental tem como objetivo reproduzir e padronizar um quadro de LRA por Isquemia/ReperfusÃo (I/R) em ratos, e analisar os possÃveis efeitos protetores renais do Extrato AlcoÃlico de PrÃpolis Vermelha (EAPV) na dose de 150mg/kg, sobre os marcadores e variÃveis desse quadro patolÃgico. Foram utilizados ratos Wistar, adultos machos, divididos em 4 grupos, utilizado um esquema de induÃÃo e de tratamento. Foram avaliados os parÃmetros bioquÃmicos indicativos da funÃÃo renal (creatinina, ureia, Clearence de creatinina) funÃÃo tubular (FENa+ e FEK+), o perfil oxidativo atravÃs do MDA (Malonaldeido) e atividade da enzima antioxidante GSH, alÃm da anÃlise histolÃgica e imunohistoquÃmica. O EAPV alterou significativamente quase todos os parÃmetros investigados. Os resultados demonstraram efeito protetor do extrato na dose de utilizada diante dos parÃmetros de nefrotoxicidade: diminuiu os nÃveis sÃricos creatinina (1,8Â0,5 vs 2,7Â0,9) e urÃia (181,1Â65,6 vs 274,3Â91,81), reverteu o aumento da FENa+ (0,58Â0,30 vs 1,03Â0,39) e da FEK+(64,74Â52,44 vs 134,4Â54,94), reverteu a diminuiÃÃo do clearence de creatinina (0,41Â0,14 vs 0,073Â0,048), diminuiu os nÃveis de MDA (90,22Â20,82 vs 133,9Â23,36) e aumentou o de GSH (1784Â297,4 vs 1267Â229,5), diminuiu o Ãndice de necrose tubular aguda (2,0Â0,7 vs 3,6Â0,5) a aumentou a expressÃo de eNOS (2,20,4 vs 0,60,5) e Heme-oxigenase (2,60,5 vs 1,40,5). Portanto, ocorreu proteÃÃo da funÃÃo renal, do dano tubular e do estresse oxidativo. Estes resultados relatam pela primeira vez o efeito da PrÃpolis Vermelha sobre um modelo de LRA induzido por I/R, demonstrando o efeito protetor do extrato na LRA.

FundaÃÃo Cearense de Apoio ao Desenvolvimento Cientifico e TecnolÃgico / As infecÃÃes relacionadas à assistÃncia à saÃde estÃo entre as principais complicaÃÃes em pacientes que se submetem a transplante de rim, em virtude, nÃo somente, dos regimes de imunossupressÃo a que estÃo submetidos, como a cuidados desempenhados pelas equipes de saÃde. O objetivo deste estudo foi analisar infecÃÃes relacionadas à assistÃncia à saÃde em pacientes transplantados renais em 2012, no MunicÃpio de Fortaleza, que possui serviÃos de transplante renal consolidados. Fez-se um estudo transversal onde foram analisadas as fichas ambulatoriais, prontuÃrios e fichas de notificaÃÃo e investigaÃÃo da CCIH de pacientes que realizaram transplante renal em 2012, no HUWC e HGF, instituiÃÃes com recordes sucessivos neste tipo de procedimento. Um total de 237 participantes, sendo, 101 (mÃdias de idade: 43,2 anos) pertencentes ao HUWC e 136 (mÃdias de idade: 45,4 anos) ao HGF, foi incluÃdo no estudo. Em ambas as instituiÃÃes, a maioria das pessoas era do sexo masculino, casada e residia na Capital do CearÃ. Grande parte delas tambÃm tinha o IMC normal, era hipertensa, tinha como principais causas de IRC: inderterminada, HAS e nefrites. A proporÃÃo de procedimentos invasivos realizados foi: biopsia do enxerto (HUWC: 45,54%; HGF: 26,47%), punÃÃo de cateter venoso central (HUWC: 98,01%; HGF: 97,06%), FAV (HUWC: 66,33%; HGF: 94,11%), e passagem de cateter duplo J (HUWC: 39,6%; HGF: 22,06%). A estimativa de prevalÃncia de IRAS nos 101 pacientes transplantados renais no HUWC foi de 50 (49,05%), e no HGF, 31 (22,79%). As IRAS mais comuns, tanto no HUWC, como no HGF, foram infecÃÃes do trato urinÃrio, e os principais agentes etiolÃgicos isolados, Klebsiella pneumoniae e Escherichia coli. No HUWC, os fatores sociodemogrÃficos, clÃnicos e epidemiolÃgicos que apresentaram associaÃÃo estatisticamente significante com o acometimento por IRAS foram: a classificaÃÃo do IMC (p<0,03), o tempo em diÃlise antes do transplante (p<0,05), o tempo de internaÃÃo total (p<0,0001), o tempo de cirurgia (p<0,001), o tempo de isquemia fria (p<0,01), a passagem de cateter duplo J (p<0,003), o tempo de uso do TOT (p<0,03) e o tempo de uso da SVD (p<0,04) no HUWC; no HGF, a classificaÃÃo do IMC (p<0,04), o LES como causa de IRC (p<0,01), a transfusÃo sanguÃnea antes do transplante (p<0,02), o tempo de internaÃÃo total (p<0,001) e o tempo de uso do CVC (p<0,04). Portanto, durante todo o perÃodo perioperatÃrio, hà necessidade de desenvolvimento de aÃÃes cautelosas por toda a equipe de saÃde, de forma a prevenir infecÃÃes e gastos desnecessÃrios do governo com internamentos e tratamentos prolongados, sustentando o crescimento da tÃcnica de transplantaÃÃo renal no Estado nos Ãltimos anos. / The healthcare-related infections are among the major complications in patients who undergo kidney transplant, by virtue not only of immunosuppression schemes to which they are subjected, as the care carried out by health teams. The aim of this study was to analyze healthcare-related infections in renal transplant recipients in 2012, in the city of Fortaleza, which has consolidated renal transplant services. A cross-sectional study where outpatient records were analyzed, charts and tokens for notification and investigation of patients who performed CCIH kidney transplant in 2012, in HUWC and HGF, institutions with successive records in this type of procedure. A total of 237 attendees, being, 101 (average age: 43.2 years) belonging to the HUWC and 136 (average age: 45.4 years) to the HGF, was included in the study. In both institutions, most people were male, married and resided in the Capital of CearÃ. Most of them also had the normal BMI was hypertensive, had as main causes of IRC: inderterminada, SAH and lupus nephritis. The proportion of invasive procedures performed were: graft biopsy (HUWC: 45.54%; HGF: 26.47%), central venous catheter puncture (HUWC: 98.01%; HGF: 97.06%), FAV (HUWC: 66.33%; HGF: 94.11%), and passage of double-J catheter (HUWC: 39.6%; HGF: 22.06%). The estimated prevalence of IRAS in 101 renal transplant recipients at HUWC was 50 (49.05%), and HGF, 31 (22.79%). The most common IRAS, both in HUWC, as in HGF, were urinary tract infections, and the main etiological agents isolated Klebsiella pneumoniae and Escherichia coli. In HUWC, the socio-demographic factors, clinicians and epidemiologists who presented statistically significant association with involvement by IRAS were: the classification of BMI (p<0.03) time on dialysis before transplantation (p<0.05), the total length of stay (p<0.0001), the time of surgery (p<0.001), cold ischemia time (p<0.01), the passage of double-J catheter (p<0.003), the speaking time of TOT (p<0.03) and time of use of the SVD (p<0.04) in HUWC; on HGF, classification of BMI (p<0.04), LES as a cause of IRC (p<0.01), blood transfusion before transplantation (p<0.02), the total length of stay (p<0.001) and time of use of the CVC (p<0.04). Therefore, throughout the perioperative period, there is a need for development of cautious actions throughout the health team, in order to prevent infections and unnecessary government spending with hospitalizations and prolonged treatments, supporting the growth of renal transplantation technique in the State in recent years.

Orientadores: Adriano Fregonesi, André Deeke Sasse / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-16T02:08:25Z (GMT). No. of bitstreams: 1 Borges_ClaudioFerreira_M.pdf: 4164710 bytes, checksum: 9f518f9bfaf2ebc75a3dd5241d26cdcf (MD5) Previous issue date: 2010 / Resumo: Propósito: Comparar a segurança e efetividade da realização de nefrolitotripsia percutânea (NPC) com e sem a inserção de nefrostomia, através de revisão sistemática e metanálise de estudos Materiais e métodos: Uma revisão sistemática da literatura foi realizada. Foi feita busca nos bancos de dados PUBMED, EMBASE, LILACS e Cochrane para identificação de estudos clínicos prospectivos randomizados que compararam a NPC com e sem inserção de nefrostomia. Os desfechos avaliados foram os índices de remoção total de cálculos, dor pós-operatória, necessidades de analgésicos, tempo cirúrgico, tempo de internação, perda de sangue e complicações. Resultados: Um total de dez estudos clínicos prospectivos randomizados (ECR) foram identificados somando 621 pacientes. Sete estudos analisaram os índices de remoção total de cálculos. A metanálise dos dados resultou em ausência de diferença entre os grupos de NPC sem nefrostomia e convencional. Quando avaliados o tempo cirúrgico, transfusão sanguínea, queda de hemoglobina e febre pós-operatória não houve diferença entre os grupos. A metanálise do tempo de internação hospitalar e da drenagem prolongada de urina pela região lombar favoreceu o grupo de NPC sem o uso de nefrostomia. Conclusão: Em pacientes selecionados, a NPC sem nefrostomia é um procedimento seguro e eficaz com taxas de remoção total de cálculo comparáveis a NPC convencional. A NPC sem nefrostomia apresentou um menor tempo de internação hospitalar e menos casos de drenagem prolongada de urina. Não foi possível realização de metanálise na avaliação da redução da dor pós-operatória e minimização das necessidades analgésicas. Entretanto, a maioria do estudos avaliados apresentaram benefícios nestes parâmetros para o grupo de NPC sem nefrostomia / Abstract: Purpose: We performed a systemic review with meta-analysis to compare tubeless versus conventional percutaneous nephrolithotripsy and assess the effectiveness and safety of this innovative procedure. Material and Methods: A systematic review of PUBMED, EMBASE, LILACS and Cochrane Library was done to identify all randomized controlled trials comparing tubeless PCNL versus conventional PCNL. The outcomes analyzed were stone free rate, pain assessment, analgesic medication requirements, operative time, hospitalization time, blood loss, stone-free rates and complications. Results: A total of 10 RCT were identified reporting 621 patients. Seven studies analyzed stone free rates. Meta-analysis of the data resulted in no difference between tubeless and conventional PCNL. Operative time, blood transfusion, hemoglobin drop and postoperative fever did not differ between the groups. Meta-analysis of length of hospitalization and prolonged urinary drainage was analyzed and favoured the tubeless PCNL group. Conclusions: Tubeless PCNL is a safe and effective procedure with a stone free rate compared to conventional PCNL. Tubeless PCNL presented a shorter hospital stay and less postoperative urinary leakage. Pain reduction and minimization of analgesic requirements also were demonstrated / Mestrado / Cirurgia / Mestre em Cirurgia

FundaÃÃo Cearense de Apoio ao Desenvolvimento Cientifico e TecnolÃgico / FundaÃÃo de Amparo à Pesquisa do Estado do Cearà / PeÃonhas de Hymenoptera, particularmente das famÃlias Apidae (abelhas) e Vespidae (vespas), sÃo uma rica fonte de peptÃdeos e proteÃnas com atividades biolÃgicas. A espÃcie Polybia Paulista à uma vespa social neotropical muito agressiva e endÃmica no Sudeste do Brasil. O objetivo deste trabalho foi estudar os efeitos renais e vasculares induzidos pelo veneno da Polybia paulista (VPp). Foram utilizados ratos (n = 4) machos Wistar pesando entre 250 e 300g, cujos rins foram isolados e perfundidos com SoluÃÃo de Krebs-Henseleit modificada contendo 6g% de albumina bovina previamente dialisada. A menor concentraÃÃo (1Âg/mL) nÃo apresentou efeito nos parÃmetros avaliados. PorÃm, a concentraÃÃo de 3Âg/mL produziu um aumentou na pressÃo de perfusÃo renal (PP), na resistÃncia vascular renal (RVR), no fluxo urinÃrio (FU) e no ritmo de filtraÃÃo glomerular (RFG). Foi tambÃm observada uma reduÃÃo aos 60â no percentual de transporte tubular de sÃdio (% TNa+). Na avaliaÃÃo histolÃgica do rim perfundido com VPp foi observado depÃsito de proteÃnas nos tÃbulos renais e nos espaÃos urinÃrios, bem como regiÃes focais de necrose/apoptose. A citotoxidade do veneno da Polybia paulista em cultura de cÃlulas MDCK foi analisada atravÃs do ensaio colorimÃtrico com sal de tetrazolium (MTT). O veneno promoveu um efeito citotÃxico dependente de concentraÃÃo com um valor de IC50 de 25.81Âg/mL. TambÃm foram mensurados os nÃveis de lactato desidrogenase (LDH) e observado um aumento significativo nas maiores concentraÃÃes estudadas. No estudo em leito mesentÃrico a pressÃo de perfusÃo foi mensurada atravÃs de um transdutor de pressÃo. O efeito vascular do VPp (3Âg/mL) nÃo alterou a pressÃo basal, na concentraÃÃo de 10Âg/mL, foi observado um aumento pressÃrico discreto e na concentraÃÃo de 100 Âg/mL, foi observado um acentuado aumento na pressÃo de perfusÃo basal. O efeito vasoconstritor observado no estudo renal e em leito mesentÃrico, tambÃm foi demonstrado no protocolo de anel de aorta, sendo observado de maneira concentraÃÃo dependente, um aumento do tÃnus basal apÃs a infusÃo do VPp. Em conclusÃo, o VPp causou nefrotoxidade, sugestivo de uma aÃÃo direta com morte celular em cÃlulas do tÃbulo renal por necrose. O efeito vascular contrÃtil que envolve influxo de cÃlcio atravÃs de canais operados por voltagem, provavelmente à mediado pela ativaÃÃo de receptores alfa adrenÃgicos.

Introduction: There is an increasing number of Canadians living with end stage renal disease (ESRD). Kidney transplantation is currently the best treatment for ESRD but long-term outcomes remain suboptimal. Identifying factors associated with better outcomes may lead to interventions or practice change that could improve patient survival or quality of life. The objectives of this thesis were to: i) systematically review the literature to examine centre variation in kidney transplantation outcomes and identify centre and provider level factors that may contribute to variation in outcomes; ii) describe differences that may exist at the patient, centre and provider level at the time of kidney transplantation across the six transplant centres in Ontario, Canada; iii) examine variation in graft and patient survival rates across transplant centres in Ontario; and iv) examine whether patient, centre and provider level characteristics contribute to variation in graft and survival rates across transplant centres. Methods: The first objective of this thesis was met by conducting a systematic review of the literature according to a predefined protocol. The last three objectives of the thesis were met by conducting a population based retrospective cohort study using administrative data from Ontario. Differences at the patient, centre and provider level were described at the time of kidney transplantation. Outcomes of interest included total graft loss; graft loss with follow-up censored at death; death with graft function; and total mortality. All outcomes were assessed at one year post transplantation and at the end of study follow up. Cox proportional hazards regression was used to obtain hazard ratios (HR) for each centre relative to the average across all centres. The independent effect of centre volume and provider characteristics on outcomes was also examined. Results: The systematic review identified 24 eligible studies. Outcomes included graft survival (n=24) and patient survival (n=9). The main characteristics evaluated were centre volume (n=17) and provider volume (n=2). Centre variation in graft survival was described in 80% (12/15) of studies, while less than half of studies (8/17) found a significant association between volume and graft survival. The population based retrospective cohort included 5092 adults (≥18 years) who received a primary solitary kidney transplant across 6 transplant centres in Ontario between January 1st 2000 and December 31st 2013. Variation in patient, centre and provider level factors existed across centres at the time of transplantation. At the end of study follow-up, case-mix adjusted HRs for total graft loss ranged from 0.84 (95% CI 0.53-1.33) to 1.16 (95% CI 1.00-1.34) across centres (p-value for between centre variation 0.46). After adjusting for centre and provider factors, differences across centres persisted. Centre volume, provider experience and provider type were not independently associated with either short or long-term outcomes (all p>0.05) with the exception of graft loss with follow-up censored at death. Discussion: This thesis suggests that there is variation in clinical outcomes across transplant centres in Ontario which is not explained by patient factors, centre volume or provider characteristics at the time of transplantation. Additionally centre volume, provider type and experience were not independently associated with outcomes. Future prospective studies with a larger sample size of transplant centres that examine follow-up care after discharge from hospital (e.g. frequency of visits) are required to better understand this phenomenon.

Abstract The mammalian metanephric kidney develops through ureteric bud branching morphogenesis and tubule formation and involves secreted inductive signals and possibly their antagonists to regulate the process. Sprouty (spry) genes encode antagonists of FGFs and the EGF signalling pathways. To get an insight to potential developmental roles of the spry genes, the expression of spry1, 2 and 4 was analyzed in developing kidney. Spry1 is expressed in the ureteric bud, and spry2 and 4 in the ureteric bud, the kidney mesenchyme and the nephrons deriving from it suggesting developmental roles for the sprys in kidney development. Spry function was addressed in vivo in the kidney by targeting hspry2 expression to the ureteric bud with a Pax2 promoter. Hspry2 expression led to development of small, ectopic and cystic kidneys. Ureter branching was reduced and there was less glomeruli in a smaller kidney compared to the wild type controls. Spry2 may antagonize signalling of FGF2 and lead to changes in FGFR1 and FGFR3 expression. In organ culture ectopic FGFs restored ureteric branching of the hSpry2 transgenic kidneys suggesting that hSpry2 may antagonize FGF signalling in embryonic kidney. In addition to changes in FGFs, hspry2 expression also lead to downregulation of GDNF and BMP4. We conclude that the Sprouty-FGFs-FGFR signaling is important for kidney development. Wnt2b is a recently identified member of the Wnt family of secreted growth factors, but its function in organogenesis is unknown. In the kidney Wnt2b is localized to the perinephric mesenchymal cells at the initiation of organogenesis. Wnt2b signalling supported ureteric bud growth and branching in vitro. Ureteric bud that was co-cultured with Wnt2b expressive cells or incubated with a known Wnt pathway regulator lithium, and then recombined with isolated kidney mesenchyme led to recovery of the expression of some ureteric epithelial marker genes and reconstitution of early kidney development. Hence, Wnt2b signalling is critical for induction of ureteric branching in vitro. Type XVIII collagen is a matrix molecule and may be involved in Wnt signalling. Roles of type XVIII collagen in kidney and lung organogenesis was analysed. Type XVIII collagen expression correlated with the differences in epithelial branching in both of these organs and its expression in the epithelial tissue was mutually exclusive. In recombinants of ureteric bud and lung mesenchyme, type XVIII collagen expression pattern shifted from kidney to lung type and was accompanied by a shift in epithelial Sonic Hedgehog (Shh) expression and by ectopic lung Surfactant Protein C in the ureteric bud. Blocking of type XVIII collagen function prevented ureteric development with lung mesenchyme and associated with reduction in the expression of Wnt2. Taken together, the findings suggest critical roles for Sprouty2, Wnt2b and type XVIII collagen in controlling pattern formation and the mode of ureteric bud branching in the embryonic kidney.

The response of Phaseolus vulgaris L. cv. Contender grown in controlled environmental conditions, at either ambient or elevated (360 and 700 μmol mol ̄¹, respectively) CO₂ concentrations ([CO₂]), was monitored from 10 days after germination (DAG) until the onset of senescence. Elevated CO₂ had a pronounced effect on total plant height (TPH), leaf area (LA), dry weight (DW) accumulation and specific leaf area (SLA). All of these were significantly increased by elevated [CO₂] with the exception of SLA, which was significantly reduced. Except for higher initial relative growth rates (RGR) in CO₂-enriched plants, RGR did not differ significantly between the two CO₂ treatments throughout the remainder of growth period. While growth parameters clearly differed between CO₂ treatments, the effects of CO₂ on many physiological processes including net assimilation rate (NAR), Rubisco activity, and some foliar nutrient concentrations were largely transient. For example, CO₂ enrichment significantly increased NAR, but from 20 DAG onward, NAR declined to levels measured on plants grown under ambient CO₂. Similarly, the decline in both foliar N concentration and Rubisco activity in CO₂-enriched plants after 20 DAG was significantly greater than the decline observed for ambient CO₂ plants. Soluble leaf protein and total chlorophylls (a+b) were also significantly reduced in plants grown under elevated CO₂. Chlorophyll (a/b) ratios increased with time underelevated CO₂, indicating that the rate of decline of chlorophyll b was higher than that of chorophyll α. No significant changes in total carotenoid (x+c) levels were observed in either CO₂ treatment. Under enhanced CO₂, the foliar concentrations of K and Mn were increased significantly, while P, Ca, Fe and Zn were reduced significantly. However, changes in Mg and Cu concentrations were not significant. High CO₂-grown plants also exhibited pronounced leaf discoloration or chlorosis, coupled with a significant reduction in leaf longevity. The levels of non-structural carbohydrates (sucrose, glucose, fructose and starch) and nitrogenous compounds (nitrogen, total soluble proteins and free amino acids) were determined for leaves and developing seeds of P. vulgaris. Leaf tissue of elevated CO₂-grown plants accumulated significantly higher levels of both soluble sugars and starch. Leaf ultrastructure revealed considerable erilargement of starch grain sizes with surface areas more than five times larger compared to those of control plants. No apparent differences in structure and membrane integrity of chloroplasts in both CO₂ treatments were noted. Although ambient CO₂-grown plants had comparatively low levels of non-structural carbohydrates (NSC), they accumulated significantly higher levels of nitrogenous compounds. The levels of NSC were consistently higher in seeds of plants grown under elevated CO₂. In comparison to plants grown at elevated [CO₂], pods and seeds of ambient CO₂-grown plants had significantly larger pools of free amino compounds and N. Stomatal conductance (gs) declined significantly, as expected for plants grown under elevated CO₂. This was accompanied by a decline in transpiration rates (E). Reduced gs and E led to high AlE ratio, which meant improved water use efficiency (WUE) values for CO₂-enriched bean plants. Leaf carbon isotope discrimination (∆) against the heavier isotope of carbon (¹³C), has been used to select for high WUE in C₃ plants. In plants grown at elevated CO₂ concentration, ,1 was significantly reduced. Although ∆ was negatively correlated with WUE in both CO₂ treatments, the correlation was steeper and highly negative for CO₂-enriched plants. These results indicate underlying differences in gas-exchange physiology, including stomatal responses between ambient and elevated CO₂-grown plants. Photosynthetic acclimation was investigated using the response of assimilation to internal carbon dioxide concentration (A/C₁ curves). At early stages of growth, the initial slope of the A/C₁ response curve did not differ with CO₂ treatment. In contrast, CO₂-saturated photosynthetic rate (Amax) was significantly higher in plants grown under elevated versus ambient CO₂ at 15 DAG. However, at subsequent stages of growth both the initial slope and Amax declined in bean plants grown in elevated CO₂. Apparent carboxylation efficiency (ACE, estimated from the initial slope of A/C₁ response) values followed a similar trend and were significantly reduced in CO₂-enriched plants. These results indicate that acclimation or negative adjustment of photosynthesis may have been caused by a combination of both stomatal and biochemical limitations. Bean plants grown under conditions of elevated atmospheric CO₂ flowered 3 to 4 days earlier, and produced significantly more flowers and pods than plants grown at ambient conditions. Plants grown at elevated CO₂ aborted 22 and 20% more flowers and pods, respectively, than plants grown at ambient CO₂. Elevated CO₂ also significantly increased the number of tillers or lateral branches produced by plants, which contributed to a significant increase in pod number and seed yield in these plants. Although plants grown at elevated CO₂ produced on average 8 seeds per pod, while plants grown under ambient CO2 conditions produced 5 seeds per pod, the greater number of seeds was offset by lower seed weights in plants grown under _ elevated CO₂. Thus, despite high seed yield in beans grown under elevated CO₂, the harvest index (HI) did not change significantly between CO₂ treatments.

Chronic kidney disease (CKD) is an important independent risk factor in the development of cardiovascular disease (CVD). Indeed, patients with CKD are far more likely to die from CVD than reach end stage renal disease. Conventional cardiovascular risk factors and co-morbidity contribute to this increased risk of CVD. However, emerging evidence suggests other novel factors including inflammation, oxidative stress, and a shift in the balance of the vasodilator nitric oxide and vasoconstrictor endothelin system, are also important contributors. Despite increasing evidence that the endothelin system plays an important role in the development of CKD and CVD, there has been little research examining possible therapeutic benefits of its modification in patients with CKD. The overall aims of the work presented within this thesis were to examine CVD risk in patients with renal impairment and then to see what impact chronic inhibition of the endothelin system would have on risk factors for CVD and CKD progression. In the first two studies I examined markers of arterial stiffness (AS) and endothelial function in a cohort of patients with immune-mediated renal disease. I was able to show in the acute setting that improvement in renal function following treatment for these conditions leads to significant improvements in AS. Interestingly, in patients who were in remission from their renal disease, only classical cardiovascular risk factors appear to be linked to AS. In the next study I was able to prove that sitaxsentan, a selective oral ETA antagonist, did not cause functional blockade of the ETB receptor in man. This was the first study of its kind to confirm that a “selective” endothelin antagonist truly is selective in vivo: a finding that will allow more accurate mechanistic investigation of the ET system. In the final studies, I showed that in subjects with stable non-diabetic proteinuric CKD, chronic selective ETA receptor antagonism reduces blood pressure and AS, and that these systemic benefits are associated with an increase in renal blood flow and reduction in proteinuria. The reduction in proteinuria is most likely haemodynamic and linked to a fall in GFR and filtration fraction, similar to what is seen with ACE inhibitors. Importantly, these benefits were seen in patients already taking maximally tolerated renin-angiotensin aldosterone system blockade, suggesting that chronic endothelin antagonism could be an important future therapy in the management of CKD. In summary, I have shown that renal impairment can directly affect markers of arterial function and by inference increase the risk of CVD. Chronic antagonism of the endothelin system with ETA receptor blockers would appear to improve many of these biomarkers, including reductions in BP, AS and proteinuria. There were no adverse effects reported in these studies, suggesting that selective ETA antagonism may be safe enough for clinical development in CKD patients. Further larger clinical trials are warranted.

Glomerular diseases are the leading cause of end stage kidney disease worldwide. Podocyte injury plays a key role in the initiation and development of such diseases, which follow a progressive course due to the limited capacity of podocytes to regenerate. Podocytes are highly specialised, terminally differentiated cells, which play a vital role in the glomerular filtration barrier. They are also the main sites of expression of the Wilms Tumour Suppressor gene, WT1, in the adult. WT1 is a complex gene, which plays an essential role in renal development by controlling the process of mesenchymal to epithelial transition that forms the nephron. Adult podocytes maintain both epithelial and mesenchymal features and continue to express high levels of WT1. Little is known about the role of WT1 in adult podocytes as previous studies have been limited due to the confounding developmental effects and embryonic lethality of existing animal models. This thesis sought to investigate the hypothesis that WT1 is an essential gene in adult kidney and plays a fundamental role in the adult podocyte. Given its role in nephron development, WT1 loss was hypothesised to result in dedifferentiation and an alteration of the epithelial-mesenchymal balance in the podocyte, affecting its specialised function. Using an inducible, conditional animal model of Wt1 loss, Wt1 was deleted from the adult, confirming its essential role in adult kidney. Wt1 deletion resulted in severe podocyte injury and failure of the glomerular filtration barrier, as well as loss of expression of key podocyte genes. Preliminary analysis suggests this was not simply due to podocyte apoptosis and/or detachment, supporting a role for Wt1 in podocyte differentiation. This was corroborated by in vitro studies that demonstrated a requirement for Wt1 for podocyte differentiation. Significantly, Wt1 loss resulted in a marked change in the expression of epithelial and mesenchymal markers in podocytes, with upregulation of mesenchymal characteristics, in keeping with a transitional stage consistent with an earlier developmental form. To investigate the mechanism behind these findings a conditionally immortalised podocyte cell line was generated as a model of Wt1 loss in vitro. In order to confirm and specifically analyse the podocyte phenotype, BAC recombineering was utilised to produce a promoter-reporter transgene construct to attempt to generate a fluorescent-labelled, podocyte specific animal model of Wt1 loss. The findings of this thesis establish that Wt1 is essential for adult podocyte function, and appears to be a key upstream regulator of podocyte differentiation. Extension of this work may allow the identification of potential targets to promote podocyte differentiation and/or regeneration in the setting of acquired and progressive glomerular disease.

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