Os rins desempenham um papel fundamental no sistema urinário e sua principal função é a filtração do sangue. Uma das causas que podem comprometer o funcionamento dos rins é a Insuficiência Renal Aguda (IRA) que é definida como a diminuição da taxa de filtração glomerular (TFG) de forma rápida e inesperada, causando a perda da função renal. Essa doença apresenta um número significativo de internações e também óbitos. O marcador padrão usado nos exames laboratoriais é a creatinina sérica, no entanto, a concentração da creatinina sérica pode variar dependendo de vários fatores, como idade, sexo, nutrição, entre outros. Além disso, sua concentração não varia consideravelmente nas primeiras indicações da lesão renal. Dessa forma, o diagnóstico é tardio e a função renal já pode estar comprometida. A proteína cistatina C (CST3) vem sendo indicada como um novo marcador para a doença, mostrando superioridade especialmente para detectar pequenas variações da TFG. A concentração da cistatina C não varia significativamente com a idade, sexo e massa muscular. Assim, foi desenvolvido um imunossensor para detectar a cistatina C, usando a configuração de transistor de efeito de campo de porta estendida e separada (SEGFET). Os eletrodos foram caracterizados por várias técnicas como MEV, microscopia de força atômica, técnicas eletroquímicas e também foi analisada a sensibilidade do ouro. As etapas de construção do imunossensor, e a interação do anticorpo com concentrações crescentes da proteína foram verificadas através de técnicas eletroquímicas, na faixa de concentração de 3 a 100 ng.mL-1. Nas medidas no SEGFET observou-se uma mudança significativa da corrente após a adição da cistatina C e para as substâncias interferentes não. O imunossensor apresentou alta sensibilidade detectando concentrações bem baixas da proteína alvo. A curva analítica ΔIDS% x [CST3] obteve-se L.D. = 0,75 ng.mL-1, L.Q. = 2,27 ng.mL-1 e r = 0,98122. A curva analítica ΔVGS% x [CST3] obteve-se L.D. = 0,28 ng.mL-1, L.Q. = 0,87 ng.mL-1 e r = 0,99846. Embora tenha algumas limitações o imunossensor é inovador e apresenta muitas vantagens podendo ser aplicado futuramente para o diagnóstico da doença. / The kidneys play a key role in the urinary system and its main function is the filtration of blood. One of the causes that can compromise the functioning of the kidneys is the Acute Kidney Injury (AKI), which is defined as the quickly and unexpected decrease in Glomerular Filtration Rate (GFR), causing the loss of kidney function. This disease presents a significant number of hospitalizations and also deaths. The standard marker used in laboratory tests is serum creatinine, however, serum creatinine concentration may vary depending on a number of factors, such as age, sex, nutrition, and so on. In addition, its concentration does not vary considerably in the first indications of renal injury. Thus, the diagnosis is delayed and renal function may already be compromised. The cystatin C protein (CST3) has been indicated as a new marker for the disease, showing superiority especially for detecting small variations in GFR. The concentration of cystatin C does not vary significantly with age, sex and muscle mass. Thus, an immunosensor was developed to detect cystatin C using the extended and a separate gate field effect transistor (SEGFET) configuration. The electrodes were characterized by several techniques such as SEM, atomic force microscopy, electrochemical techniques and also the sensitivity of gold was analyzed. The construction of the immunosensor, and the interaction of the antibody with increasing concentrations of the protein were verified by electrochemical techniques, in the concentration range of 3 to 100 ng.mL-1. In the measurements in the SEGFET a significant change of the current was observed after the addition of cystatin C and for the interfering substances there was no difference. The immunosensor showed high sensitivity detecting very low concentrations of the target protein. The analytical curve ΔIDS% x [CST3] was obtained L.D. = 0,75 ng.mL-1, L.Q. = 2,27 ng.mL-1 and r = 0,98122. The analytical curve ΔVGS% x [CST3] was obtained L.D. = 0,28 ng.mL-1, L.Q. = 0,87 ng.mL-1 and r = 0,99846. Although it has some limitations the immunosensor is innovative and presents many advantages and can be applied in the future to diagnose the disease.

Background. This study aims to establish age- and gender-specific cystatin C (CysC) reference values for healthy infants, children, and adolescents and to relate them to pubertal stage, height, weight, and body mass index (BMI). Methods. Serum CysC and creatinine levels of 6217 fasting, morning venous blood samples from 2803 healthy participants of the LIFE Child study (age 3 months to 18 years) were analyzed by an immunoassay. Recruitment started in 2011; 1636 participants provided at least one follow-up measurement. Percentiles for CysC were calculated. Age- and gender-related effects of height, weight, BMI, and puberty status were assessed through linear regression models. Results. Over the first 2 years of life, median CysC levels decrease depending on height (ß = − 0.010 mg/l/cm, p < 0.001) and weight (ß = − 0.033 mg/l/kg, p < 0.001) from 1.06 to 0.88 mg/l for males and from 1.04 to 0.87 mg/l for females. Following the second year of age, the levels remain stable for eight years. From 11 to 14 years of age, there is an increase of median CysC levels in males to 0.98 mg/l and a decrease in females to 0.86 mg/l. The change is associated with puberty (ß = 0.105 mg/l/Tanner stage, p < 0.001 in males and ß = − 0.093 mg/l/Tanner stage, p < 0.01 in females) and in males with height (ß = 0.003 mg/l/cm, p < 0.001). Conclusions. CysC levels depend on age, gender, and height, especially during infancy and puberty. We recommend the use of age- and gender-specific reference values for CysC serum levels for estimating kidney function in clinical practice.:1 Contents ........................................................................................................... 1 2 Introduction ...................................................................................................... 2 GFR measurement ............................................................................................. 2 Technically advanced methods ........................................................................ 2 Serum creatinine................................................................................................ 3 Serum Cystatin C............................................................................................... 3 Current state of research..................................................................................... 5 Reference values of Cystatin C ......................................................................... 5 Cystatin C in healthy test persons .................................................................... 6 Validity of Cystatin C for kidney diseases.......................................................... 6 Post-transplant validity of Cystatin C ............................................................... 7 Validity of Cystatin C in extra-renal diseases .................................................... 7 GFR-equations................................................................................................... 8 Confounders ...................................................................................................... 9 Relevance of the topic ......................................................................................... 11 Hypothesis ........................................................................................................... 13 3 Publication – manuscript................................................................................... 14 4 Summary and interpretation ............................................................................. 16 5 References ........................................................................................................ 20 6 Appendix ........................................................................................................... I 7 Description of the own contributions ................................................................ VII 8 Erklärung über die eigenständige Abfassung der Arbeit .................................. VIII 9 Curriculum vitae ................................................................................................ IX 10 Scientific publications and presentations ....................................................... XI 11 Acknowledgements.......................................................................................... XII

Acute Kidney Injury (AKI) is a prevalent systemic disorder that has an extremely high rate of mortality even after detection. Historically, the diagnosis and treatment of AKI was marred by the lack of universally accepted criteria defining AKI. Therefore, reports of incidence and mortality varied widely depending on location and the criteria used at the time, but all reports indicated a poor prognosis for the patient. Until recently, the only modes of detecting AKI were primarily through measurements of three clinical findings: serum creatinine concentration, blood urea nitrogen concentration, and urine output. While these measurements are still widely used as standard practice, they have limitations in their utility because their values can fluctuate depending on a person's age, gender, race, diet, and other comorbid conditions. Nevertheless, as these were the only universally accepted units of measurement for kidney function, the Acute Dialysis Quality Initiative (ADQI) used them to create the Risk, Injury, Failure, Loss, and End stage kidney disease (RIFLE) criteria to classify the severity of kidney injury across clinical settings. Eventually, modifications were made by the Acute Kidney Injury Network (AKIN) to increase the sensitivity of AKI diagnosis. It was not until the last decade that new biomarkers of kidney injury began to be researched that provided earlier detection of physical kidney injury before functional manifestations would present themselves. Some of these new biomarkers include cystatin C, kidney injury molecule-1 (KIM-1), and neutrophil gelatinase associated lipocalin (NGAL). This study will investigate how the properties of these new biomarkers are superior when compared to those of serum creatinine in early detection of AKI and specification as to the local site of injury within the nephron. The conclusion is that cystatin C has the potential to indicate damage to glomerular filtration while KIM-1 and NGAL have the ability to indicate damage to the proximal tubule. Along with the ability to provide information as to the specific site of renal injury, the levels of cystatin C, KIM-1, and NGAL increase much more rapidly and to a much higher value than serum creatinine once physical renal damage has occurred. These characteristics along with future research will allow for earlier detection of AKI, more personalized treatment plans, and an overall better prognosis for the patient.

Cardiovascular diseases (CVD) are the number one cause of death worldwide. About one fifth of those who survived a myocardial infarction will suffer a recurrent cardiovascular event (CVE). Given the low participation in recommended cardiac rehabilitation, there is interest in early risk stratification after a primary CVE. This dissertation evaluates leisure time physical activity (LTPA), N-Terminal pro-Brain Natriuretic Peptide (NT-proBNP) and cystatin C as predictors of a secondary CVE in a German cohort of cardiac rehabilitation patients with stable coronary heart disease followed from 1999 to 2008. Study 1 evaluated self-reported LTPA at one-year follow-up. Those reporting seldom/never practice of LTPA showed a higher risk (Hazard Ratio (HR) 1.30 [95% Confidence Interval (CI) 0.62, 2.69]), while those reporting LTPA at least 5-6 times/week had a reduced risk (HR 0.88 [95% CI 0.54, 1.43]) for a subsequent CVE, when compared to the reference group (1-4 times/month). Study 2 examined LTPA trajectories during the age period 20-49 years. Compared to those with a gradual decline of LTPA, the highest risk was observed among those with a steeper decrease of LTPA (HR 1.59 [95% CI 0.97, 2.62]). A continuous increase of LTPA was associated with a risk reduction (HR 0.71 [95% CI 0.41, 1.22]) with respect to a recurrent CVE. Studies 3 and 4 evaluated the prognostic value of two novel biomarkers, when added to a model containing well-established CVD risk factors. In Study 3, NT-proBNP levels at one-year follow-up and a 10% increase in the slope of a NT-proBNP three-year trajectory were associated with a subsequent CVE ,with HRs of 1.63 [95% CI 1.17, 2.27] and 1.24 [95% CI 1.12, 1.37], respectively. One-year, but not baseline, levels of NT-proBNP showed an improvement in risk reclassification. Study 4 examined cystatin C versus creatinine. Although both were associated with a recurrent CVE, only the addition of cystatin C improved model performance, discrimination and reclassification. In conclusion, in patients with stable coronary heart disease, LTPA, NT-proBNP, and cystatin C might help to identify individuals at high risk for a recurrent CVE. Further research is needed to evaluate treatment modalities for secondary prevention in this group.

No description available.

Cysteinproteaset cathepsin B har en trolig roll i vävnadsnedbrytningen vid kronisk parodontit då det visats kunna bryta ner extracellulära komponenter. Cystatin C är en fysiologisk inhibitor av cathepsin B och båda har kopplats till parodontal sjukdom. Syftet med studien var att undersöka närvaro av samt lokalisera cathepsin B och cystatin C i gingivala biopsier från patienter med kronisk parodontit. Närvaro och lokalisation av cathepsin Bs och cystatin Cs demonstrerades genom immunohistokemisk metod av kryostatsnittad vävnad. Polyklonal kanin anti-human primär antikropp för både enzym och inhibitor användes. För identifiering av primära antikroppar användes Dako REAL™ EnVision™ Detection System. Granskning skedde i ljusmikroskop. Studien visar på en närvaro av både enzym och inhibitor i epitel och bindväv i parodontalt sjuk vävnad. Cathepsin B lokaliserades i stor utsträckning till bindväven, främst perivaskulärt. I epitelet lokaliserades endast ett fåtal infärgade celler. Cystatin C hade i bindväven en diffus utbredning vilket gjorde lokalisation till specifika cellpopulationer svår. I epitelet sågs en tydlig infärgning kring/i celler med dendritiskt utseende. Dessa celler identifierades som sannolika langerhanska celler. / The cysteine protease cathepsin B has been shown to have a possible role in tissue destruction in chronic periodontitis, as it has been shown to degrade extracellular components. Cystatin C is a physiological inhibitor of cathepsin B and both are associated with periodontal disease. The aim with this study was to investigate the presence and localization of cathepsin B and cystatin C in gingival biopsies from patients with chronic periodontitis. Cathepsin B's and cystatin C’s presence and localization was demonstrated by immunohistochemical method of cryostat sectioned tissue from patients with chronic periodontitis. Polyclonal rabbit anti-human primary antibody for both the enzyme and the inhibitor were used. Identification of the primary antibodies were confirmed with Dako REAL ™ EnVision ™ Detection System. The sectioned tissue was analysed by using light microscopy. Our study indicates a presence of both enzyme and inhibitor in epithelial and connective tissue from patients with periodontal disease. Cathepsin B was localized mainly to the connective tissue, primarily to the perivascular space. In the epithelium only a few stained cells were identified. In the connective tissue, cystatin C had a diffuse distribution. Therefor it was difficult to distinguish the specific cell populations associated with the inhibitor. The epithelium showed a clear staining around/in dendritic cells. These cells were identified as probable Langerhans cells.

It is suggested that Alzheimer’s disease (AD) is caused by an imbalance between production, degradation and clearance of the amyloid-β (Aβ) protein. This imbalance leads to aggregation of Aβ and tau proteins and neurodegeneration in the brain. Today there is increasing evidence that the balance between the protease cathepsin B and the protease inhibitor cystatin C affects the tendency for Aβ to aggregate. The primary aim of this thesis was to investigate Aβ, cystatin C and cathepsin B levels in blood and cerebro-spinal fluid (CSF) in relation to the risk of AD. Studies I &amp; II were based on the re-examinations of participants, at ages 70 and 77, in the Uppsala Longitudinal Study of Adult Men (ULSAM), a community-based prospective study initiated in 1970 (participants then being 50 years of age). In ULSAM, low plasma Aβ1-40 (Study I) and low serum cystatin C levels (Study II) were associated with a higher risk of AD. Studies III &amp; IV were based on a cross-sectional sample of people with AD, mild cognitive impairment and healthy controls, recruited at three Swedish Memory Disorder units: Uppsala University Hospital, Uppsala, Skåne University Hospital, Malmö, and Karolinska University Hospital, Huddinge, Stockholm. In Study III, CSF cystatin C levels were positively correlated with both Aβ1-42 and tau levels. In Study IV, individuals with AD had higher mean plasma cathepsin B levels than healthy controls. In conclusion, low plasma Aβ1-40 and low serum cystatin C levels may precede clinically manifest AD in elderly men, cystatin C levels are positively correlated with Aβ1-42 and tau levels in CSF, and mean plasma cathepsin B levels are higher in people with AD compared to healthy controls. In addition to Aβ1-42 and tau levels in CSF, Aβ1-40, cystatin C and cathepsin B levels in blood may reflect the risk of AD.

The thesis aimed to investigate cystatin C and creatinine-based estimates of glomerular filtration rate (eGFR), both at admission and during follow-up, on the combined endpoint of cardiovascular death and myocardial infarction in patients with acute coronary syndrome (ACS). We also evaluated two cystatin C assays and assessed genetic determinants of cystatin C concentrations. We used the PLATelet inhibition and Patient Outcomes study, where all types of ACS patients (n=18624) were randomized to ticagrelor or clopidogrel treatment. Multivariable Cox regression models, including clinical variables and biomarkers (troponin and NT-proBNP), and c-statistics were calculated. Cystatin C and the creatinine-based CKD-EPI equation exhibited similar significant prognostic impact on the combined endpoint, with Area Under Curves (AUC) 0.6923 and 0.6941, respectively. Follow-up samples of renal biomarkers did not improve risk prediction. Patients randomized to ticagrelor treatment were associated with a non-sustained larger increase in renal markers at discharge, but neither the change nor the difference between the randomized groups affected cardiovascular risk. Two different cystatin C assays exhibited good correlation 0.86 (95% confidence interval 0.85-0.86), however moderate level of agreement. Risk prediction with a combination of creatinine and cystatin C did not outperform the creatinine-based CKD-EPI equation, AUC: 0.6913 and 0.6924, respectively (n=13050). The genetic polymorphism rs6048952 independently affected the cystatin C concentration with mean levels of 0.85mg/L, 0.80mg/L and 0.73mg/L for the A/A, A/G, and G/G genotypes, respectively. The genetic polymorphism did not affect outcome overall, however in the non-ST-elevation ACS subgroup a signal that genetic polymorphism may be associated with cardiovascular death was observed (p=0.002). In conclusion: cystatin C or eGFR, irrespective of equation or assay, are important cardiovascular risk factors in ACS patients. Nonetheless, the incremental value of adding any renal variable, to a multivariable risk model, is small. Further research on the impact of cystatin C genetic polymorphism is warranted. / <p>PhD, i medicin.</p>

Lors de la fibrose pulmonaire idiopathique (FPI), la différenciation fibroblastique s’accompagne d’une accumulation excessive des composants de la matrice extracellulaire ainsi qu’à un dérèglement de la balance protéases / antiprotéases. Nous avons étudié le rôle des cathepsines à cystéine dans la myofibrogenèse et leur contribution potentielle à la physiopathologie de la fibrose pulmonaire chez l’Homme. Pour cela, le profil d’expression des cathepsines ainsi que de leurs inhibiteurs naturels a été évalué dans un modèle cellulaire expérimental, puis dans des myofibroblastes primaires et enfin dans des liquides de lavage broncho-Alvéolaires (LBA) de patients atteints de FPI. Nos résultats montrent que lors de la FPI la cystatine C (inhibiteur naturel des protéases à cystéine) régule les activités protéolytiques des cathepsines extracellulaires et pourrait ainsi contribuer à l’accumulation de collagènes. Elle serait un biomarqueur potentiel de la FPI. D’autre part la cathepsine B participe à la différentiation fibroblastique et son inhibition retarde la myofibrogenèse. / During idiopathic pulmonary fibrosis (IPF), fibroblast differentiation is accompanied by an excessive accumulation of extracellular matrix components as well as an imbalance between proteases and theirs inhibitors. We evaluated the role of human cysteine cathepsins in myofibrogenesis and their potential contribution to the pathogenesis of IPF. Expression of cathepsins and their natural inhibitors have been studied in an experimental cell model, but also in primary myofibroblasts and in bronchoalveolar lavage fluids (BALF) of patients suffering from IPF. Our results show that cystatin C (a natural inhibitor of cysteine proteases) regulates the extracellular proteolytic activities of cathepsins and could contribute to the accumulation of collagens. Cystatin C could also be a potential biomarker of IPF. On the other hand, cathepsin B participates in fibroblast differentiation and its inhibition delays myofibrogenesis.

Submitted by Fabio Sobreira Campos da Costa (fabio.sobreira@ufpe.br) on 2016-06-14T13:17:26Z No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Tese Doutorado Milton Cezar.pdf: 1351763 bytes, checksum: 943b1f06e1c61f7137dcc7b3c7413adf (MD5) / Made available in DSpace on 2016-06-14T13:17:26Z (GMT). No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Tese Doutorado Milton Cezar.pdf: 1351763 bytes, checksum: 943b1f06e1c61f7137dcc7b3c7413adf (MD5) Previous issue date: 2013-02-27 / A esquistossomose é uma doença crônica e debilitante que afeta cerca de 240 milhões de pessoas em todo o mundo e outras 700 milhões vivem em áreas endêmicas de países em desenvolvimento, o que tem sido um problema de saúde pública. Em 2009, havia 6.780.683 casos confirmados da doença no Brasil. A esquistossomose crônica pode levar a processos inflamatórios hepáticos graves, desencadear distúrbios renais e danos celulares que incluem o aumento do estresse oxidativo e a disfunção endotelial. Isto contribui para o surgimento da microalbuminúria, uma perda de proteínas que pode ser um preditor precoce de doenças cardiovasculares e renais. Neste estudo, foi investigado em 130 pacientes com esquistossomose hepatoesplênica ou hepatointestinal a ocorrência de microalbuminúria, e sua correlação com o aumento dos níveis pressóricos. A função renal também foi avaliada utilizando biomarcadores (Cistatina C / Creatinina) e estimada através da medida da taxa de filtração glomerular. Os valores obtidos foram comparados com 40 pessoas (Grupo Controle) saudáveis, advindos das mesmas classes sociais. Por fim, foi investigado em ratos infectados pelo parasita Schistosoma mansoni, a elevação da pressão arterial, a ocorrência de proteinúria, o estresse oxidativo renal e o estresse oxidativo hepático. Dos 130 pacientes (grupo Schistosoma mansoni) avaliados, 14,6% (11 homens e 8 mulheres) apresentaram microalbuminúria (valores entre 30 e 300 mg / g de creatinina) em comparação ao grupo controle, que mostrou apenas 5 % (um homem e uma mulher) com microalbuminúria. Dos 19 pacientes que apresentaram microalbuminúria, 15 tinham esquistossomose hepatoesplênica, e 4 tinham esquistossomose hepatointestinal. A taxa de filtração glomerular renal e a pressão arterial aumentaram no grupo Schistossoma mansoni em relação ao Grupo Controle. O resultado obtido na avaliação da taxa de filtração glomerular com a Cistatina C sérica foi superior ao resultado obtido com a Creatinina sérica. No estudo realizado com animais, os ratos infectados pelo parasita Schistossoma mansoni tiveram aumento da pressão arterial e perda de proteína na urina em comparação ao grupo controle. Este processo foi mais intenso em animais infectados e tratados com sobrecarga de sódio. Os animais não infectados pelo parasita Schistossoma mansoni e que não foram tratados com a sobrecarga de sódio, não tiveram proteinúria, nem aumento dos níveis pressóricos. Todos os resultados obtidos permitem sugerir que a microalbuminúria pode ser utilizada como um novo marcador não invasivo para o diagnóstico precoce de hipertensão arterial e de glomerulopatia esquistossomótica. / Schistosomiasis is a debilitating chronic disease that affects nearly 240 million people around the world and another 700 million live in endemic areas in developing countries, representing a serious public health issue. In 2009, there were 6,780,683 confirmed cases of the disease in Brazil. Chronic schistosomiasis can lead to severe hepatic inflammatory processes, and trigger kidney dysfunction and cellular damage, including an increase of oxidative stress and endothelial dysfunction. This contributes to the appearance of microalbuminuria, a loss of proteins that may serve as an early predictor of cardiovascular and kidney diseases. This study investigated the occurrence of microalbuminuria in 130 patients with hepatosplenic or hepatointestinal schistosomiasis, and its correction with the increase of pressure levels. Kidney function was also evaluated using biomarkers (Cystatin C / Creatinine) and estimated through the measurement of the glomerular filtration rate. The values obtained were compared with 40 healthy persons (Control Group) from the same social classes. Finally, the study investigated – in rats infected by the Schistosoma mansoni parasite – the elevation of arterial pressure, the occurrence of proteinuria, kidney oxidative stress and hepatic oxidative stress. From the 130 patients (Schistosoma mansoni group) evaluated, 14.6% (11 men and 8 women) presented microalbuminuria (values between 30 and 300 mg / g of creatinine) in comparison to the Control Group, which showed only 5% (one man and one woman) as having microalbuminuria. From the 19 patients who presented microalbuminuria, 15 had hepatosplenic schistosomiasis, and 4 had hepatointestinal schistosomiasis. The kidney glomerular filtration rate and the arterial pressure increased in the Schistossoma mansoni group in relation to the Control Group. The result obtained from the evaluation of the glomerular filtration rate with serum Cystatin C, was higher than that obtained with serum Creatinine. In the study carried out with animals, the rats infected by the Schistossoma mansoni parasite had an increase in arterial pressure and loss of protein in the urine, compared with the Control Group. The animals that were not infected with the Schistossoma mansoni parasite and that were not treated with a sodium overload, did not have proteinuria or an increase in the pressure levels. All the results obtained suggest that microalbuminuria may be used as a new non-invasive marker for early diagnosis of arterial hypertension and schistosomal glomerulopathy.