Diabetes is closely associated with increased oxidative stress, especially originating from the mitochondria. A mechanism to reduce increased mitochondria superoxide production is to reduce the mitochondria membrane potential by releasing protons across the mitochondria membrane. This phenomenon is referred to as mitochondria uncoupling since oxygen is consumed independently of ATP being produced and can be mediated by Uncoupling Proteins (UCPs). However, increased oxygen consumption is potentially detrimental for the kidney since it can cause tissue hypoxia. Therefore, this thesis aimed to investigate the role of mitochondria uncoupling for development of diabetic nephropathy.      UCP-2 was demonstrated to be the only isoform expressed in the kidney, and localized to tubular segments performing the majority of tubular electrolyte transport. Streptozotocin-induced diabetes in rats increased UCP-2 protein expression and correlated to increased non-transport dependent oxygen consumption in isolated proximal tubular cells. These effects were prevented by intense insulin treatment to the diabetic animals demonstrating a pivotal role of hyperglycemia. Importantly, elevated UCP-2 protein expression increased mitochondria uncoupling in mitochondria isolated from diabetic kidneys. Mitochondria uncoupling and altered morphology was also evident in kidneys from db/db-mice, a model of type-2 diabetes, together with proteinuria and glomerular hyperfiltration which are both clinical manifestations of diabetic nephropathy. Treatment with the antioxidant coenzyme Q10 prevented mitochondria uncoupling as well as morphological and functional alterations in these kidneys. Acute knockdown of UCP-2 paradoxically increased mitochondria uncoupling in a mechanism involving the adenosine nucleotide transporter. Increased uncoupling via adenosine nucleotide transporter decreased mitochondria membrane potential and kidney oxidative stress but did not affect glomerular filtration rate, renal blood flow, total kidney oxygen consumption or intrarenal tissue oxygen tension.      The role of increased mitochondria oxygen consumption was investigated by administering the chemical uncoupler dinitrophenol to healthy rats. Importantly, increased mitochondria oxygen consumption resulted in kidney tissue hypoxia, proteinuria and increased staining of the tubular injury marker vimentin, demonstrating a crucial role of increased oxygen consumption per se and the resulting kidney tissue hypoxia for the development of nephropathy.      Taken together, the data presented in this thesis establishes an important role of mitochondria uncoupling for the development of diabetic nephropathy.

Diabetic long-term complications, despite intensive treatment, cause serious handicaps at relatively young age in diabetic patients. Diabetic nephropathy (DN) develops in up to 30% of patients with type 1 diabetes (T1D). Besides the eventual loss of kidney function, with need for dialysis treatment and transplantation, this complication also increases the risk of early death from cardiovascular disease. In addition to hyperglycaemia, the risk of developing DN is influenced by a number of life-style related factors, such as smoking and diet, but the mechanisms of action of these factors are largely unknown. The incidence of DN is not linearly related to diabetes duration. There is a peak incidence of DN at 15-20 years and this, together with results from family studies, shows that genetic factors are important contributors. Possible candidate genes are those involved in regulation of intraglomerular pressure and blood pressure, oxidative stress and inflammation. The main aims of this thesis were: ● To investigate the risk of DN associated with polymorphisms in; A) the endothelial NO-synthase gene (NOS3) and genes in the renin-angiotensin-system (RAAS) (all involved in the regulation of intraglomerular pressure). B) the manganese superoxide dismutase gene (SOD2) (involved in the regulation of oxidative stress). C) the ICAM1 gene (involved in activation and migration of lymphocytes) ● To investigate gene-smoking interactions ● To investigate the influence of normal diet on risk of microalbuminuria. The aims were addressed in different case-control settings, including 347 T1D patients from Sweden and 1163 patients from Finland, with or without DN, defined as; overt DN – having albumin excretion rate (AER) ≥200 μg/min, incipient DN – AER between 20 and 200 μg/min, non-DN controls – having AER <20 μg/min and at least 20 years of diabetes duration. In one study also non-diabetic healthy individuals were included to asses the risk of T1D associated with the ICAM1 gene. Results: The RAAS genes were investigated in the Swedish sample set and there was an association between a polymorphism in the angiotensin II type 1 receptor (AGTR1) gene and overt DN, when adjusting for age, duration of diabetes, HbA1c, sex and smoking (adjusted OR=3.04, 99% CI=1.02-9.06). Also a synergistic interaction with smoking was indicated. The ICAM1 gene was investigated in the Swedish sample set, but no association with DN was found. There were, however, associations between T1D and two polymorphisms in this gene, rs281432 (OR=1.64, 95% CI=1.14-2.38) and rs5498 (OR=2.46, 95% CI=1.59-3.80). In the combined Swedish/Finnish sample set, the Glu/Glu genotype of the Glu298Asp polymorphism in the NOS3 gene was associated with DN when age at diabetes onset, duration of diabetes, HbA1c, blood pressure, sex and smoking were taken into account (adjusted OR=1.46, 95% CI=1.12-1.91). There was also association between a polymorphism in the MnSOD gene and DN in this sample set. Homozygosity for the valine-allele of the Val16Ala polymorphism was associated with increased risk of DN in a model including age at diabetes onset, duration of diabetes, HbA1c, sex and smoking (adjusted OR=1.32, 95% CI=1.00-1.74). Smoking was associated with DN (OR=2.00, 95% CI=1.60-2.50) and in the Swedish sample set there were indications of interactions between smoking and the NOS3 and SOD2 genes, but these results could not be confirmed in the Finnish sample set. A high protein intake can enhance glomerular filtration rate and accelerate progression to DN, also other dietary components such as fat, fibres, vitamins and the ratio red/white meat have been discussed as important for DN development. In a nested case-control study including young T1D patients, the normal dietary intakes of protein and other nutrients were assessed using a semiquantitative questionnaire. The results showed that T1D patients consuming more than 6.5 g fish protein (>75th percentile) per day were at slightly lower risk to have microalbuminuria in both crude (OR=0.49, 95% CI=0.25-0.97) and adjusted analyses (OR=0.26, 95% CI=0.09-0.76, adjusted for age, duration of diabetes, sex, HbA1c, mean arterial pressure, BMI, region, smoking, energy intake and fish fat intake). Conclusions: The risk of having diabetic nephropathy is influenced by at least two genes controlling blood pressure and one gene protecting against oxidative stress. Smoking also increases the risk of DN and our findings indicate that smoking may accentuate the effect of the AGTR1, NOS3 and SOD2 genes. Normal dietary intake of protein was not associated with risk of having microalbuminuria in young T1D patients, on the other hand, an intake of fish protein above the 75th percentile decreased the risk of microalbuminuria.

<b>Background:</b> 'Competing risks' are events that either preclude or alter the probability of experiencing the primary study outcome(s). Many standard survival models fail to account for competing risks, introducing an unknown level of bias in their measures of absolute and relative risk. Individuals with type 2 diabetes mellitus (T2DM) and albuminuria are at increased risk of multiple competing causes of mortality, including cardiovascular disease (CVD), cancer and renal disease, yet studies to date have not implemented competing risks methodology. <b>Aim:</b> Using albuminuria in T2DM as a case study, this Thesis set out to quantify differences between standard- and competing-risks-adjusted survival analysis estimates of absolute and relative risk for the outcomes of cardiovascular and cancer mortality. <b>Methods:</b> 86,962 patients aged &ge;35 years with T2DM present on or before 2005 were identified in the Clinical Practice Research Datalink. To quantify differences in measures of absolute risk, cumulative risk estimates for cardiovascular and cancer mortality from standard survival analysis methods (Kaplan-Meier estimator) were compared to those from competing-risks-adjusted methods (cumulative incidence competing risk estimator). Cumulative risk estimates were stratified by patient albuminuria level (normoalbuminuria vs albuminuria). To quantify differences in measures of relative risk, estimates for the effect of albuminuria on the relative hazards of cardiovascular and cancer mortality were compared between standard cause-specific hazard (CSH) models (Cox-proportional-hazards regression), competing risk CSH models (unstratified Lunn-McNeil model), and competing risk subdistribution hazard (SDH) models (Fine-Gray model). <b>Results:</b> Patients with albuminuria, compared to those with normoalbuminuria, were older (p&LT;0.001), had higher systolic blood pressure (p&LT;0.001), had worse glycaemic control (p&LT;0.001), and were more likely to be current or ex-smokers (p&LT;0.001). Over the course of nine years of follow-up 22,512 patients died; 8,800 from CVD, 5,239 from cancer, and 8,473 from other causes. Median follow-up was 7.7 years. In patients with normoalbuminuria, nine-year standard and competing-risks-adjusted cumulative risk estimates for cardiovascular mortality were 11.1% (95% confidence interval (CI): 10.8-11.5%) and 10.2% (95% CI: 9.9-10.5%), respectively. For cancer mortality, these figures were 8.0% (95% CI: 7.7-8.3%) and 7.2% (95% CI: 6.9-7.5%). In patients with albuminuria, standard and competing-risks-adjusted estimates for cardiovascular mortality were 21.8% (95% CI: 20.9-22.7%) and 18.5% (95% CI: 17.8-19.3%), respectively. For cancer mortality, these figures were 10.7% (95% CI: 10.0-11.5%) and 8.6% (8.1-9.2%). For the effect of albuminuria on cardiovascular mortality, hazard ratios from multivariable standard CSH, competing risks CSH, and subdistribution hazard ratios from competing risks SDH models were 1.75 (95% CI: 1.63-1.87), 1.75 (95% CI: 1.64-1.87), and 1.58 (95% CI: 1.48-1.69), respectively. For the effect of albuminuria on cancer mortality, these values were 1.27 (95% CI: 1.16-1.39), 1.28 (95% CI: 1.17-1.40), and 1.11 (95% CI: 1.01-1.21). <b>Conclusions:</b> When evaluating measures of absolute risk, differences between standard and competing-risks-adjusted methods were small in absolute terms, but large in relative terms. For the investigation of epidemiological relationships using relative hazards models, standard survival analysis methods produced near-identical risk estimates to the CSH competing risks methods for the clinical associations evaluated in this Thesis. For the evaluation of risk prediction using relative hazards models, CSH models produced consistently higher risk estimates than SDH models, and their use may lead to over-estimation of the predictive effect of albuminuria on either outcome. Where outcomes are less common (like cancer) CSH models provide poor estimates of risk prediction, and SDH models should be used. This research demonstrates that differences can be present between risk estimates derived using CSH and SDH methods, and that the two are not necessarily interchangeable. Moreover, such differences may be present in other clinical areas.

A nefropatia diabética (ND) é uma complicação microvascular freqüente, que acomete cerca de 40% dos indivíduos com diabete melito (DM). A ND associa-se a significativo aumento de morte por doença cardiovascular. É a principal causa de insuficiência renal terminal em países desenvolvidos e em desenvolvimento, representando, dessa forma, um custo elevado para o sistema de saúde. Os fatores de risco para o desenvolvimento e a progressão da ND mais definidos na literatura são a hiperglicemia e a hipertensão arterial sistêmica. Outros fatores descritos são o fumo, a dislipidemia, o tipo e a quantidade de proteína ingerida na dieta e a presença da retinopatia diabética. Alguns parâmetros de função renal também têm sido estudados como fatores de risco, tais como a excreção urinária de albumina (EUA) normal-alta e a taxa de filtração glomerular excessivamente elevada ou reduzida. Alguns genes candidatos têm sido postulados como risco, mas sem um marcador definitivo. O diagnóstico da ND é estabelecido pela presença de microalbuminúria (nefropatia incipiente: EUA 20-199 μg/min) e macroalbuminúria (nefropatia clínica: EUA ≥ 200 μg/min). À medida que progride a ND, aumenta mais a chance de o paciente morrer de cardiopatia isquêmica. Quando o paciente evolui com perda de função renal, há necessidade de terapia de substituição renal e, em diálise, a mortalidade dos pacientes com DM é muito mais significativa do que nos não-diabéticos, com predomínio das causas cardiovasculares. A progressão nos diferentes estágios da ND não é, no entanto, inexorável. Há estudos de intervenção que demonstram a possibilidade de prevenção e de retardo na evolução da ND principalmente com o uso dos inibidores da enzima conversora da angiotensina, dos bloqueadores da angiotensina II e do tratamento intensivo da hipertensão arterial. Os pacientes podem entrar em remissão, ou até mesmo regredir de estágio. A importância da detecção precoce e da compreensão do curso clínico da ND tem ganhado cada vez mais ênfase, porque a doença renal do DM é a principal causa de diálise no mundo e está associada ao progressivo aumento de morte por causas cardiovasculares. / Diabetic nephropathy (DN) is a frequent microvascular complication, which affects about 40% of diabetes mellitus (DM) patients. DN is associated with an increased cardiovascular death rate. DN is the major cause of kidney failure in developing as well as in developed countries, and it is, therefore, associated with increased health system costs. The more defined risk factors for the development and progression of DN are sustained hyperglycemia and hypertension. Other putative risk factors are smoking, dyslipidemia, the amount and source of protein in the diet, and the presence of diabetic retinopathy. Some renal function parameters have also been studied as risk factors, such as high normal urinary albumin excretion (UAE) and extremely high or low glomerular filtration rate levels. Some candidate genes have been analyzed as risk factors, but without any definitive marker. DN diagnosis is established by the presence of microalbuminuria (incipient nephropathy: UAE 20-199 μg/min), and macroalbuminuria (overt nephropathy: UAE ≥200 μg/min). As DN progress, the chance of death from coronary artery disease increases. When patients progress to kidney failure with uremia, renal replacement therapy becomes necessary, and when on dialysis, diabetic patients have higher mortality rates in comparison to non-diabetic ones, primarily from cardiovascular causes. DN progression through stages is not always the rule. Intervention studies demonstrate that DN prevention and remission are possible, mainly with angiotensinconverting enzyme inhibitors, angiotensin-II receptor blockers, and intensive hypertension treatment. The importance of the earlier detection, and the understanding of clinical course of DN, have progressively grown, because it is the leading cause of dialysis in the world, and is associated with increased cardiovascular mortality.

A nefropatia diabética (ND) é uma complicação microvascular freqüente, que acomete cerca de 40% dos indivíduos com diabete melito (DM). A ND associa-se a significativo aumento de morte por doença cardiovascular. É a principal causa de insuficiência renal terminal em países desenvolvidos e em desenvolvimento, representando, dessa forma, um custo elevado para o sistema de saúde. Os fatores de risco para o desenvolvimento e a progressão da ND mais definidos na literatura são a hiperglicemia e a hipertensão arterial sistêmica. Outros fatores descritos são o fumo, a dislipidemia, o tipo e a quantidade de proteína ingerida na dieta e a presença da retinopatia diabética. Alguns parâmetros de função renal também têm sido estudados como fatores de risco, tais como a excreção urinária de albumina (EUA) normal-alta e a taxa de filtração glomerular excessivamente elevada ou reduzida. Alguns genes candidatos têm sido postulados como risco, mas sem um marcador definitivo. O diagnóstico da ND é estabelecido pela presença de microalbuminúria (nefropatia incipiente: EUA 20-199 μg/min) e macroalbuminúria (nefropatia clínica: EUA ≥ 200 μg/min). À medida que progride a ND, aumenta mais a chance de o paciente morrer de cardiopatia isquêmica. Quando o paciente evolui com perda de função renal, há necessidade de terapia de substituição renal e, em diálise, a mortalidade dos pacientes com DM é muito mais significativa do que nos não-diabéticos, com predomínio das causas cardiovasculares. A progressão nos diferentes estágios da ND não é, no entanto, inexorável. Há estudos de intervenção que demonstram a possibilidade de prevenção e de retardo na evolução da ND principalmente com o uso dos inibidores da enzima conversora da angiotensina, dos bloqueadores da angiotensina II e do tratamento intensivo da hipertensão arterial. Os pacientes podem entrar em remissão, ou até mesmo regredir de estágio. A importância da detecção precoce e da compreensão do curso clínico da ND tem ganhado cada vez mais ênfase, porque a doença renal do DM é a principal causa de diálise no mundo e está associada ao progressivo aumento de morte por causas cardiovasculares. / Diabetic nephropathy (DN) is a frequent microvascular complication, which affects about 40% of diabetes mellitus (DM) patients. DN is associated with an increased cardiovascular death rate. DN is the major cause of kidney failure in developing as well as in developed countries, and it is, therefore, associated with increased health system costs. The more defined risk factors for the development and progression of DN are sustained hyperglycemia and hypertension. Other putative risk factors are smoking, dyslipidemia, the amount and source of protein in the diet, and the presence of diabetic retinopathy. Some renal function parameters have also been studied as risk factors, such as high normal urinary albumin excretion (UAE) and extremely high or low glomerular filtration rate levels. Some candidate genes have been analyzed as risk factors, but without any definitive marker. DN diagnosis is established by the presence of microalbuminuria (incipient nephropathy: UAE 20-199 μg/min), and macroalbuminuria (overt nephropathy: UAE ≥200 μg/min). As DN progress, the chance of death from coronary artery disease increases. When patients progress to kidney failure with uremia, renal replacement therapy becomes necessary, and when on dialysis, diabetic patients have higher mortality rates in comparison to non-diabetic ones, primarily from cardiovascular causes. DN progression through stages is not always the rule. Intervention studies demonstrate that DN prevention and remission are possible, mainly with angiotensinconverting enzyme inhibitors, angiotensin-II receptor blockers, and intensive hypertension treatment. The importance of the earlier detection, and the understanding of clinical course of DN, have progressively grown, because it is the leading cause of dialysis in the world, and is associated with increased cardiovascular mortality.

Diabetic nephropathy (DN) is a multifactorial patholgy, characterized by the increased presence of albumin in urine. Currently, urinary albumin (uAlb) is a marker for glomerular damage mostly used for the diagnosis of DN; however, some reports in the literature showed that patients might exhibit histological signs of DN and normal uAlb. Markers of tubular damage have shown great ability to diagnose DN prior to onset of uAlb stages. Urinary markers can be adjusted by urinary creatinine (uCr), to compensate for the daily excretion rates; nevertheless, these standards are not fully established. Thus, the aim of this study was to evaluate the diagnostic ability of the markers of tubular damage NAG, GGT, NAP, KIM-1 and NGAL and the influence of the correction for uCr in DN. Glycemic control, lipid and hepatic enzyme profile as well urinary markers of glomerular and tubular damage were assessed in type 2 DM patients stratified into two groups regarding the existence of DN. The tubular markers evaluated in this study were higher in type 2 DM patients with DN compared to type 2 diabetes without DN for both markers expressed in absolute values or as a ratio to the uCr. When analyzing the areas under the curve (AUROC) obtained, we find that all markers, except for GGT expressed in absolute values, have the ability to identify the ND. NGAL and KIM-1, when expressed in absolute values had better diagnostic ability (AUROC> 0.9, sensitivity and specificity> 90%). NAG, GGT and NAP were when expressed in ratio had the best diagnostic capability, with AUROC equal to 0.683, 0.783 and 0.850, respectively. Stratifying patients into groups according to urinary albumin excretion (UEA), we found that NGAL and KIM-1 already showed increased levels in UEA range 10 - 30 mg/g cr. In addition to being, GGT showed an association with glomerular hyperfiltration Individuals in type 2 DM without nephropathy. Urinary tubular markers used in the study have potential value in diagnosis, especially NGAL and KIM-1 that showed the best diagnostic features, and are early markers of DN type 2 DM patients. / A nefropatia diabética (ND) é uma desordem multifatorial, caracterizada pelo aumento da presença da albumina na urina. A albumina urinária (uALb) é um marcador de dano glomerular mais utilizado para o diagnóstico da ND, no entanto, existem relatos na literatura que demonstraram que pacientes já apresentam sinais histológicos de ND mas com a uALb dentro da faixa de normalidade. Tem sido demonstrado uma grande capacidade dos marcadores de dano tubular em diagnosticar a ND em estágios anteriores ao aparecimento da uAlb. Os marcadores urinários na maioria são corrigidos pela creatinina urinária (uCr), para compensar as taxas de excreção diária, apesar de não haver padronização a respeito. Assim o objetivo deste estudo foi avavliar a capacidade diagnóstica dos marcadores de dano tubular NAG (N-β-acetil-glucosaminidase), GGT (Gama-glutamiltransferase), NAP (Protein nonalbumin), KIM-1(Kidney molecule injury 1) e NGAL (Neutrophil gelatinase associated lipocalin) em relação à ND e verificar a influência da correção pela uCr nas características diagnósticas dos mesmos em pacientes com DM tipo 2. Foram mensurados indicadores do controle glicêmico, perfil lipídico e hepático, marcadores urinários de dano glomerular e tubular nos pacientes com DM do tipo 2 estratificados em dois grupos, com e sem ND. Observou-se que os marcadores tubulares avaliados foram significativamente mais elevados nos pacientes com DM tipo 2 com ND em relação ao grupo sem ND, tanto para os marcadores expressos em valores absolutos ou na forma de razão com a uCr. Quando foram analisadas as áreas sob a curva (AUROC) obtidas, verificamos que todos os marcadores, com exceção de GGT expressa em valores absolutos, tiveram a capacidade de identificar a ND. NGAL e KIM-1, quando expressos em valores absolutos tiveram a melhor capacidade diagnóstica (AUROC > 0,9, sensibilidade e especificidade > 90%). NAG, GGT e NAP quando expressos na razão tiveram a melhor capacidade de diagnóstico, com AUROC igual a 0,683, 0,783 e 0,850, respectivamente. Estratificando os pacientes em grupos de acordo com a excreção urinária de albumina (EUA), verificamos que a NGAL e o KIM-1 já apresentaram níveis aumentados no intervalo EUA 10 - 30 mg/g cr. Além disso, a GGT demonstrou estar associada com a hiperfiltração glomerular em indivíduos DM tipo 2 sem nefropatia. NGAL e KIM-1 foram os marcadores tubulares urinários que demonstraram as melhores características diagnósticas, além de ser marcadores precoces da ND em pacientes DM tipo 2.

A nefropatia diabética (ND) é uma complicação microvascular freqüente, que acomete cerca de 40% dos indivíduos com diabete melito (DM). A ND associa-se a significativo aumento de morte por doença cardiovascular. É a principal causa de insuficiência renal terminal em países desenvolvidos e em desenvolvimento, representando, dessa forma, um custo elevado para o sistema de saúde. Os fatores de risco para o desenvolvimento e a progressão da ND mais definidos na literatura são a hiperglicemia e a hipertensão arterial sistêmica. Outros fatores descritos são o fumo, a dislipidemia, o tipo e a quantidade de proteína ingerida na dieta e a presença da retinopatia diabética. Alguns parâmetros de função renal também têm sido estudados como fatores de risco, tais como a excreção urinária de albumina (EUA) normal-alta e a taxa de filtração glomerular excessivamente elevada ou reduzida. Alguns genes candidatos têm sido postulados como risco, mas sem um marcador definitivo. O diagnóstico da ND é estabelecido pela presença de microalbuminúria (nefropatia incipiente: EUA 20-199 μg/min) e macroalbuminúria (nefropatia clínica: EUA ≥ 200 μg/min). À medida que progride a ND, aumenta mais a chance de o paciente morrer de cardiopatia isquêmica. Quando o paciente evolui com perda de função renal, há necessidade de terapia de substituição renal e, em diálise, a mortalidade dos pacientes com DM é muito mais significativa do que nos não-diabéticos, com predomínio das causas cardiovasculares. A progressão nos diferentes estágios da ND não é, no entanto, inexorável. Há estudos de intervenção que demonstram a possibilidade de prevenção e de retardo na evolução da ND principalmente com o uso dos inibidores da enzima conversora da angiotensina, dos bloqueadores da angiotensina II e do tratamento intensivo da hipertensão arterial. Os pacientes podem entrar em remissão, ou até mesmo regredir de estágio. A importância da detecção precoce e da compreensão do curso clínico da ND tem ganhado cada vez mais ênfase, porque a doença renal do DM é a principal causa de diálise no mundo e está associada ao progressivo aumento de morte por causas cardiovasculares. / Diabetic nephropathy (DN) is a frequent microvascular complication, which affects about 40% of diabetes mellitus (DM) patients. DN is associated with an increased cardiovascular death rate. DN is the major cause of kidney failure in developing as well as in developed countries, and it is, therefore, associated with increased health system costs. The more defined risk factors for the development and progression of DN are sustained hyperglycemia and hypertension. Other putative risk factors are smoking, dyslipidemia, the amount and source of protein in the diet, and the presence of diabetic retinopathy. Some renal function parameters have also been studied as risk factors, such as high normal urinary albumin excretion (UAE) and extremely high or low glomerular filtration rate levels. Some candidate genes have been analyzed as risk factors, but without any definitive marker. DN diagnosis is established by the presence of microalbuminuria (incipient nephropathy: UAE 20-199 μg/min), and macroalbuminuria (overt nephropathy: UAE ≥200 μg/min). As DN progress, the chance of death from coronary artery disease increases. When patients progress to kidney failure with uremia, renal replacement therapy becomes necessary, and when on dialysis, diabetic patients have higher mortality rates in comparison to non-diabetic ones, primarily from cardiovascular causes. DN progression through stages is not always the rule. Intervention studies demonstrate that DN prevention and remission are possible, mainly with angiotensinconverting enzyme inhibitors, angiotensin-II receptor blockers, and intensive hypertension treatment. The importance of the earlier detection, and the understanding of clinical course of DN, have progressively grown, because it is the leading cause of dialysis in the world, and is associated with increased cardiovascular mortality.

Orientador: Jose Butori Lopes de Faria / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-15T03:50:13Z (GMT). No. of bitstreams: 1 Peixoto_ElisaBoucadaMauroInacio_D.pdf: 16977054 bytes, checksum: fc0226c54ac90b577209ce94906bf208 (MD5) Previous issue date: 2010 / Resumo: Hipertensão arterial (HA) e diabetes mellitus (DM) são os dois maiores fatores de risco independentes para o desenvolvimento da doença renal crônica. Porém, HA e DM frequentemente coexistem em humanos e sua combinação acarreta aumento na incidência e gravidade da nefropatia e conseqüente progressão para doença renal terminal. Tanto a hiperglicemia quanto a HA induzem estresse oxidativo no rim, que está implicado na patogênese da nefropatia diabética. O aumento do estresse oxidativo, pela produção de superóxido dependente da via NADPH oxidase e pela diminuição da defesa antioxidante, acarreta danos renais, aumento da albuminúria e progressão da nefropatia diabética. Na presente série de estudos, investigamos os efeitos do tempol, um antioxidante mimético da superóxido desmutase endógena (SOD), no estado redox via NADPH oxidase e em danos renais induzidos pelo estresse oxidativo em um modelo experimental que combina HA genética (ratos espontaneamente hipertensos - SHR) e DM induzido por estreptozotocina. Os estudos foram descritos nos seguintes artigos: Artigo I: Neste artigo, investigamos se o tratamento com o antioxidante tempol melhora o estresse oxidativo e os conseqüentes danos renais, na presença de HA e fase inicial de DM. Parâmetros de estresse oxidativo e índices de lesão renal foram avaliados em ratos SHR pré-hipertensos de 4 semanas de idade, com 20 dias de DM. Ratos diabéticos receberam ou não tempol intraperitonealmente por 20 dias. O tratamento com tempol proporcionou o aumento da expressão renal do antioxidante SOD extracelular, diminuição da produção renal de superóxido via NADPH oxidase e redução na expressão de 8-hidroxi-2'-deoxiguanosina (8-OHdG), marcador de dano ao DNA induzido por estresse oxidativo, comparado com os animais diabéticos não-tratados. Além disso, o tratamento com tempol melhorou danos renais característicos da nefropatia diabética, como albuminúria e expressão de colágeno IV. Portanto, concluímos que o tratamento com o antioxidante tempol reestabelece o estado redox e previne danos renais induzidos pelo estresse oxidativo em estágio precoce de DM e HA. Artigo II: No segundo artigo investigamos como o tempol melhora a albuminúria em ratos diabéticos e hipertensos. Córtex renal e glomérulos isolados foram coletados de ratos SHR com 4 semanas de idade, após 20 dias de DM e tratamento com tempol. O tratamento com tempol restaurou os componentes da barreira de filtração glomerular em ratos SHR diabéticos, pois preveniu a depleção de podócitos por apoptose, além de evitar a redução da expressão de nefrina induzidas pelo DM. Tempol também reduziu a expressão da proteína poli(ADP-ribose) polimerase (PARP-1) ribosilada em glomérulos de SHR diabéticos, uma enzima nuclear ativada por danos ao DNA induzidos por estresse oxidativo, sugerindo redução na atividade de PARP-1. In vitro, glomérulos isolados incubados com H2O2 (espécies reativas de oxigênio) apresentaram elevado número de células em apoptose, e os tratamentos com tempol e com inibidor de PARP-1 evitaram a apoptose de células glomerulares. Portanto, concluímos nesse artigo que o tempol reduz albuminúria em ratos diabéticos e hipertensos pela diminuição da apoptose de podócitos induzida, em parte, por PARP-1. Em resumo, a presente tese fornece evidência de que o antioxidante tempol é renoprotetor na nefropatia diabética experimental, uma vez que restaura o estado redox, reduz o acúmulo de matriz extracelular, previne apoptose de podócitos induzida por PARP-1 e reduz albuminúria, sem alterar a glicose sangüínea e a pressão arterial. / Abstract: Hypertension (HTN) and diabetes mellitus (DM) are the two major independent risk for development of chronic renal disease. However, HTN and DM frequently coexist in human and the combination of these conditions increases the frequency and severity of nephropathy and end-stage renal disease. Both, hyperglycemia and HTN, can induce renal oxidative stress, which strongly contributes to the pathogenesis of diabetic nephropathy. Elevation in oxidative stress, by increasing NADPH oxidase-dependent superoxide production and by decreasing antioxidant defense, leads to renal injury, albuminuria and diabetic nephropathy progression. The present series of studies were therefore undertaken to investigate the effect of tempol, an antioxidant mimetic of the superoxide dismutase (SOD), in the redox status via NADPH oxidase and in oxidative stress-induced renal injury in an experimental model that combines genetic HTN (spontaneously hypertensive rats -SHR) and streptozotocin-induced DM. The studies were described in the following papers: Paper I: In this study we investigated whether the treatment with tempol improve the redox imbalance and consequent renal injury, in the presence of HTN and early stage of DM. Oxidative-stress parameters and indices of renal injury were evaluated in 4-week-old prehypertensive SHR with 20 days of DM. The diabetic rats either did or did not receive tempol intraperitoneally for 20 days. Treatment with tempol provided an elevation on the expression of renal antioxidant extracellular SOD, decreased the production of renal NADPH-dependent superoxide production and diminished 8-hydroxy-2'-deoxyguanosine (8-OHdG) expression, a marker of oxidative stress-induced DNA damage, compared with untreated diabetic group. Additionally, tempol treatment reduced renal damage parameters associated with diabetic nephropathy, such as albuminuria and collagen IV expression. Therefore, in the paper I we concluded that treatment with an antioxidant tempol reestablished the redox status and prevented oxidative stress-induced renal damage in early stage of DM and HTN. Paper II: In this study we investigated the mechanism by which tempol corrects albuminuria in early stage of experimental DM and HTN. Renal cortex and isolated glomeruli of 4-week-old prehypertensive SHR were collected after 20 days of DM and treatment with tempol. Tempol ameliorates the components of glomerular filtration barrier in the diabetic SHR by reducing podocyte apoptosis and depletion, and by restoring nephrin expression. Tempol also diminished the amount of ribosilated poly(ADP-ribose) polimerase PARP-1, a nuclear enzyme activated by DNA strand breaks due to oxidative stress, in the diabetic kidney of SHR, suggesting reduction of PARP-1 activity. In vitro, isolated glomeruli exposed to reative oxygen species by incubation with hydrogen peroxide showed a higher number of apoptotic cells that was prevented by tempol or a PARP-1 inhibitor. Therefore, in the paper II we concluded that tempol reduces albuminuria in experimental model of DM and HTN at least in part by diminish PARP-induced podocyte apoptosis and oxidative stress. In summary, the present thesis provides evidence that the antioxidant tempol is renal protective in experimental diabetic nephropathy by reestablishing redox status, reducing accumulation of kidney extracellular matrix, reducing PARP-1-induce podocyte apoptosis and albuminuria. These effects were independent of blood glucose and blood pressure reduction, the two most effective approaches for the treatment of diabetic nephropathy. / Doutorado / Ciencias Basicas / Doutor em Clínica Médica

Background: Diabetic nephropathy (DN) is defined as a rise in urinary albumin excretion rate, often associated with an increase in blood pressure. It is the leading cause of end-stage renal disease and carries an increased risk for cardiovascular mortality. Microalbuminuria is the first sign of diabetic renal impairment or incipient nephropathy and is generally considered the best noninvasive predictor for the development of DN. Urinary markers of tubular damage are mainly composed of enzymes or plasma proteins of low molecular weight that are normally freely filtered by the glomerulus, and these biomarkers can be useful for diagnosis of DN. Thus, the aim of this study was to test the diagnostic accuracy of the urinary excretion of gama-glutamyltransferase (GGT) and alkaline phosphatase (ALP) for diagnosis of diabetic nephropathy (DN). Methods: Fasting glucose, fructosamine, serum creatinine, glomerular filtration rate (GFR), serum uric acid, serum albumin, and urinary albumin, creatinine, GGT and ALP were assessed in 74 type 2 diabetic patients without nephropathy and 38 type 2 diabetic patients with nephropathy. Results: Urinary GGT and ALP were threefold higher in type 2 diabetic patients with nephropathy. Significant correlations were observed between urinary albumin and GGT (r=0.439, P<0.001) and urinary albumin and ALP (r=0.305, P<0.01). Areas under the curve for GGT and ALP were 0.7696 (P<0.001) and 0.7233 (P<0.001), respectively. At a cut-off value of 72 U/g creatinine, GGT demonstrated a sensitivity of 96.0% and a specificity of 52.6%. At a cut-off value of 20 U/g creatinine, ALP demonstrated a sensitivity and specificity of 83.8% and 36.8%, respectively. Conclusions: Urinary GGT and ALP have potential value in the diagnosis of nephropathy in type 2 diabetic patients, but GGT has a slightly higher ability to discriminate nephropathy than ALP. / Introdução: A nefropatia diabética (ND) é definida como um aumento na taxa de excreção urinária de albumina, sendo esta frequentemente associada ao aumento da pressão sanguínea. A ND é a principal causa de doença renal em estágio final, estando também associada ao aumento do risco de mortalidade cardiovascular. A microalbuminúria é o primeiro sinal de dano renal ou nefropatia incipiente, sendo geralmente considerado o melhor preditor não-invasivo para o desenvolvimento de ND. Os marcadores urinários de dano tubular são principalmente compostos por enzimas ou proteínas plasmáticas de baixo peso molecular que são normalmente filtradas pelo glomérulo, sendo que estes biomarcadores podem ser úteis para o diagnóstico da ND. Assim, o objetivo deste estudo foi avaliar as características diagnósticas dos níveis urinários de gama-glutamiltransferase (GGT) e fosfatase alcalina (FAL) para o diagnóstico da ND. Métodos: Glicemia de jejum, frutosamina, creatinina sérica, taxa de filtração glomerular (TFG), ácido úrico, albumina, além dos níveis urinários de albumina, GGT e FAL foram mensurados em 74 pacientes diabéticos tipo 2 sem nefropatia e 38 pacientes diabéticos tipo 2 com nefropatia. Resultados: As enzimas GGT e FAL, mensuradas em amostras de urina, foram três vezes mais elevadas nos pacientes diabéticos tipo 2 com nefropatia. Foram observadas correlações significativas entre a albumina urinária e GGT (r=0,439, P<0,001) e albumina urinária e FAL (r=0,305, P<0,01). As áreas sob a curva para GGT e FAL foram 0,7696 (P<0,001) e 0,7233 (P<0,001), respectivamente. Ao considerar o ponto de corte de 72 U/g de creatinina, a GGT demonstrou uma sensibilidade de 96,0% e especificidade de 52,6%. Considerando o ponto de corte de 20 U/g de creatinina, a FAL demonstrou uma sensibilidade e especificidade de 83,8% e 36,8%, respectivamente. Conclusões: As enzimas GGT e FAL mensuradas em amostras de urina apresentaram potencial valor para o diagnóstico de nefropatia em pacientes com diabetes tipo 2, mas a GGT apresentou uma habilidade discretamente superior à FAL nesta diferenciação.

Type 1 diabetes (T1D) is etiologically considered as an autoimmune disease, where insulin-producing β-cells are damaged by autoimmune attacks. Regulatory T (Treg) cells are immune homeostasis cells. In the present thesis I aimed to investigate the role of Treg cells and other immune cells in the early development of T1D. In order to do that, we first determined which immune cells that are altered at an early stage of the T1D development. We found that dendritic cells and plasmacytoid dendritic cells induce the initial immune response. Next, we investigated the role of Treg cells in multiple low dose streptozotocin (MLDSTZ) induced T1D and in NOD mice. We found that the numbers of Treg cells were increased in both MLDSTZ and NOD mice when the MLDSTZ mice were hyperglycemic. However, the increased Treg cells showed a decreased production of anti-inflammatory cytokines (IL-10, IL-35 and TGF-β) and an increased expression of pro-inflammatory cytokines (IFN-γ and IL-17a). These results revealed that Treg cells switch their phenotype under T1D conditions. IL-35 administration effectively prevented the development of, and reversed established MLDSTZ induced T1D. Treg cells from IL-35 treated mice showed an increased expression of the Eos transcription factor, accompanied by an increased expression of IL-35 and a decreased expression of IFN-γ and IL-17a. These data indicate that IL-35 administration counteracted the early development of T1D by maintaining the phenotype of the Treg cells. Furthermore, IL-35 administration reversed established T1D in the NOD mouse model by maintaining the phenotype of Treg cells, seemingly by inducing the expression of Eos. Moreover, the circulating level of IL-35 was significantly lowered in both new onset and long-standing T1D patients compared to healthy controls. In addition, patients with T1D with remaining C-peptide had significantly higher levels of IL-35 than patients lacking C-peptide, suggesting that IL-35 might prevent the loss of β-cell mass. In line with this hypothesis, we found that LADA patients had a higher proportion of IL-35+ tolerogenic antigen presenting cells than T1D patients. Subsequently, we determined the proportions of IL-35+ Treg cells and IL-17a+ Treg cells in T1D patients with diabetic nephropathy (DN), which were age, sex and BMI matched with healthy controls and T1D patients. The proportion of IL-35+ Treg cells was decreased in DN and T1D patients, but IL-17a+ Treg cells were more abundant than in healthy controls. Furthermore, we found that the number of Foxp3+ Treg cells was increased in the kidneys of MLDSTZ mice. However, infiltration of mononuclear cells was seen in kidneys of these mice. In addition, kidney tissues of IL-35 treated MLDSTZ mice did not show any mononuclear cell infiltration. These results demonstrate that IL-35 may be used to prevent mononuclear cell infiltration in kidney diseases. Our findings indicate that the numbers of Foxp3+ Treg cells are increased in T1D, but that these Treg cells fail to counteract the ongoing immune assault in islets and kidneys of hyperglycemic mice. This could be explained by a phenotypic shift of the Treg cells under hyperglycemic conditions. IL-35 administration reversed established T1D in two different animal models of T1D and prevented mononuclear cell infiltration in the kidneys by maintaining the phenotype of Treg cells.