Ageing and inflammation with focus on end-stage renal diseases : genetic and epigenetic factors

The presence of ageing-associated disorders at a relatively young age in patients suffering from chronic kidney disease (CKD) has led to the hypothesis that CKD is characterized by accelerated ageing, resulting in a marked discrepancy between chronological and biological age. Factors that accelerate biological ageing, such as inflammation, oxidative stress, and toxins, impact the processes of cellular senescence and/or apoptosis, thereby shortening the life span of cells, and consequently, of the organism as a whole. Numerous studies have linked increased cellular senescence and apoptosis to disorders commonly associated with ageing, such as cardiovascular disease (CVD), osteoporosis, and cognitive dysfunction – all of which are common in the uremic phenotype. In Study I, we demonstrate that increased arterial gene expression of cyclin-dependent kinase inhibitor 2A (CDKN2A), a known inducer of cellular senescence, is associated with the presence of CVD and vascular calcification (VC) in CKD patients. Furthermore, there is a positive correlation between CDKN2A expression and the expression of matrix Gla protein (MGP) and runt-related transcription factor 2 (RUNX2), both of which are involved in osteogenesis. We also show a tentative relationship between a higher degree of VC and increasing p16INK4a expression, a cognate protein of CDKN2A. In Study II, we use telomere length as a biomarker of biological age, showing that CKD patients have shorter telomeres than non-CKD controls. In addition, our results indicate a possible association between longitudinal telomere length, folate, and immunosuppressive treatment in patients undergoing renal transplantation (RTx). This suggests that anti- metabolite therapy may have an impact on biological ageing in RTx patients. In Study III, we show that the global methylation status in dialysis and RTx patients at baseline and after 12 months of renal replacement therapy (RRT) differs at several sites in the genome from that of age- and gender-matched healthy controls. Furthermore, differences in methylation between patients and controls can be found at CpG sites located in genes with known functional relevance to CKD, cellular ageing, CVD and/or metabolic disease. Continuing our investigations of factors affecting epigenetic status, Study IV investigates the association between the degree of self-reported physical activity and global DNA methylation in Swedish seniors. In this study, we demonstrate that individuals who reported higher physical activity had less global DNA methylation than those who were less physically active. Study V describes the application of a multifactorial mathematical model for predicting the presence of inflammation in a dataset generated from 225 incident dialysis patients. Eight of the ten features with the highest predictive factor were single nucleotide polymorphisms (SNPs), suggesting a large genetic influence on inflammation in CKD patients. In Study VI, the interplay between inflammatory status, genotype, and mortality is demonstrated in two cohorts of incident dialysis patients. The mortality was reduced in inflamed individuals carrying a 32 base-pair deletion in the C-C motif chemokine receptor 5 (CCR5) gene compared to individuals who were inflamed but lacked the deletion

Development of a bioartificial renal tubular device based on primary, human, renal epithelial cells

Bei einem chronischen oder akuten Ausfall der Niere stehen im klinischen Alltag extrakorporale Nierenersatzverfahren, wie die Hämodialyse, Hämofiltration und Hämodiafiltration zur Verfügung. Die genannten medizinischen Verfahren bilden nur die physikalischen Prinzipien des Glomerulus ab und können die, über eine reine Stofftrennung hinausgehenden, im Besonderen die komplexen, physiologischen Funktionen des renalen Tubulussystems nicht nachbilden. Die mit dem Ausfall der Nierenfunktion verbundenen metabolischen und endokrinen Störungen werden somit nicht ausreichend korrigiert und sind Ursache der hohen Morbidität und Mortalität dieser Patientengruppe durch kardio-vaskuläre sowie inflammatorische Komplikationen. Mit der Anwendung des Tissue Engineerings, der Integration von Zellen bzw. Geweben und deren biologischer Funktionen in form- und funktionsgebende Substrate stünden für diese Problematik Methoden zur Verfügung, die fehlenden biologischen Funktionen der Tubulusepithelien der Niere in die bereits vorhandenen, modernen Nierenersatztherapien zu integrieren und den Patienten zur Verfügung zu stellen. Im Rahmen dieser Arbeit wurden Membranmaterialien, Kulturbedingungen wie Sauerstoffbedarf, Medienformulierungen und Strömungszustände identifiziert, Systeme und Methoden entwickelt, die es ermöglichen, ein BTK mit primären, humanen Tubuluszellen zu bilden und über mikroskopische Methoden den Differenzierungsstatus der Zellen innerhalb der Membrankapillaren zu erfassen. Der Sauerstoffbedarf distaler und proximaler Tubulusepithelzellen wurde unter verschiedenen Bedingungen ermittelt und führte zusammen mit einem Literaturvergleich sowie theoretischen Betrachtungen der maximalen Scherkräfte zur Auslegung der in vitro Perfusionsparameter des entwickelten Kultursystems. Es konnte ein klinisch etabliertes Membranmaterial identifiziert werden, welches den primären Zellen ohne vorherige Bioaktivierung als ein optimales Substrat dient und somit spätere Zulassungsverfahren für den klinischen Einsatz vereinfacht. Die Entwicklung eines serumarmen Mediums für die Kultur der primären Zellen stellte sich als wichtigster Schritt in der Übertragung der zuvor mit permanenten Zelllinien entwickelten Systeme und Methoden heraus. Mittels mikroskopischer Methoden (REM & CLSM) konnte die Differenzierung der verwendeten Tubulusepithelzellen innerhalb der Hohlfasermodule anhand spezifischer Marker nachgewiesen und somit der Erfolg der in dieser Arbeit entwickelten Systeme und Methoden dokumentiert werden. Trotz des Nachweises der prinzipiellen Machbarkeit im Labormaßstab, stellt die verwendete Zellquelle aufgrund der geringen Verfügbarkeit im Upscaling Prozess vom Labor hin zu einem klinischen Therapieverfahren, die größte Hürde dar. Aktuelle Entwicklungen bei der Identifizierung adulter Stammzellfraktionen der Niere, im Besonderen der Beitrag CD133+ Zellen bei der Tubulusregeneration wurden in dieser Arbeit aufgegriffen. Die Ergebnisse des durchgeführten CD133 Screenings der verwendeten Zellfraktionen zeigen, dass gerade die hochaufgereinigten, proximalen und distalen Fraktionen einen verminderten Anteil dieser potentiellen Stammzellfraktion enthalten. Weniger aufgereinigte Fraktionen könnten somit eine Alternative bei der zukünftigen Entwicklung von bioartifiziellen Tubuluskonstrukten darstellen.In case of acute or chronic renal failure there are several renal replacement therapies like haemodialysis, haemofiltration and haemodiafiltration. Mimicking the filtering function of the glomerulus only, these membrane based therapies do not provide the important and complex physiological functions of the renal tubular system. Therefore, metabolic and endocrine disorders are corrected insufficiently which is the cause for high mortalities and morbidities among this group of patients. The integration of renal tubular epithelial cells into common hollow-fibre devices by applying Tissue Engineering techniques might be one possibility to deliver these lacking functions. These bioartifical renal tubular devices could extend existing renal replacement therapies to provide a more complete artificial kidney replacement. In the scope of this work, the suitability of different membrane materials to act as a substrate for primary, human, renal tubular epithelial cells has been tested. Furthermore, culture conditions like oxygen uptake rates, medium compositions and flow conditions inside hollow-fibre cultures have been evaluated. Based on the results of the aforementioned studies, a perfusion setup along with its related methods as well as protocols for microscopic evaluations have been established successfully. The lab-scale feasibility of a bioartificial renal tubular device was shown together with the successful epithelial differentiation inside the capillaries by Scanning Electron- and Confocal Laser Scanning Microscopy. However, as discussed in this thesis, the cell source remains the major obstacle in upscaling such devices for clinical use in adequate amounts. This stays unsolved as long as alternative cell sources like adult renal stem cells, responsible for the regeneration of the renal tubular system are not identified and isolated

Increased telomere attrition following renal transplantation: impact of anti-metabolite therapy

Background: The uremic milieu exposes chronic kidney disease (CKD) patients to premature ageing processes. The impact of renal replacement therapy (dialysis and renal transplantation [RTx]) or immunosuppressive treatment regimens on ageing biomarkers has scarcely been studied. Methods: In this study telomere length in whole blood cells was measured in 49 dialysis patients and 47 RTx patients close to therapy initiation and again after 12 months. Forty-three non-CKD patients were included as controls. Results: Non-CKD patients had significantly (P <= 0.01) longer telomeres than CKD patients. Telomere attrition after 12 months was significantly greater in RTx patients compared to dialysis patients (P = 0.008). RTx patients receiving mycophenolate mofetil (MMF) had a greater (P = 0.007) degree of telomere attrition compared to those treated with azathioprine. After 12 months, folate was significantly higher in RTx patients than in dialysis patients (P < 0.0001), whereas the opposite was true for homocysteine (P < 0.0001). The azathioprine group had lower levels of folate after 12 months than the MMF group (P = 0.003). Conclusions: The associations between immunosuppressive therapy, telomere attrition, and changes in folate indicate a link between methyl donor potential, immunosuppressive drugs, and biological ageing. The hypothesis that the increased telomere attrition, observed in the MMF group after RTx, is driven by the immunosuppressive treatment, deserves further attention

CDKN2A/p16INK4a expression is associated with vascular progeria in chronic kidney disease

Patients with chronic kidney disease (CKD) display a progeric vascular phenotype linked to apoptosis, cellular senescence and osteogenic transformation. This has proven intractable to modelling appropriately in model organisms. We have therefore investigated this directly in man, using for the first time validated cellular biomarkers of ageing (CDKN2A/p16INK4a, SA-β-Gal) in arterial biopsies from 61 CKD patients undergoing living donor renal transplantation. We demonstrate that in the uremic milieu, increased arterial expression of CDKN2A/p16INK4a associated with vascular progeria in CKD, independently of chronological age. The arterial expression of CDKN2A/p16INK4a was significantly higher in patients with coronary calcification (p=0.01) and associated cardiovascular disease (CVD) (p=0.004). The correlation between CDKN2A/p16INK4a and media calcification was statistically significant (p=0.0003) after correction for chronological age. We further employed correlate expression of matrix Gla protein (MGP) and runt-related transcription factor 2 (RUNX2) as additional pathognomonic markers. Higher expression of CDKN2A/p16INK4a, RUNX2 and MGP were observed in arteries with severe media calcification. The number of p16INK4a and SA-β-Gal positive cells was higher in biopsies with severe media calcification. A strong inverse correlation was observed between CDKN2A/p16INK4a expression and carboxylated osteocalcin levels. Thus, impaired vitamin K mediated carboxylation may contribute to premature vascular senescence

CDKN2A/p16INK4a expression is associated with vascular progeria in chronic kidney disease

Patients with chronic kidney disease (CKD) display a progeric vascular phenotype linked to apoptosis, cellular senescence and osteogenic transformation. This has proven intractable to modelling appropriately in model organisms. We have therefore investigated this directly in man, using for the first time validated cellular biomarkers of ageing (CDKN2A/p16INK4a, SA-β-Gal) in arterial biopsies from 61 CKD patients undergoing living donor renal transplantation. We demonstrate that in the uremic milieu, increased arterial expression of CDKN2A/p16INK4a associated with vascular progeria in CKD, independently of chronological age. The arterial expression of CDKN2A/p16INK4a was significantly higher in patients with coronary calcification (p=0.01) and associated cardiovascular disease (CVD) (p=0.004). The correlation between CDKN2A/p16INK4a and media calcification was statistically significant (p=0.0003) after correction for chronological age. We further employed correlate expression of matrix Gla protein (MGP) and runt-related transcription factor 2 (RUNX2) as additional pathognomonic markers. Higher expression of CDKN2A/p16INK4a, RUNX2 and MGP were observed in arteries with severe media calcification. The number of p16INK4a and SA-β-Gal positive cells was higher in biopsies with severe media calcification. A strong inverse correlation was observed between CDKN2A/p16INK4a expression and carboxylated osteocalcin levels. Thus, impaired vitamin K mediated carboxylation may contribute to premature vascular senescence

Longitudinal genome-wide DNA methylation changes in response to kidney failure replacement therapy

Chronic kidney disease (CKD) is an emerging public health priority associated with high mortality rates and demanding treatment regimens, including life-style changes, medications or even dialysis or renal transplantation. Unavoidably, the uremic milieu disturbs homeostatic processes such as DNA methylation and other vital gene regulatory mechanisms. Here, we aimed to investigate how dialysis or kidney transplantation modifies the epigenome-wide methylation signature over 12 months of treatment. We used the Infinium HumanMethylation450 BeadChip on whole blood samples from CKD-patients undergoing either dialysis (n = 11) or kidney transplantation (n = 12) and 24 age- and sex-matched population-based controls. At baseline, comparison between patients and controls identified several significant (PFDR < 0.01) CpG methylation differences in genes with functions relevant to inflammation, cellular ageing and vascular calcification. Following 12 months, the global DNA methylation pattern of patients approached that seen in the control group. Notably, 413 CpG sites remained differentially methylated at follow-up in both treatment groups compared to controls. Together, these data indicate that the uremic milieu drives genome-wide methylation changes that are partially reversed with kidney failure replacement therapy. Differentially methylated CpG sites unaffected by treatment may be of particular interest as they could highlight candidate genes for kidney disease per se

Integration of environmental assessment in a PLM context: a case study in luxury industry

Nowadays, the environment becomes a major issue in our society. It gives rise to regu-lations, market demand and stakeholder's pressure which are concerning companies. These latter have to reduce the negative impact of their new product by eco-design and adopting a continuous improvement for their existing product portfolio. To do so, environmental assessment system is needed. Life Cycle Assessment (LCA) is the most known and recognized. However, this method is complex, requires significant resources and a large amount of accurate data. We propose a methodology to connect a simplified LCA tool with PLM system and ERP to evaluate an entire product portfolio at any time. This will allow design teams to consider the environmental issues in early design phase and gives the companies a global vision of their product portfolio. This methodology is experimented with packaging products of luxury brand, using the Teamcenter PLM system and a Simplified LCA Too

Being 'Green and Competitive': The Impact of Environmental Actions and Collaborations on Firm Performance

In this paper, we seek to enhance the understanding of the link between environmental management and firm performance, so contributing to the debate of being "green and competitive". Relying on the resource-based view, we study the effect of different environmental management capabilities on a firm's market and image performance. In particular, we analyze the capabilities to implement product and process-related environmental actions with different types of environmental focus (materials, energy, pollution) and the capabilities to develop environmental collaborations with different types of actors (both business actors and non-business actors). To this aim we conducted a survey on 122 Italian companies. Results show that market performance and image performance have partially different antecedents. Specifically, a firm's market performance is positively affected by the capabilities to implement environmental actions with a focus on energy and pollution and to develop environmental collaborations both with business and with non-business actors. On the other hand, a firm's image performance is positively affected by the capabilities to implement environmental actions with a focus on materials and to develop environmental collaborations with non-business actors. © 2013 John Wiley & Sons, Ltd and ERP Environment