Orientadores: Eliana Pereira de Araújo, Lício Augusto Velloso / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-22T10:03:16Z (GMT). No. of bitstreams: 1 Nunez_CarlaEvelynCoimbra_D.pdf: 6141570 bytes, checksum: e9e12c78d1c319b6c83160c2ebf9976b (MD5) Previous issue date: 2013 / Resumo: A obesidade e caracterizada pelo acumulo excessivo de gordura no organismo, podendo resultar em dano a saúde. Mudança socioeconômica, ocorrida nos últimos cinquenta anos tem contribuído para o aumento da prevalência da obesidade, a qual e hoje considerada um dos principais problemas de saúde publica no mundo. O acumulo progressivo de ácidos graxos no tecido adiposo, e eventualmente, em outros sítios anatômicos não especializados na estocagem de energia sob a forma de gordura como, por exemplo, o fígado e o músculo, e associado à ativação de uma resposta inflamatória subclinica que desempenha papel importante na indução da resistência a insulina. Esta, por sua vez, e considerada o mecanismo fisiopatogênico unificador de uma serie de doenças comumente associadas à obesidade, tais como o diabetes mellitus, a aterosclerose, a esteatohepatite nao-alcoolica, entre outros. A inflamação subclinica desempenha um papel central na indução da resistência a insulina em obesos. Atualmente o estresse de reticulo endoplasmático e a ativação da sinalização do TLR4 vêm sendo identificados como potenciais mecanismos ativadores da inflamação sub-clinica associada à obesidade. No ambiente intracelular a ativação dos sinais inflamatórios disparados por ambos, estresse de reticulo endoplasmático ou TLR4, podem associar-se, modulando ou sendo modulado por outros eventos. Um desses eventos e a autofagia que se caracteriza como um processo celular finamente regulado e desempenha um papel importante no controle de varias funções da célula, tais como, reciclagem de organelas, disponibilidade de nutrientes e diferenciação celular. Um estudo recente demonstrou a existência de aumento na atividade autofágica em tecido adiposo de pessoas obesas e propôs a associação causal entre autofagia e resistência a insulina. A redução da adiposidade e o mecanismo mais eficiente para reduzir à resistência a insulina em pessoas obesas. Entretanto, o impacto da redução de adiposidade sobre a regulação da autofagia no tecido adiposo não e conhecido. Neste estudo, a regulação da autofagia no tecido adiposo durante o emagrecimento foi observada em duas etapas distintas. Inicialmente, um modelo animal de obesidade induzida por dieta, submetido posteriormente, a restrição calórica de 40% durante quinze dias. Animais obesos alimentados ad libitum, apresentaram aumento dos marcadores de autofagia no tecido adiposo, o que foi revertido na restrição calórica. De forma diversa, a restrição ocasionou o aumento da autofagia nos animais magros. A reintrodução de alimentação ad libitum foi suficiente para reduzir a autofagia nos animais magros, mas não nos obesos, cuja supra-regulacao da autofagia foi mais uma vez observada. Na segunda parte do estudo, autofagia foi avaliada em fragmentos de tecido adiposo subcutâneo de pacientes obesos selecionados para cirurgia bariátrica colhidos no ato da cirurgia e apos um ano, aproximadamente. Foram incluídos no estudo nove pacientes obesos não-diabeticos e seis pacientes obesos diabéticos. Assim como no modelo animal, obesidade em humanos foi associada a um aumento dos marcadores de autofagia no tecido adiposo os quais foram reduzidos apos a perda de peso. Assim, na vigência da obesidade ocorre uma regulação anômala da autofagia, estando aumentada durante alimentação ad libitum e reduzindo-se com a restrição alimentar / Abstract: Obesity, defined as abnormal or excessive fat accumulation that may impair life quality, is one of the major public health problems in modern world. It results from an imbalance between food intake and energy expenditure leading to the progressive accumulation of fatty acids in the adipose tissue and in some tissues that are not specialized in energy storage, such as liver and muscle. Insulin resistance is one of the main outcomes of obesity and is regarded as the main mechanism connecting diseases that are commonly associated with obesity, such as, type 2 diabetes mellitus, atherosclerosis, and non-alcoholic steatohepatitis, among others. Subclinical inflammation plays a major role in the induction of insulin resistance in obesity. Recently, endoplasmic reticulum stress and the activation of TLR4 signaling have been identified as potential triggering mechanisms for obesity-associated subclinical inflammation. At the intracellular environment activation of inflammatory signaling triggered by either endoplasmic reticulum stress or TLR4 signaling can integrate and modulate or be modulated by other cellular events. One such event is autophagy which is a highly regulated process that plays an important role in the control of a wide range of cellular functions such as organelle recycling, nutrient availability and tissue differentiation. A recent study has shown an increased autophagic activity in the adipose tissue of obese subjects, and a role for autophagy in obesity associated insulin resistance was proposed. Body mass reduction is the most efficient approach to tackle insulin resistance in over-weight subjects; however, the impact of weight loss in adipose tissue autophagy is unknown. In this study we used a two-step approach to evaluate adipose tissue autophagy during body mass reduction. First, a mouse model of diet-induced obesity and diabetes was submitted to a fifteen-day, 40% caloric restriction. At base-line, markers of autophagy were increased in obese mice as compared to lean controls. Upon caloric restriction, autophagy increased in the lean mice, while decreasing in the obese mice. The reintroduction of ad libitum feeding was sufficient to rapidly reduce autophagy in the lean mice and increase autophagy in the obese mice. In the second part of the study, autophagy was evaluated in the subcutaneous adipose tissue of nine obese-non-diabetic and six obese-diabetic subjects undergoing bariatric surgery for body mass reduction. Specimens were collected during the surgery, and approximately one year later. As in the mouse model, human obesity was associated with increased autophagy and body mass reduction led to an attenuation of autophagy in the adipose tissue. Thus, while caloric restriction leads to increased autophagy in the adipose tissue in lean subjects, in obesity, autophagy is defectively regulated, being increased during ad libitum feeding and reduced upon caloric restriction / Doutorado / Clinica Medica / Doutora em Ciências

A Síndrome de Marfan (SMF) é a enfermidade hereditária mais comum dentre as que afetam o sistema conjuntivo, causada por mutações da glicoproteína fibrilina-1, o principal componente estrutural das microfibrilas elásticas da matriz extracelular. As manifestações fenotípicas da SMF são sistêmicas e acometem tipicamente os sistemas ocular, esquelético e cardiovascular, este uma importante causa de morbi-mortalidade. Entretanto, não está claro como a mutação induz a doença. Estudos anteriores sugerem anomalias morfológicas do retículo endoplasmático (RE) ou retenção intracelular da fibrilina-1 nos estágios avançados da SMF. Entretanto, a contribuição do enovelamento da fibrilina-1 mutada e do estresse do RE na fisiopatologia celular da SMF não é conhecida. Proteínas mal-enoveladas podem levar à retenção intracelular e/ou aumento da degradação através da via de degradação associada ao RE (ERAD), além da indução da resposta a proteínas mal-enoveladas (UPR), ambas com potencial contribuição à fisiopatologia de doenças, incluindo a SMF. Assim, estudamos em fibroblastos embrionários isolados de camundongos (MEFs) com SMF se a fibrilina-1 mutada é reconhecida pelo controle de qualidade do RE pelo seu mal- enovelamento e induz estresse do RE por sua retenção intracelular. Demonstramos que a mutação na fibrilina-1 per se não promoveu chaperonas marcadoras de UPR ou geração de oxidantes. Além disso, não levou a uma maior sensibilização das células à indução exógena de estresse do RE, nem promoveu maior morte celular após inibição do proteassoma. Além disso, não foi observada retenção intracelular da fibrilina-1 nas células SMF, e mesmo após inibição da via secretora ou indução de estresse do RE, a inibição da secreção da fibrilina-1 foi similar nos MEFs SMF e wild-type (WT). A dissulfeto isomerase proteica (PDI), uma importante chaperona redox do RE, interage com fibrilina-1, e seu silenciamento levou a um aumento na secreção da fibrilina-1 pelos MEFs WT, mas não SMF. Além disso, o silenciamento da PDI promoveu a desorganização da matriz extracelular depositada de fibrilina-1 nos MEFs WT, enquanto nos MEFs SMF, a desorganização basal da matriz não foi adicionalmente alterada. Em paralelo, investigações in vivo mostraram que o estresse do RE não é induzido em camundongos SMF com 1 ou 3 meses de idade, apesar de manifestações fenotípicas evidentes. Entretanto, concomitante à progressão da doença, detectamos a ocorrência de estresse do RE nas aortas ascendentes dos camundongos aos 6 meses. Esta detecção foi exclusiva desta região da aorta e não ocorreu em outros órgãos afetados ou não afetados pela SMF. Assim, a manifestação do fenótipo clássico da SMF não requer uma perda da homeostase do RE diretamente induzida pela fibrilina-1 mutada. Ao contrário, esta é capaz de evadir mecanismos de controle de qualidade mediados pela PDI, sendo secretada normalmente. Assim, esta evasão do controle de qualidade pela PDI é uma condição permissiva essencial para o fenótipo da SMF. Por outro lado, o estresse do RE é uma característica evolutiva do aneurisma da aorta ascendente na SMF concomitante ao agravamento do fenótipo neste tecido / Marfan syndrome (MFS) is the most common connective tissue hereditary disease, caused by mutations in the glycoprotein fibrillin-1, the main structural component of extracellular matrix elastic microfibrils. MFS phenotypic manifestations are systemic and typically involve the ocular, skeletal and cardiovascular systems, the latter a major cause of morbidity/mortality. However, how gene mutation induxes disease is yet unclear. Previous studies suggest endoplasmic reticulum (ER) morphological abnormalities or fibrillin-1 intracellular retention in advanced MFS stages. However, the contribution of mutated fibrillin-1 folding and ER stress to MFS cellular pathophysiology is unknown. Un/misfolded proteins may associate with their intracellular retention and/or increased degradation through ER-associated degradation (ERAD), in addition to inducing the unfolded protein response (UPR), both sharing potential contributions to disease pathophysiology, including MFS. Thus, we studied in embryonic fibroblasts (MEFs) isolated from WT and MFS mice, if mutated fibrillin-1 can be recognized by ER quality control as a misfolded protein, able to induce ER stress due to its intracellular retention. We showed that fibrillin-1 mutation by itself did not promote UPR chaperone markers or oxidant generation. Moreover, it did not sensitize cells to exogenous ER stress nor affected cell survival curves after proteasome inhibition. Furthermore, no intracellular retention of fibrillin-1 was observed in MFS cells, and even after secretory pathway inhibition or ER stress induction, fibrillin-1 secretion inhibition was similar in MFS and wild-type (WT) MEFs. Protein disulfide isomerase (PDI), an important ER redox chaperone, interacts with fibrillin-1 and its silencing induced an increased fibrillin-1 secretion in WT, but not MFS MEFs. Besides, PDI silencing promoted fibrillin-1 extracellular matrix disorganization in WT MEFs, whereas in MFS MEFs, the basal matrix disorganization was not further modified. Parallel in vivo evaluations demonstrated that ER stress is also not induced in 1 and 3 month-old mice MFS, despite evident phenotypical manifestations. However, concomitant to accelerated disease progression at 6 months, ER stress was detectable in ascendant aorta, but not in other disease-affected or unaffected organs. Thus, classic MFS phenotype manifestations do not require loss of ER homeostasis directly induced by mutated fibrillin-1. Contrarily, the latter can evade a PDI-mediated quality control mechanism to be normally secreted. Therefore, evading such PDI-mediated quality control is an essential permissive condition for enabling the MFS phenotype. On the other hand, ER stress is an evolutive feature of MFS ascendant aorta aneurysm concomitant to phenotype progression in this tissue

Dans la mucoviscidose (CF), Pseudomonas aeruginosa colonise les voies respiratoires, conduisant à une inflammation chronique de l’épithélium bronchique. Une analyse transcriptomique antérieure nous a permis d’identifier CHAC1 comme un gène différentiellement exprimé entre les cellules épithéliales bronchiques primaires de patients CF et non-CF, au niveau basal et au cours de l’infection à P. aeruginosa. CHAC1 est une protéine dégradant le glutathion et associée au stress du réticulum endoplasmique et à l’apoptose. L’objectif principal de ce travail était de comprendre la contribution de CHAC1, en particulier dans la réponse inflammatoire et l’apoptose des cellules épithéliales pulmonaires. Nous avons donc, dans un premier temps, confirmé que CHAC1 est surexprimé au niveau ARNm dans les cellules épithéliales bronchiques primaires non-CF par rapport aux cellules CF. Nous avons observé que P. aeruginosa et deux de ses facteurs de virulence, le LPS et la flagelline, induisent l’expression de CHAC1 dans les cellules non-CF. L’expression de CHAC1 induite par le LPS est indépendante de PERK mais implique ATF4. De plus, nous avons observé qu’une réduction de l’expression de CHAC1 est associée, après stimulation par du LPS et de la flagelline, à une modulation des marqueurs inflammatoires notamment l’IL-8, l’IL-6, CCL2 et PGE2. Enfin, nous avons montré que P. aeruginosa n’est pas capable d’induire de l’apoptose dans la lignée de cellules épithéliales bronchiques NCI-H292. Ces résultats suggèrent que la régulation de l’expression de CHAC1 dans les cellules CF pourrait contribuer à la réponse inflammatoire excessive et chronique observée chez les patients atteints de mucoviscidose. / In cystic fibrosis (CF), Pseudomonas aeruginosa colonizes the airways, leading to chronic inflammation of the bronchial epithelium. A previous transcriptomic analysis allowed us to identify CHAC1 as a gene differentially expressed between primary bronchial epithelial cells of CF and non-CF patients at the basal level and during P. aeruginosa infection. CHAC1 is a glutathione-degrading protein associated with endoplasmic reticulum stress and apoptosis. The main objective of this work was to understand the contribution of CHAC1, particularly in the inflammatory response and apoptosis of pulmonary epithelial cells. We therefore first confirmed that CHAC1 is overexpressed at the mRNA level in non-CF primary bronchial epithelial cells relative to CF cells. We observed that P. aeruginosa and two of its virulence factors, LPS and flagellin, induce CHAC1 expression in non-CF cells. The expression of CHAC1 induced by LPS is independent of PERK but involves ATF4. Moreover, we have observed that a reduction in the expression of CHAC1 is associated, after stimulation by LPS and flagellin, with a modulation of the inflammatory markers, in particular IL-8, IL-6, CCL2 and PGE2. Finally, we have shown that P. aeruginosa is not capable of inducing apoptosis in the NCI-H292 bronchial epithelial cell line. These results suggest that CHAC1 is involved in the regulation of bronchial cell inflammation during P. aeruginosa infection and the regulation of CHAC1 expression in CF cells may contribute to the observed excessive and chronic inflammatory response in patients with cystic fibrosis.

Glioblastoma multiforme (GBM) is a deadly grade IV brain tumor characterized by a heterogeneous population of cells that are drug resistant, aggressive, and infiltrative. The current standard of care, which has not changed in over a decade, only provides GBM patients with 12-14 months survival post diagnosis. We asked if the addition of a novel endoplasmic reticulum (ER) stress inducing agent, JLK1486, to the standard chemotherapy, temozolomide (TMZ), which induces DNA double strand breaks (DSBs), would enhance TMZ’s efficacy. Because GBMs rely on the ER to mitigate their hypoxic environment and DNA repair to fix TMZ induced DSBs, we reasoned that DSBs occurring during heightened ER stress would be deleterious. Treatment of GBM cells with TMZ+JLK1486 decreased cell viability and increased cell death due to apoptosis. We found that TMZ+JLK1486 prolonged ER stress induction, as indicated by elevated ER stress marker BiP, ATF4, and CHOP, while sustaining activation of the DNA damage response pathway. This combination produced unresolved DNA DSBs due to RAD51 reduction, a key DNA repair factor. The combination of TMZ+JLK1486 is a potential novel therapeutic combination and suggests an inverse relationship between ER stress and DNA repair pathways.

Einleitung: Verschiedene Erkrankungen gehen bei Pferden mit einer erhöhten Entzündungsbereitschaft einher. Das entzündliche Geschehen kann dabei in engem Zusammenhang mit dem Auftreten von Stress des endoplasmatischen Retikulums (ER) stehen. Im Zuge der Immunantwort entsteht eine Signalkaskade, die über Entzündungsmarker quantifiziert werden kann. Bei der Expression verschiedener Entzündungsmediatoren rückt insbesondere die Leber in den Fokus. Die anti-inflammatorische und ER-Stress-modifizierende Wirkung von oralen Polyphenolen aus einem Grüntee-Kurkuma-Extrakt (GKE) wurde bereits in anderen Tierspezies, wie z.B. Wiederkäuern oder Schweinen nachgewiesen. Ziel der Studie: Das Ziel der vorliegenden Studie war die Überprüfung der Effekte einer GKE-Supplementierung auf eine Lipopolysaccharid (LPS)-induzierte Entzündung bei Pferden und Ponys. Die Hypothese war, dass die Supplementierung des GKE zu einer Reduktion der entzündlichen Reaktion und des Zellstresses bei Pferden und Ponys führt. Tiere, Material und Methoden: In einer randomisierten, placebo-kontrollierten Studie wurden fünf gesunde adulte Warmblutpferde (Angaben in Mittelwert ± Standardabweichung, Alter 19 ± 5 Jahre; Körpermasse (KM) 589 ± 81 kg) und sechs gesunde adulte Shetlandponys (Angaben in Mittelwert ± Standardabweichung, Alter 9 ± 3 Jahre; KM 126 ± 8 kg) einbezogen. Die tägliche Basisration bestand aus Heu (2 kg/100 kg KM). Im Cross-over erhielten die Tiere für 21 Tage 10 g eines GKE (20% Polyphenolgehalt) nach Angaben des Herstellers, oder alternativ ein Placebo (Kalziumcarbonat) zusammen mit 1 kg (Pferde) oder 0,2 kg (Ponys) eines kommerziellen Ergänzungsfuttermittels für Pferde. Nach der 21-tägigen Supplementierungsphase wurde bei allen Tieren zur Induktion eines moderaten, generalisierten Entzündungsgeschehens LPS (10 ng/kg KM) intravenös appliziert. 24 Stunden vor und 12 Stunden nach der LPS-Challenge wurden Blutproben und Lebergewebe entnommen. Die Entnahme des Lebergewebes erfolgte minimalinvasiv unter Ultraschallkontrolle. Während der anschließenden 3-monatigen Wash-out Phase erhielten alle Tiere ausschließlich Heu zur freien Aufnahme. Hiernach fand im Cross-over Verfahren ein Wechsel der Fütterungsgruppen statt. Eine erneute LPS-Challenge und die anschließende Probennahme wurden nach den oben genannten Kriterien durchgeführt. In allen Blutproben wurden die Parameter Serum Amyloid A (SAA), Haptoglobin (Hp) und Retinol-bindendes Protein 4 (RBP4) gemessen. Mittels RT-qPCR wurden in allen Leberbioptaten die mRNA-Level ausgewählter Entzündungsmarker bestimmt (Hp, TNF-α, IL-1β, IL-6, CD68, FGF21, NF-kB, ATF4). Zusätzlich erfolgte eine histologische Untersuchung auf entzündliche Reaktionen im Gewebe. Die Daten, mit Ausnahme der immunhistochemischen Auswertungen, wurden unter Verwendung der kommerziellen Software Statistica® statistisch ausgewertet. Die Daten wurden mittels des Shapiro-Wilks-Testes auf Normalverteilung überprüft. Da die Daten nicht normalverteilt waren, wurde der Wilcoxon-Test für nicht-parametrische Daten angewendet. Das Signifikanzniveau lag bei p < 0,05. Die Darstellung der Daten erfolgt als Median und dem 25./75. Perzentil. Ergebnisse: Vor der LPS-Challenge lagen alle Werte für SAA ≤ 2,6 mg/l. Nach der LPS-Challenge stiegen die Werte signifikant an (Angabe Median und in Klammern 25./75. Perzentil, Placebo-Gruppe: 98,4 (70,2/118) μg/ml, Zeit p = 0,008; GKE-Gruppe: 70,7 (46,8/111) μg/ml, Zeit p = 0,003). Innerhalb der GKE-Gruppe stieg der Wert für Hp im Serum nach der LPS-Challenge signifikant an (Zeit p = 0,005). Es konnten keine weiteren signifikanten Unterschiede in den Blutparametern im Vergleich der GKE-Gruppe zur Placebogruppe nach LPS Stimulation gefunden werden. Im Lebergewebe zeigte sich nach der LPS-Challenge für die GKE-Gruppe eine 2,6-fach geringere mRNA-Expression für das pro-inflammatorische Zytokin IL-1β im Vergleich zur Placebogruppe (Supplementierung p = 0,04). Die mRNA-Level von CD68 (Zeit p = 0,04) und Hp (Zeit p = 0,03) zeigten nach der LPS-Challenge im Lebergewebe einen signifikanten Anstieg innerhalb der Placebogruppe. Innerhalb der GKE-Gruppe zeigten sich keine Veränderungen durch die LPS-Challenge. Ein signifikanter Unterschied zwischen der Placebo- und der GKE Gruppe nach LPS wurde nicht gefunden. Andere Parameter im Lebergewebe, wie z.B. Transkriptionsfaktor NF-κB und die Zytokine IL-6 und TNF-α, zeigten keine signifikanten Veränderungen nach der LPS-Challenge in beiden Fütterungsgruppen. Für die Ergebnisse der Immunhistochemie konnten keine signifikanten Unterschiede in Abhängigkeit zur LPS-Challenge oder der Fütterung gezeigt werden. Schlussfolgerung: Die LPS-Challenge induzierte eine generalisierte Entzündung bei Pferden und Ponys. Die Supplementierung eines GKE für 21 Tage zeigte über die Reduktion des Entzündungsmarkers IL-1β in der Leber ein anti-inflammatorisches Potential. Andere Blut- und Leberparameter zeigten keinen Supplementierungseffekt. Somit konnte kein umfassender anti-inflammatorischer Effekt der GKE Supplementierung festgestellt werden.:1 Introduction 2 Literature review 2.1 Inflammation 2.1.1 Acute phase reaction 2.1.1.1 Serum amyloid A 2.1.1.2 Haptoglobin 2.1.1.3 Retinol-binding protein 4 2.1.2 Transcription factor NF-κB 2.1.2.1 Pro-inflammatory cytokines (Il-1β, IL-6, TNF-α) 2.1.3 Macrophages 2.1.3.1 Cluster of differentiation 68 2.2 Stress of the endoplasmic reticulum 2.2.1 Fibroblast growth factor 21 2.2.2 Activating transcription factor 4 2.3 Oxidative stress 2.4 Polyphenols 2.4.1 Green tea 2.4.2 Curcuma 2.4.3 Bioavailability 2.4.4 Anti-inflammatory effects 2.4.5 Antioxidative effects 3 Publication 3.1 Effects of a blend of green tea and curcuma extract supplementation on lipopolysaccharide-induced inflammation in horses and ponies 4 Discussion 4.1 Effects of the LPS challenge 4.1.1 Induction of inflammation by LPS 4.1.2 Induction of ER stress and oxidative stress by LPS 4.2 Effects of the GCE supplementation 4.2.1 Anti-inflammatory effects 4.2.2 Reduction of ER stress and oxidative stress 4.2.3 Comparison of in vivo effects in equines and farm animals 4.3 Daily dosages of polyphenols 4.4 Conclusion 5 Zusammenfassung 6 Summary 7 References 8 Appendix 8.1 List of presentations as part of this thesis 8.2 Further publications 8.2.1 Published articles 8.2.2 Published conference contributions 9 Acknowledgement / Introduction: Different medical conditions are linked with an inflammatory status in horses and ponies. Inflammation can be induced by endoplasmic reticulum (ER) stress, leading to an immune response which can be measured by several markers of the signalling cascade. The liver plays an important role as production site of these inflammatory markers. The influence of feeding a polyphenol-rich supplement consisting of green tea and curcumin extract (GCE) on ER stress and inflammation has been already described in several species such as cattle and pigs. Aim of the study: The aim of the study was to investigate the effect of a GCE supplementation on lipopolysaccharide (LPS)-induced inflammation in horses and ponies. It was hypothesized that the GCE supplementation will reduce inflammatory reactions and cell stress compared to the placebo group. Animals, material and methods: In a randomized, placebo controlled study, five healthy adult warmblood horses (mean (±SD) age 19 ± 5 years, mean (±SD) body weight (BW) 589 ± 81 kg) and six healthy adult Shetland ponies (mean (±SD) age 9 ± 3 years and mean (±SD) BW 126 ± 8 kg) were included. Animals were fed daily with meadow hay (2 kg/ 100 kg BW). In a cross-over design, animals were supplemented for 21 days with 10 g of a GCE (20% total polyphenol content) according to manufacturer’s instructions or a placebo (calcium carbonate) in a mixture with 1 kg (horses) or 0.2 kg (ponies) of a commercial feed for horses. After supplementation for 21 days, all animals underwent an intravenous LPS (10 ng/kg BW) challenge to induce a moderate systemic inflammation. 24 hours before and 12 hours after the LPS challenge, blood and liver samples were collected. Liver tissue was taken transcutaneously under ultrasound control. Subsequently, a 3-months-lasting wash-out period was conducted where the animals were fed with meadow hay ad libitum. A change of feeding groups according to the cross-over design was performed. A second LPS challenge and sample collection were performed according to the above-mentioned protocol. All blood samples were analysed for serum amyloid A (SAA), haptoglobin (Hp) and Retinol-binding protein 4 (RBP4). RT-qPCR was used to analyse liver mRNA levels of selected markers of inflammation (Hp, TNF-α, IL-1β, IL-6, CD68, FGF21, NF-kB, ATF4). In addition, liver tissue was histologically examined for inflammatory responses. All data except for immunohistochemical results were statistically assessed with a commercial software package (Statistica). Data were checked for normal distribution using the Shapiro-Wilks test. As data set was not normally distributed the Wilcoxon test for non-parametric data was used for analyses. Level of significance was set at P < 0.05. Data are presented as median and 25./75. percentile. Results: All SAA values before LPS were ≤ 2.6 mg/L. After LPS challenge, all SAA values were significantly higher compared to baseline (data expressed as medians and 25./75. Percentile in brackets, placebo group: 98.4 (70.2/118) μg/mL, time P = 0.008; GCE group: 70.7 (46.8/111) μg/mL, time P = 0.003). GCE group showed a significant increase for serum Hp after LPS challenge (time P = 0.005). Other significant differences in blood parameters comparing placebo and GCE group after LPS could not be found. In the liver, the pro-inflammatory cytokine IL-1β showed a 2.6-fold lower mRNA level after LPS challenge in the GCE group compared to placebo group (supplementation P = 0.04). Hepatic mRNA levels of CD68 (time P = 0.04) and Hp (time P = 0.03) increased significantly in the placebo group, but not in the GCE supplementation group. A significant difference between the two feedings groups after LPS could not be found. Other liver parameters, such as transcription factor NF-κB and the cytokines IL-6 and TNF-α, were not different after LPS challenge in both feeding groups. Immunohistochemical results showed no difference between GCE and placebo group after LPS challenge. Conclusion: The LPS challenge induced inflammation in the horses and ponies. Feeding a GCE for 21 days showed some potential to alter LPS-induced systemic inflammatory responses by reducing mRNA levels of the inflammatory marker IL-1β in liver tissue. Nevertheless, an overall anti-inflammatory effect by GCE was missing, since other blood and liver parameters were not affected by the GCE supplementation.:1 Introduction 2 Literature review 2.1 Inflammation 2.1.1 Acute phase reaction 2.1.1.1 Serum amyloid A 2.1.1.2 Haptoglobin 2.1.1.3 Retinol-binding protein 4 2.1.2 Transcription factor NF-κB 2.1.2.1 Pro-inflammatory cytokines (Il-1β, IL-6, TNF-α) 2.1.3 Macrophages 2.1.3.1 Cluster of differentiation 68 2.2 Stress of the endoplasmic reticulum 2.2.1 Fibroblast growth factor 21 2.2.2 Activating transcription factor 4 2.3 Oxidative stress 2.4 Polyphenols 2.4.1 Green tea 2.4.2 Curcuma 2.4.3 Bioavailability 2.4.4 Anti-inflammatory effects 2.4.5 Antioxidative effects 3 Publication 3.1 Effects of a blend of green tea and curcuma extract supplementation on lipopolysaccharide-induced inflammation in horses and ponies 4 Discussion 4.1 Effects of the LPS challenge 4.1.1 Induction of inflammation by LPS 4.1.2 Induction of ER stress and oxidative stress by LPS 4.2 Effects of the GCE supplementation 4.2.1 Anti-inflammatory effects 4.2.2 Reduction of ER stress and oxidative stress 4.2.3 Comparison of in vivo effects in equines and farm animals 4.3 Daily dosages of polyphenols 4.4 Conclusion 5 Zusammenfassung 6 Summary 7 References 8 Appendix 8.1 List of presentations as part of this thesis 8.2 Further publications 8.2.1 Published articles 8.2.2 Published conference contributions 9 Acknowledgement

<p>The endoplasmic reticulum (ER) is a specialized organelle that facilitates correct protein folding and maturation. Disruptions in ER homeostasis lead to ER stress and activation of a series of signal transduction cascades known as the unfolded protein response (UPR), which acts to restore ER homeostasis. In recent years, ER stress and UPR dysfunction have been linked to obesity, fatty liver and insulin resistance. Lipid-laden adipocytes, the main cellular component of white adipose tissue (WAT), play a critical role in whole body energy homeostasis as well as lipid and carbohydrate metabolism. Mature adipocytes, which are metabolically active endocrine cells, differentiate from precursor fibroblast-like preadipocytes, through a process called adipogenesis, leading to formation of cells capable of secreting numerous proteins, cytokines and hormones. ER homeostasis and UPR activation are essential to the function/differentiation of highly secretory cells, however, the role of ER stress/UPR activation in adipogenesis had previously not been examined. We hypothesized that<em> adipogenesis may rely on physiological UPR activation to accommodate the demand on the ER for increased folding and secretion of proteins.</em></p> <p>Initial experiments examining UPR activation during 3T3-L1 adipogenesis identified that expression of ER stress/UPR markers was modulated during adipocyte differentiation. Furthermore, inhibition of ER stress/UPR activation by the chemical chaperone, 4-phenyl butyric acid (4-PBA), inhibited adipogenesis and blunted high fat-diet induced weight gain in 4-PBA supplemented mice. These findings suggested that UPR activation modulates adipogenesis and adipose tissue metabolism.</p> <p>Subsequently, we sought to identify novel candidate ER stress/UPR responsive genes that may be involved in adipogenesis and WAT metabolism. The expression of a recently recognized ER stress-responsive gene, T-cell death associated gene 51 (TDAG51) was identified to be differentially regulated during adipogenesis. However, the function of TDAG51 in adipogenesis or energy regulation was not known. Studies from this thesis showed that TDAG51 protein expression is attenuated by ER stress/UPR activation in preadipocytes and declines during adipogenesis. Based on these results, and given the importance of adipogenesis in WAT function and whole body energy metabolism, it was<em> </em>hypothesized that<em> TDAG51 may be a novel regulator of adipogenesis and energy homeostasis.</em> Indeed, as reported here, knock-down or absence of TDAG51 (<em>TDAG51^-/-</em>) in pre-adipocytes increased lipogenesis and lead to earlier and more potent expression of adipogenic markers.</p> <p>Finally, we investigated whether absence of TDAG51 in mice affected adiposity and metabolic outcomes. Consistent with the <em>in vitro </em>results, we found that <em>TDAG51^-/-</em>mice fed a standard chow diet, exhibited an age-associated increase in WAT, developed fatty liver, and exhibited insulin resistance as compared to wild-type mice.</p> <p>Taken together, the findings in this thesis indicate that physiological UPR activation and the UPR-responsive gene TDAG51 play important roles in regulating adipogenesis, lipogenesis and whole-body energy metabolism. Thus, therapeutic approaches aimed at modulating ER folding capacity, UPR activation and/or TDAG51 expression may have great potential in the treatment of obesity and its co-morbidities.</p> / Doctor of Philosophy (PhD)

Les gènes TDP-43 (TAR DNA Binding Protein 43) et FUS/TLS (Fused in Sarcoma/Translocated in Liposarcoma) sont actuellement à l’étude quant à leurs rôles biologiques dans le développement de diverses neuropathies telles que la Sclérose Latérale Amyotrophique (SLA). Étant donné que TDP-43 et FUS sont conservés au cours de l’évolution, nous avons utilisé l’organisme modèle C. elegans afin d’étudier leurs fonctions biologiques. Dans ce mémoire, nous démontrons que TDP-1 fonctionne dans la voie de signalisation Insuline/IGF pour réguler la longévité et la réponse au stress oxydatif. Nous avons développé des lignées C. elegans transgéniques mutantes TDP-43 et FUS qui présentent certains aspects de la SLA tels que la dégénérescence des motoneurones et la paralysie adulte. La protéotoxicité causée par ces mutations de TDP- 43 et FUS associées à la SLA, induit l’expression de TDP-1. À l’inverse, la délétion de tdp-1 endogène protège contre la protéotoxicité des mutants TDP-43 et FUS chez C. elegans. Ces résultats suggèrent qu’une induction chronique de TDP-1/TDP-43 sauvage propagerait la protéotoxicité liée à la protéine mutante. Nous avons aussi entrepris un criblage moléculaire pilote afin d’isoler des suppresseurs de toxicité neuronale des modèles transgéniques mutants TDP-43 et FUS. Nous avons ainsi identifié le bleu de méthylène et le salubrinal comme suppresseurs potentiels de toxicité liée à TDP-43 et FUS via réduction de la réponse au stress du réticulum endoplasmique (RE). Nos résultats indiquent que l’homéostasie de repliement des protéines dans le RE représente une cible pour le développement de thérapies pour les maladies neurodégénératives. / Two recently discovered causative genes for ALS, TDP-43 (TAR DNA Binding Protein 43) and FUS/TLS (Fused in Sarcoma/Translocated in Liposarcoma) are under further investigation regarding their biological roles in neuropathies such as Amyotrophic Lateral Sclerosis (ALS). Since TDP-43 and FUS are evolutionarily conserved we turned to the model organism C. elegans to learn more about their biological functions. Here we report that TDP-1 functions in the Insulin/IGF pathway to regulate longevity and the oxidative stress response. We have generated mutant TDP-43 and FUS transgenic lines in C. elegans that recapitulate certain aspects of ALS including motor neuron degeneration and adult-onset paralysis. Proteotoxicity caused by ALS- associated mutations in TDP-43 or FUS also induce TDP-1 expression and consistently, deletion of endogenous tdp-1 rescues mutant TDP-43 and FUS proteotoxicity in C. elegans. These results suggest that chronic induction of wild type TDP-1/TDP-43 by proteotoxicity may actively promote neurodegeneration. We also screened for small- molecule suppressors of mutant TDP-43 and FUS neuronal toxicity in transgenic C. elegans and identified methylene blue and salubrinal as potent suppressors of TDP-43 and FUS toxicity in our models through induction of the endoplasmic reticulum (ER) stress response. Our results indicate that protein folding homeostasis in the ER may be an important target for therapeutic development in neurodegenerative diseases.

Introdução: O hiperparatireoidismo primário (HP) é a terceira maior causa de doenças endocrinológicas na população, sendo a principal causa de hipercalcemia. Caracteriza-se por hipersecreção do paratormônio (PTH) pelas células principais da paratireoide, levando a um distúrbio da homeostase do cálcio (Ca). O adenoma de paratireoide é a principal causa do HP, com 80-85% dos casos, sendo o objeto deste estudo. Mutações genéticas podem corresponder a mais de 11% da origem deste tumor benígno hipersecretor. Apesar dos avanços dos métodos de diagnóstico, o tratamento é essencialmente cirúrgico, havendo carência de tratamentos alternativos eficientes, que podem ser aprimorados com maior conhecimento fisiopatológico. Como as células do adenoma são hipersecretoras de proteínas, possuem abundante quantidade de retículo endoplasmático (RE), onde as proteínas sofrem dobramento, adquirindo sua conformação nativa. Em células hipersecretoras é comum o aumento da atividade da maquinaria de tratamento protéico, gerando sinais de sobrecarga no RE e citoplasma, sendo denominado de estresse do retículo endoplasmático (ERE). O ERE e as suas vias efetoras, a UPR (resposta a proteínas não-dobradas) e o ERAD (degradação associada ao retículo endoplasmático), estão presentes na fisiopatologia de diversas doenças ou de células hipersecretoras, servindo como importante alvo terapêutico. Objetivos: Analisar a atividade do ERE nas células do adenoma de paratireoide. Casuística e Métodos: Análise da expressão dos principais genes do ERE por RealTime-PCR, em 14 pacientes portadores de adenoma de paratireoide operados no Serviço de Cirurgia de Cabeça e Pesoco do Hospital das Clínicas da Faculdade de Medicina de Ribeirão Preto da Universidade de São Paulo, comparando-se a expressão do adenoma em relação ao controle (tecido normal). Resultados: Houve aumento da expressão de vários genes relacionados ao ERE, ERAD e à UPR, com significância estatística, principalmente da via de PERK (Pancreatic endoplasmic reticulum kina), do ERAD e da autofagia, evidenciando um quadro de cronicidade e eficência da maquinaria do ERE, com finalidade antiapoptótica. O resultado foi compatível com a manifestação clínica do adenoma de paratireoide, que cursa com raros casos de remissão espontânea e necrose central. Conclusão: Verificou-se que há uma atividade do ERE na fisiopatologia do adenoma de paratireoide, com efetividade na síntese e seleção protéica do PTH, trazendo longevidade às células do adenoma por meio da prevalência dos mecanismos de citoproteção, em detrimento da via da apoptose. O estudo traz importantes informações que podem ser úteis na elaboração de novos tratamentos medicamentosos, fazendo com que o ERE seja um futuro alvo terapêutico também no adenoma de paratireoide, assim como já é a realidade em outras patologias. / Background: Primary Hyperparathyroidism (PHP) is the third most common cause of endocrinologic disorders in the world, and the main cause of hypercalcemia. It is manifested by PTH (parathormone) hypersecretion by the parathyroid chief cells and consecutive calcium (Ca) homeostasis disturbance. Parathyroid adenoma (PA) is present in 80-85% of patients and, for this reason, is the aim of this study. Gene mutations is associated with at least 11% of this hypersecretory benign tumor. Even though the clinical presentation is much better than the past few decades, therapeutic options beside surgery do not advance along increasing efficiency in diagnostic tools. This is partly because there are many lacks in the pathophysiology of Pas, that would give new possibilities of medical treatment. PA is composed of hypersecretory cells and rich endoplasmic reticulum cytoplasm, where the main protein treatment and selection factors are situated. Considering that PA cells have protein hypersecretory activities, it is expected an abundant ER mass, that provides the machinery to treat and fold the great amount of nascent protein, to turn it to native form. As protein traduction increases, more energy is needed to keep ER function and this may result in the endoplasmic reticulum stress (ERE) in the cells. ERE and the downstream effectors, UPR (unfolded response protein) and ERAD (endoplasmic reticulum associated degradation), are acting in the physiology of several diseases and others hypersecretory cell types, providing important treatment targets. Objectives: To analyze ERE activity in PA cells. Casuistic and Methods: Evaluation of the main ERE genes expressed with Real Time-PCR analysis, in 14 patients with PA PHP and that were treated with conventional surgery, with further comparison between PA and control groups. Results: There were significant expression elevation of ERE, UPR and ERAD related genes, with statistical significance, specially of PERK downstream, ERAD and autophagy induction, suggesting efficient, though chronic, ERE activity level, with stimulated anti-apoptosis pathway. The final results confirmed our hypothesis that there is lower pro-apoptosis activity than expected by some authors, but this is compatible with low incidence of spontaneous remission or PA necrosis. Conclusion: There is contundent ERE activity in the PA pathophysiology, with great protein metabolism effectiveness expressed by PTH bioactivity, increasing cell longevity by stimulating cytoprotection pathways, instead of pro-apoptosis one. We believe this outcome will influence others research on this challenging and gratifying field of investigation, that will certainly provide new treatment options to PA, as ERE has been playing a significant role as therapeutic target with others hypersecretory diseases.

L’impératif d’une aquaculture durable conduit à orienter l’alimentation des poissons vers la substitution de la farine de poisson par des produits végétaux renouvelables. Toutefois, ce remplacement est souvent limité par des niveaux trop faibles en méthionine dans les matières premières végétales. Ainsi la supplémentation en méthionine de ces nouveaux régimes à base de végétaux est essentielle, mais requière une bonne connaissance de son rôle pour adapter les apports aux conditions physiologiques des poissons tout en prenant en compte les contraintes économiques et environnementales de production. Dans ce contexte, cette thèse avait pour principal objectif de caractériser les effets induits par une carence en méthionine sur le métabolisme mitochondrial de la truite arc en ciel. Les résultats obtenus dans notre première étude montrent que l'alimentation de truites avec un régime déficient en méthionine entraîne une baisse des performances de croissance associée à une baisse de l’intégrité mitochondriale et une diminution du statut oxydatif dans le foie. Nous démontrons également que ces perturbations s’accompagnent de l’induction d’un processus de dégradation des mitochondries par autophagie (appelé mitophagie) ainsi que d’une augmentation du stress du Réticulum Endoplasmique (RE) et de l’apoptose. Ces données originales publiées dans le journal Scientific Reports mettent ainsi en évidence les liens étroits qui existent entre différentes fonctions cellulaires pour faire face à un déséquilibre nutritionnel en méthionine. Outre cet effet à court terme, nous démontrons également, dans un seconde étude, qu’une carence en méthionine alimentaire pendant une courte période (2 semaines) lors des premiers repas des alevins entraîne une induction à long terme de facteurs liés à la mitophagie. Ces résultats, soumis à publication dans Journal of Experimental Biology, démontrent ainsi pour la première fois la mise en place d’un processus de programmation de cette fonction cellulaire par une carence précoce en méthionine. L’enrichissement en H3K4me3 et H3K36me3 des foies des poissons issus d’alevins carencés en méthionine par rapport aux poissons témoins suggère une implication de mécanismes épigénétiques dans ces effets. Enfin, dans une troisième étude qui se détache de la thématique principale de la thèse et qui a fait l’objet d’une publication dans le journal Frontiers in Physiology, nous nous sommes attachés à préciser les interactions existante entre l’autophagie, l’homéostasie du RE et le métabolisme intermédiaire. Dans l’ensemble, ces données approfondissent notre compréhension du rôle de la méthionine alimentaire au niveau cellulaire et soulignent le potentiel de cet acide aminé en tant que levier pour appliquer de nouvelles stratégies alimentaires, comme la programmation nutritionnelle, afin d’optimiser la nutrition et la santé des poissons d'élevage. / The expansion of the aquaculture industry in combination with limited availability and high prices of fishmeal has prompted feed producers to include more plant proteins in the aquaculture feeds. However, replacement of fish meal with plant proteins is often limited by the level of methionine in the alternative plant protein sources. Understanding of the different roles of methionine in fish is therefore essential to develop new diets and/or feeding strategies that are in tune with optimal fish growth, environmental and economic constraints. In this context, the main objective of this thesis was to characterize the effects induced by methionine deficiency on the hepatic mitochondrial metabolism in rainbow trout. The results obtained in our first study show that feeding trout with a methionine deficient diet leads to a decrease in growth performance associated with a decrease in both mitochondrial integrity and oxidative stress in the liver. We also demonstrate that these defects are accompanied by the induction of an autophagy-dependent mitochondrial degradation process (called mitophagy) as well as an increase in Endoplasmic Reticulum (ER)-stress and apoptosis. These original data published in Scientific Reports thus highlight the existence of close interactions between different cellular functions to cope to a dietary methionine deficiency. In addition to this short-term effect, we also demonstrate in a second study (submitted for publication in the Journal of Experimental Biology), that early nutritional stimulus during two weeks with a methionine deficient diet resulted in a long term programming of mitophagy. The enrichment of H3K4me3 and H3K36me3 in the liver of fish from methionine-deficient fry compared to their control counterparts suggests that epigenetic mechanisms are involved in these effects. Finally, in a third and last study, recently accepted for publication in Frontiers in Physiology, we sought to clarify, in primary culture of trout hepatocytes, the existing interactions between autophagy, ER homeostasis and intermediate metabolism under amino acid deprived conditions. Together, the results obtained in the present thesis extended our understanding of the role of dietary methionine at cellular level and emphasize the potential of this amino acid to apply new feeding strategies, such as nutritional programming, to optimize the nutrition and health of farmed fish.

Le stress du Réticulum Endoplasmique (RE) est impliqué dans la physiopathologie des maladies rénales, et la réponse UPR (Unfolded Protein Response), qui est activée en réponse à ce stress, joue un rôle important dans l'homéostasie des cellules tubulaires rénales et des podocytes. L’étude des mécanismes moléculaires et des conséquences de l'activation de cette voie est donc importante dans la compréhension de la physiopathologie des maladies rénales et dans la caractérisation de biomarqueurs de lésions évolutives. L’Angiogénine (ANG, appelée également RNase 5) est une ribonucléase secrétée, qui est impliquée dans la réponse à certains stress cellulaires, et permet une adaptation cellulaire et tissulaire.
L'objectif de ce travail a été de mettre en évidence les mécanismes de régulation et les fonctions biologiques de l'ANG en réponse au stress du RE. A partir d'un modèle de cellules tubulaires rénales humaines en culture, nous avons montré que le stress du RE induisait l’expression de l’Angiogénine ainsi que sa sécrétion. Cette observation a été également faite sur différents modèles murins de lésions rénales. Le facteur transcriptionel sXBP1, activé par le transducteur de la réponse UPR, IRE1a, est directement impliqué dans la régulation de l'expression de l'Angiogénine.
Nous avons mis en évidence que l'Angiogénine participait à l’inhibition de la traduction protéique en réponse au stress du RE en produisant des fragments d'ARN de transfert appelés tiRNAs (stress-induced tRNA fragments) qui répriment la traduction des protéines en interférant avec le complexe initiateur de la traduction. L'Angiogénine favorise la survie cellulaire en réduisant l'apoptose induite par le stress du RE, et des souris invalidées pour le gène codant l'Angiogénine sont plus sensibles aux lésions de nécrose tubulaire aigues induites par la Tunicamycine. Outre les propriétés cellulaires "intrinsèques" de l'Angiogénine, nous avons également caractérisé les mécanismes de sécrétion de l'Angiogénine par l'épithélium rénal en situation de stress du RE. La sécrétion épithéliale de l'Angiogénine est sous le contrôle des facteurs transcriptionnels NF-κB et sXBP1, et se produit sous un mode conventionnel, c’est-à-dire dépendant du transit par l'appareil de Golgi. A ce titre, la régulation de l'Angiogénine est similaire à celle de l'Interleukine 6. L'Angiogénine induit une polarisation des macrophages vers un phénotype pro-inflammatoire. Enfin, considérant que l'Angiogénine est secrétée par l'épithélium rénal en situation de stress, nous avons montré que l’Angiogénine peut être un marqueur non invasif de souffrance rénale. L'Angiogénine peut être quantifiée dans les urines de patients porteurs de maladies rénales, et sa concentration est corrélée à la concentration urinaire de Retinol Binding Protein (une protéine de petit poids moléculaire, marqueur de dysfonction tubulaire), mais pas avec celle de l'Albumine. En outre, la concentration urinaire d'Angiogénine est significativement plus élevée dans les urines de patients transplantés rénaux dont la biopsie rénale met en évidence des lésions de tubulite (rejet aigu cellulaire et néphropathie associée au BK virus) que dans les urines de patients indemnes de lésions tubulaires (rejet humoral, ou absence de lésions histologiques). Nous avons mis en évidence par immuno-histochimie un marquage nucléaire du facteur transcriptionnel sXBP1 dans les tubules de reins porteurs de lésions de tubulite, suggérant un lien potentiel entre sécrétion d'Angiogénine et activation du facteur transcriptionnel sXBP1 dans un environnement inflammatoire. En conclusion, nous avons intégré la régulation l'Angiogénine dans la réponse épithéliale rénale au stress du RE, et caractérisé ses fonctions biologiques intracellulaires et paracrines. Notre travail a identifié l'Angiogénine urinaire en étant que potentiel marqueur de lésions rénales tubulaires. / The Endoplasmic Reticulum (ER) stress is involved in the pathophysiology of renal diseases ; the UPR (Unfolded Protein Response), which is activated in response to that stress plays an important role in renal tubular cells and podocytes homeostasis and consequently in tissu homeostasis. Understanding the molecular mechanisms and the consequences of the activation of this pathway is important to characterize the pathophysiology of renal diseases and identification of biomarkers of ongoing lesions. Angiogenin (ANG, also known as RNase 5) is a secreted ribonuclease, which is involved in the cellular stress response, it allows cell and tissue adaptation. The goal of this work was to clarify and identify the mechanisms regulating Angiogenin’s expression and its biological functions during ER stress. Using a human renal tubular cell line, we have shown that ER stress induces the expression of angiogenin and its secretion. This observation was also made on several murine models of renal injury. The transcriptional factor sXBP1 activated by the UPR transducer, IRE1α, is directly involved in regulating the expression of angiogenin. We have shown that angiogenin participates in the inhibition of protein translation in response to ER stress by cleaving transfer RNA and generating tiRNAs (stress-induced tRNA fragments) that suppress protein translation by interfering with the translation initiation complex. Angiogenin promotes cell survival by reducing ER stress-induced apoptosis, ANG knockout mice are more sensitive to acute tubular necrotic lesions induced by tunicamycin. In addition to the cell-autonomous effects of angiogenin, we also characterized the mechanisms by which Angiogenin is secreted by the renal epithelium under ER stress. Angiogenin is secreted in a conventional manner under the control of the transcriptional factors NF-kB and sXBP1. As such, the regulation of angiogenin is similar to Interleukin-6. We also demonstrated that Angiogenin induces macrophage polarization to a pro-inflammatory phenotype. Finally, considering that angiogenin is secreted by the renal epithelium under stress, we have shown that angiogenin may be a noninvasive marker of kidney injury. Angiogenin can be quantified in the urine of patients with kidney disease, its urinary concentration is correlated to the urinary concentration of Retinol Binding Protein (a low molecular weight protein marker of tubular dysfunction), but not with that of Albumin . In addition, the urinary concentration of angiogenin is significantly higher in the urine of renal transplant patients whose renal biopsy highlights tubulitis lesions (cell acute rejection and BK virus associated nephropathy) than in the urine of patients without histological tubular damage (antibody-mediated rejection, or no visible histological lesions). We have demonstrated by immuno-histochemistry a tubular nuclear localization of the activated transcriptional factor sXBP1 in the biopsies of patients with high tubulitis score, suggesting a potential relationship between the secretion of Angiogenin and the activation of transcriptional factor sXBP1 within an inflammatory environment. To conclude, we have described Angiogenin as a new mediator of the integrated ER stress response, and characterized its cell- and non-cell-autonomous biological functions. Our study have identified urinary angiogenin as a potential marker of ongoing kidney tubular injuries.