Expression profile of the cyclin-dependent kinase inhibitors p21CIP1/WAF1 and p27KIP1 in spontaneous canine mammary tumors

Deckblatt-Impressum Abkürzungen und Definitionen Inhaltsverzeichnis Einleitung Literaturübersicht Material und Methoden Ergebnisse Diskussion Zusammenfassung Summary Anhang Literaturverzeichnis Danksagung SelbständigkeitserklärungIm Rahmen der Karzinogenese von epithelialen Tumoren kommt dem Zellzyklus und dessen Kontrolle entscheidende Bedeutung zu. Die verschiedenen Regulatoren sind verantwortlich für den kontrollierten Ablauf des Zellzyklus, aus dem letztlich eine fehlerfreie Verdoppelung der DNA resultieren soll. Humanmedizinische Forschungsarbeiten belegen die besondere Bedeutung des Expressionsprofils der Cyclin-abhängigen Cyclinkinase-Inhibitoren (CKIs) p21 und p27 im Rahmen der Karzinogenese von humanen Mammatumoren. Diese beiden CKIs wurden in veterinärmedizinischen Studien bei Caniden bislang nur unzureichend und vorwiegend auf Proteinebene untersucht. Ziel dieser Arbeit war es somit, einen Einblick in die Expressionsprofile von p21 und p27 auf molekularer Ebene sowie auf Proteinebene zu erhalten. Vor den quantitativen Untersuchungen von p21 und p27 in caninen Mammatumoren sollte zunächst die bei humanen Mammatumoren beschriebene Regulation der beiden CKIs auch bei der Spezies Hund bestätigt werden. Aus diesem Grund wurde zur Bestimmung differentieller Genexpression eine Affymetrixchip-Analyse an Proben von Mammakarzinomen der Spezies Hund (einfache, solide Karzinome) durchgeführt. Die Ergebnisse der Analyse zeigten eine Überexpression für p21 und eine verminderte Expression für p27 in den untersuchten Mammakarzinomen im Vergleich zu neoplastisch unverändertem Mammagewebe des gleichen Individuums. Die Veränderungen des Expressionsprofils von p21 und p27 ließen auf eine möglicherweise bedeutende Rolle dieser beiden CKIs auch im Rahmen der Karzinogenese caniner Mammatumoren schließen. Zur Validierung dieser Ergebnisse wurden im Anschluss an die Affymetrixchip-Analyse 25 Proben caniner Mammakarzinome und korrespondierende Proben von neoplastisch unverändertem Mammagewebe des gleichen Individuums intra operationem entnommen, histologisch klassifiziert und mittels real-time PCR quantitativ analysiert. p21 zeigte im Mittel eine 1,38-fache Expression in den untersuchten Mammakarzinomen im Vergleich zum korrespondierenden neoplastisch unveränderten Mammagewebe, was bedeutet, dass keine zuverlässige Aussage über eine Genregulation möglich ist. Für p27 lag die relative Expression in den Mammatumoren im Mittel beim 0,77-fachen der Expression, die im korrespondierenden neoplastisch unveränderten Mammagewebe des gleichen Individuums ermittelt wurde. Somit ist für beide Cyclin-abhängigen Cyclinkinase-Inhibitoren keine zuverlässige Aussage bezüglich einer Genregulation auf Transkriptionsebene möglich. Es zeigen sich hierfür allenfalls Tendenzen. Für p21 liegt tendenziell eine erhöhte und für p27 eine verminderte Genexpression in caninen Mammakarzinomen im Vergleich zu neoplastisch unverändertem Mammagewebe des gleichen Individuums vor. Im Anschluss an die Untersuchungen auf Transkriptionsebene wurde repräsentativ ein immunhistologischer Nachweis der beiden Kandidatengene durchgeführt, um eine semiquantitative Beurteilung der exprimierten p21- und p27-Proteinmengen zu ermöglichen. Für p21 zeigte sich im Tumorgewebe semiquantitativ eine deutlich erhöhte Proteinmenge im Vergleich zu neoplastisch unverändertem Mammagewebe, im Falle von p27 hingegen lag eine geringere Proteinexpression in den Mammatumoren im Vergleich zum korrespondierenden neoplastisch unveränderten Mammagewebe vor. Die Ergebnisse der Untersuchungen auf Proteinebene und Transkriptionsebene zeigen somit erhebliche Differenzen. Diese deuten auf eine mögliche posttranskriptionelle Regulation der beiden Cyclin-abhängigen Cyclinkinase-Inhibitoren hin. Sollten sich die semiquantitativen Ergebnisse der Untersuchungen auf Proteinebene auch durch quantitative Untersuchungen bestätigen lassen, existieren mit hoher Wahrscheinlichkeit posttranskriptionelle Mechanismen, die zur Regulation der Proteinexpression von p21 und p27 beitragen, die in Folgestudien genauer zu untersuchen wären.The cell cycle and its mechanisms of control play an important role concerning the carcinogenesis of epithelial neoplasia. The different regulators of the cell cycle are responsible for its proper progress to assure the accurate doubling of the DNA. These issues in human research document the importance of the expression profile of the cyclin-dependent kinase inhibitors (CKIs) p21 and p27 concerning the carcinogenesis of human mammary tumors. To date the investigation of these two CKIs has been insufficiently in veterinary research and was mainly focused on the protein expression of the two candidates. The goal of this study was to provide insight in the expression profile of p21 and p27. Mammary carcinomas (simple, solid carcinomas) were investigated in microarray analysis. The results revealed overexpression of p21 and a limited expression of p27 in canine mammary tumors compared with non-neoplastic mamma of the same individual. 25 canine mammary carcinomas and corresponding non- neoplastic mammary tissue of the same individual were taken intra operationem, followed by histological classification of tumor species and their quantitative analysis of transcriptional expression concerning p21 and p27. p21 showed an 1,38-fold higher expression level in canine mammary tumors as in non-neoplastic mammary tissue of the same dog what means that a reliable predication of gene-regulation is impossible. The expression level for p27 was 0,77-fold in the carcinomas compared to non-neoplastic mammary tissue of the same dog. Both cyclin-dependent kinase inhibitors did not show regulation in expression concerning their transcription. Only tendential regulation was obvious. p21 showed the tendency for a higher, p27 for a lower gene-expression in the mammary carcinomas compared to non-neoplastic mammary tissue of the same dog. The expression-analysis at transcriptional level of the two candidates was followed by the randomized immunohistochemical analysis of their protein expression to provide semiquantitative information about the protein expression of p21 and p27. Immunohistochemical staining concerning p21 indicated a higher expression level of this protein in canine carcinomas whereas for p27 the staining of p27- protein was limited in canine carcinomas compared to non-neoplastic mammary tissue of the same individual. Thus the results of immunhistochemical investigation and the protein expression of the two candidates differ from expression at the transcriptional level (mRNA expression) shown by real-time PCR. The results indicate a possible posttranscriptional regulation of these two CKIs. If it is possible to approve the semiquantitative investigation of the protein level also in quantitative investigations posttranscriptional pathways are likely contributing in the regulation of p21 and p27 protein expression and should be investigated in further studies properly

Associations between depressive symptoms and disease progression in older patients with chronic kidney disease: results of the EQUAL study

Background Depressive symptoms are associated with adverse clinical outcomes in patients with end-stage kidney disease; however, few small studies have examined this association in patients with earlier phases of chronic kidney disease (CKD). We studied associations between baseline depressive symptoms and clinical outcomes in older patients with advanced CKD and examined whether these associations differed depending on sex. Methods CKD patients (>= 65 years; estimated glomerular filtration rate <= 20 mL/min/1.73 m(2)) were included from a European multicentre prospective cohort between 2012 and 2019. Depressive symptoms were measured by the five-item Mental Health Inventory (cut-off <= 70; 0-100 scale). Cox proportional hazard analysis was used to study associations between depressive symptoms and time to dialysis initiation, all-cause mortality and these outcomes combined. A joint model was used to study the association between depressive symptoms and kidney function over time. Analyses were adjusted for potential baseline confounders. Results Overall kidney function decline in 1326 patients was -0.12 mL/min/1.73 m(2)/month. A total of 515 patients showed depressive symptoms. No significant association was found between depressive symptoms and kidney function over time (P = 0.08). Unlike women, men with depressive symptoms had an increased mortality rate compared with those without symptoms [adjusted hazard ratio 1.41 (95% confidence interval 1.03-1.93)]. Depressive symptoms were not significantly associated with a higher hazard of dialysis initiation, or with the combined outcome (i.e. dialysis initiation and all-cause mortality). Conclusions There was no significant association between depressive symptoms at baseline and decline in kidney function over time in older patients with advanced CKD. Depressive symptoms at baseline were associated with a higher mortality rate in men

Kidney Failure Prediction Models: A Comprehensive External Validation Study in Patients with Advanced CKD

Background: Various prediction models have been developed to predict the risk of kidney failure in patients with CKD. However, guideline-recommended models have yet to be compared head to head, their validation in patients with advanced CKD is lacking, and most do not account for competing risks.Methods: To externally validate 11 existing models of kidney failure, taking the competing risk of death into account, we included patients with advanced CKD from two large cohorts: the European Quality Study (EQUAL), an ongoing European prospective, multicenter cohort study of older patients with advanced CKD, and the Swedish Renal Registry (SRR), an ongoing registry of nephrology-referred patients with CKD in Sweden. The outcome of the models was kidney failure (defined as RRT-treated ESKD). We assessed model performance with discrimination and calibration.Results: The study included 1580 patients from EQUAL and 13,489 patients from SRR. The average c statistic over the 11 validated models was 0.74 in EQUAL and 0.80 in SRR, compared with 0.89 in previous validations. Most models with longer prediction horizons overestimated the risk of kidney failure considerably. The 5-year Kidney Failure Risk Equation (KFRE) overpredicted risk by 10%-18%. The four- and eight-variable 2-year KFRE and the 4-year Grams model showed excellent calibration and good discrimination in both cohorts.Conclusions: Some existing models can accurately predict kidney failure in patients with advanced CKD. KFRE performed well for a shorter time frame (2 years), despite not accounting for competing events. Models predicting over a longer time frame (5 years) overestimated risk because of the competing risk of death. The Grams model, which accounts for the latter, is suitable for longer-term predictions (4 years)

Predicting Kidney Failure, Cardiovascular Disease and Death in Advanced CKD Patients

Introduction: Predicting the timing and occurrence of kidney replacement therapy (KRT), cardiovascular events, and death among patients with advanced chronic kidney disease (CKD) is clinically useful and relevant. We aimed to externally validate a recently developed CKD G4+ risk calculator for these outcomes and to assess its potential clinical impact in guiding vascular access placement. Methods: We included 1517 patients from the European Quality (EQUAL) study, a European multicentre prospective cohort study of nephrology-referred advanced CKD patients aged ≥65 years. Model performance was assessed based on discrimination and calibration. Potential clinical utility for timing of referral for vascular access placement was studied with diagnostic measures and decision curve analysis (DCA). Results: The model showed a good discrimination for KRT and “death after KRT,” with 2-year concordance (C) statistics of 0.74 and 0.76, respectively. Discrimination for cardiovascular events (2-year C-statistic: 0.70) and overall death (2-year C-statistic: 0.61) was poorer. Calibration was fairly accurate. Decision curves illustrated that using the model to guide vascular access referral would generally lead to less unused arteriovenous fistulas (AVFs) than following estimated glomerular filtration rate (eGFR) thresholds. Conclusion: This study shows moderate to good predictive performance of the model in an older cohort of nephrology-referred patients with advanced CKD. Using the model to guide referral for vascular access placement has potential in combating unnecessary vascular surgeries

The association between TMAO, CMPF and clinical outcomes in advanced CKD; results from the EQUAL study

Background Trimethylamine N-oxide (TMAO), a metabolite from red meat and fish consumption, plays a role in promoting cardiovascular events. However, data regarding TMAO and its impact on clinical outcomes are inconclusive, possibly due to its undetermined dietary source. Objectives We hypothesized that circulating TMAO derived from fish intake might cause less harm compared with red meat sources by examining the concomitant level of 3-carboxy-4-methyl-5-propyl-2-furanpropionate (CMPF), a known biomarker of fish intake, and investigated the association between TMAO, CMPF, and outcomes. Methods Patients were recruited from the European QUALity (EQUAL) Study on treatment in advanced chronic kidney disease among individuals aged >= 65 y whose estimated glomerular filtration rate (eGFR) had dropped for the first time to <= 20 mL/min per 1.73 m(2) during the last 6 mo. The association between TMAO, CMPF, and outcomes including all-cause mortality and kidney replacement therapy (KRT) was assessed among 737 patients. Patients were further stratified by median cutoffs of TMAO and CMPF, suggesting high/low red meat and fish intake. Results During a median of 39 mo of follow-up, 232 patients died. Higher TMAO was independently associated with an increased risk of all-cause mortality (multivariable HR: 1.46; 95% CI: 1.17, 1.83). Higher CMPF was associated with a reduced risk of both all-cause mortality (HR: 0.79; 95% CI: 0.71, 0.89) and KRT (HR: 0.80; 95% CI: 0.71, 0.90), independently of TMAO and other clinically relevant confounders. In comparison to patients with low TMAO and CMPF, patients with low TMAO and high CMPF had reduced risk of all-cause mortality (adjusted HR: 0.49; 95% CI: 0.31, 0.73), whereas those with high TMAO and high CMPF showed no association across adjusted models. Conclusions High CMPF conferred an independent role in health benefits and might even counteract the unfavorable association between TMAO and outcomes. Whether higher circulating CMPF concentrations are due to fish consumption, and/or if CMPF is a protective factor, remains to be verified

Symptom Burden before and after Dialysis Initiation in Older Patients

For older patients with kidney failure, lowering symptom burden may be more important than prolonging life. Dialysis initiation may affect individual kidney failure-related symptoms differently, but the change in symptoms before and after start of dialysis has not been studied. Therefore, we investigated the course of total and individual symptom number and burden before and after starting dialysis in older patients