L'insuffisance rénale chronique (IRC) est associée à une importante morbidité et mortalité cardio-vasculaire, à laquelle participent la dysfonction endothéliale. Les patients IRC présentent de plus une susceptibilité accrue aux thromboses qu’elles soient veineuses ou artérielles. Les toxines urémiques sont des solutés s'accumulant dans le sérum et les tissus des patients IRC. Parmi elles, les toxines urémiques liées aux protéines ont une toxicité endothéliale démontrée in vitro. Nous avons démontré que l’indoxyl sulfate (IS) et l’indole acetic acide (IAA), deux toxines liées aux protéines provoquent un phénotype pro coagulant de cellules endothéliales en culture via une production accrue de facteur tissulaire (FT). Le facteur tissulaire est un facteur membranaire procoagulant qui initie la cascade de la coagulation en activant le facteur VII via la voie extrinsèque. Nous avons aussi démontré que la production de FT passe par une voie cellulaire préalablement connue pour son implication dans les processus de detoxification : la voie de l’aryl hydrocarbon receptor (AHR). Dans notre travail, nous montrons que l’IS et l’IAA ont un comportement similaire à l’intoxication par la dioxine. Le lien entre FT et AHR n’avait jamais été démontré auparavant. En conclusion, l’IS et l’IAA participent à la dysfonction endothéliale des patients IRC et à la surmortalité cardiovasculaire, en augmentant la production endothéliale de FT et ainsi en provoquant un phénotype pro coagulant des cellules endothéliales. La voie AHR constitue une cible thérapeutique très intéressante dans la problématique de la surmortalité cardiovasculaire des patients IRC. / Chronic kidney disease (CKD) is associated with significant morbidity and cardiovascular mortality, which involves chronic inflammation, oxidative stress and endothelial dysfunction. CKD patients have a higher risk of venous or arterial thrombosis compared to general population. Uremic toxins are molecules that accumulate in the serum and organs of CKD patients. Among them, protein-bound uremic toxins are poorly removed by dialysis, and their endothelial toxicity had been well demonstrated in vitro. In this thesis, we demonstrated that indoxyl sulfate (IS) and indole acetic acid (IAA ), two protein-bound toxins can cause a pro coagulant phenotype of cultured endothelial cells through an increased production of tissue factor (TF ) Tissue factor is a membrane procoagulant factor that initiates the coagulation cascade by activating factor VII via the extrinsic pathway. We also demonstrated that TF increase was produced via a cellular pathway previously known to be involved in the detoxification processes: the aryl hydrocarbon receptor pathway (AHR) . The canonical ligand of AHR is dioxin, well known for its cardiovascular adverse effects. In this work, we showed that IS and IAA had a “dioxin- like effect”. The link between FT and AHR had never been shown earlier. CKD constitutes an endogenous situation similar to dioxin poisoning. In conclusion, the IS and IAA are involved in endothelial dysfunction in CKD patients and cardiovascular mortality by increasing the endothelial production of TF and thus causing a pro- coagulant phenotype of endothelial cells. AHR pathway is a very interesting therapeutic target in the problematic of cardiovascular mortality in CKD patients .

Die fortschreitende Vermehrung extrazellulärer Matrixproteine ist zentrales Kennzeichen von chronisch-progressiver Niereninsuffizienz. L-Arginin ist eine semi-essentielle Aminosäure und über seinen endogenen Metaboliten Stickoxid (NO) in komplexer Weise mit renaler Matrixvermehrung verbunden. In dieser Arbeit wurde untersucht, wie sich die Gabe von L-Arginin auf die Matrixexpansion bei experimenteller, immun-vermittelter Nierenerkrankung auswirkt. Im Modell der akuten Anti-Thy1-Glomerulonephritis der Ratte und der chronischen Lupusnephritis der MRL/lpr-Maus wurde gezeigt, daß die Aktivierung des L-Arginin-NO-Stoffwechsels sowohl mit günstigen als auch mit ungünstigen Wirkungen auf die renale Matrixakkumulation verbunden ist. Diese "duale" Wirkung von L-Arginin ist im wesentlichen als Ausdruck der "ambivalenten" Wirkung von NO zu deuten. Antifibrotische Wirkungen von L-Arginin stehen in enger Verbindung mit einer gesteigerten endothelialen NO-Synthese. Neben renaler Blutdrucksenkung vermittelt die endotheliale NO-Synthase auch parakrin wichtige antifibrotische Effekte. Profibrotische Wirkungen von L-Arginin stehen in engem Zusammenhang mit einer gesteigerten NO-Synthese durch induzierbare, destruktive NO-Synthasen. Folgen sind verstärkte Organschädigung und beschleunigte Progression von renaler Funktionseinschränkung. / Ongoing expansion of extracellular matrix proteins is a hallmark of progressive chronic renal insufficiency. L-Arginine is a semi-essential amino acid and alters renal matrix accumulation via its endogenous metabolite nitric oxide (NO) in a complex manner. The present study analyzed how administration of L-arginine affects renal matrix accumulation in experimental immune-mediated disease. In acute anti-Thy1 glomerulonephritis of the rat and chronic lupus nephritis of MRL/lpr-mice, activation of the L-arginine-NO-pathway was related to both beneficial and detrimental actions on renal matrix accumulation. This "dual" effect of L-arginine administration essentially reflects the "ambivalent" nature of NO. Antifibrotic actions of L-arginine are associated with increased endothelial NO synthesis. In addition to lowering glomerular blood pressure, endothelial NO production mediates important paracrine antifibrotic actions. Profibrotic effects of L-arginine are related to increased NO production by inducible, destructive NO synthases, resulting in increased organ damage and accelerated progression of chronic kidney insufficiency.

RESUME Contexte La maladie rénale chronique (MRC) constitue un problème mondial majeur de Santé publique. Son ampleur réelle en Afrique demeure inconnue. Malgré, les progrès réalisés dans lidentification et la prévention de la MRC et le traitement de la phase terminale de la maladie, ces domaines restent un grand défi en Afrique Sub-saharienne à cause du manque cruel des ressources nécessaires. Objectif Ce travail a pour objectif de cerner lépidémiologie de la MRC à Kinshasa en vue délaborer des stratégies de dépistage précoce et de prévention adaptées. Le but ultime est de contribuer à la réduction de la morbidité et la mortalité rénales mais aussi cardiovasculaires. Méthodes : Le présent travail est une revue synthétique de 4 études menées à Kinshasa : Une étude documentaire des 412 cas réalisée aux Cliniques Universitaires de Kinshasa (CUK), durant la période allant de Janvier 2001 à Décembre 2004 pour identifier le profil épidémiologique et clinique des patients atteints de la MRC. Les résultats de cette étude ont motivé le besoin dévaluer lampleur de la maladie dans la population et dans les structures de santé existantes. Il en a résulté trois études. Une étude épidémiologique de type transversal effectuée à partir de 503 ménages sélectionnés de manière aléatoire selon un plan de sondage à plusieurs degrés dans 10 des 35 Zones de santé composant Kinshasa, capitale de la République Démocratique du Congo (RDC). Une seconde étude, aussi de type transversal, réalisée à partir de 527 patients à risque de MRC, fréquentant neuf Centres de santé (CS) de niveau primaire et quatre hôpitaux de référence de la ville de Kinshasa. Une campagne de dépistage de la protéinurie et des facteurs de risque de la MRC chez 3.018 sujets. Résultats : Lanalyse des données enregistrées en milieu hospitalier a montré : Une augmentation annuelle progressive et inquiétante des proportions (60,6%, 65,9%, 67,4% et 70,5%) de la MRC admises aux CUK quasi exclusivement au stade terminal de la maladie nécessitant une prise en charge rapide par la dialyse péritonéale. Malheureusement, 11% seulement pouvaient accéder à ce traitement onéreux. La majorité des malades à prédominance masculine (sexe ratio 2,2/1) décèdent prématurément à un âge moyen (45,8±14,5 ans), à un moment de leur vie où ils sont encore économiquement très productifs. Les causes probables de la MRC chez ces patients sont la glomérulonéphrite chronique (37%), lhypertension artérielle (27%) et le diabète sucré (26%). Les études transversales dans la population générale et les institutions de santé traditionnelles de la ville de Kinshasa ont mis en évidence les caractéristiques épidémiologiques suivantes: La prévalence globale (tous les stades confondus) de la MRC est de 12% dans la population générale, mais 3% seulement sont conscients de leur état de rein. Celle de linsuffisance rénale chronique (IRC) estimée par le débit de filtration glomérulaire (DFGe) < 60 ml/min/1,73 m² est de 8%. Cette MRC touche particulièrement les adultes (52±15 ans). Les facteurs de risque potentiels de la MRC, liés à des maladies non transmissibles (MNT) sont en progression comparativement aux études antérieures. Ces facteurs sont lhypertension (28%), le diabète sucré (12%) et lobésité (15%). Dans les Centres de santé de Kinshasa, la prévalence globale de la MRC méconnue parmi les sujets à risque est le triple de celle rapportée dans la population générale de la même ville. Parmi cette population malade, les proportions de la MRC atteignent 44% chez les hypertendus, 39% chez les diabétiques ; 16% chez les obèses et 12% chez les sujets infectés par le Virus de limmunodéficience humaine (VIH). 82% des diabétiques avaient une glycémie à jeun non contrôlée (> 126 mg/dl) et 78% dhypertendus navaient pas une pression artérielle sous la cible la moins stricte, cest à dire contrôlée à moins de 140/90 mmHg. Les déterminants identifiés de lIRC ont été lhypertension (OR ajusté 3,3), le diabète sucré (OR 2) et la protéinurie (OR 2,9). Les principaux déterminants de DFGe < 60 ml/min/1,73 m² chez les diabétiques étaient lâge et la durée du diabète sucré. Les résultats de la campagne de dépistage de la protéinurie et des facteurs de risque de la MRC ont révélé ce qui suit : La prévalence de la protéinurie a été de 17%. Les autres facteurs de risque de la MRC identifiés chez les sujets en bonne santé apparente ont été: lhypertension (37%), le diabète sucré (9%), lobésité (11%) et le syndrome métabolique (5%). Pour identifier un cas de protéinurie, il est nécessaire de dépister 4 diabétiques, 5 hypertendus, 4 sujets avec syndrome métabolique, 5 sujets âgés de plus de 50 ans et 9 personnes ne présentant aucune des conditions susmentionnées. Les déterminants majeurs de la protéinurie étaient lâge > 50 ans (OR ajusté 1,4), le diabète sucré (OR 1,3), le surpoids (OR 1,2) et le niveau socio-économique bas (OR 1,4). Conclusion : Ces études établissent pour la toute première fois dans une population africaine la forte prévalence de la MRC et ses facteurs de risque notamment lhypertension, le diabète sucré, lobésité, lâge > 50 ans et linfection à VIH. La maladie affecte ladulte encore jeune comparée aux Etats-Unis où elle prédomine à la vieillesse. Nos études ont montré aussi à la fois la forte prévalence de la protéinurie chez les sujets sans facteurs de risque traditionnels précités, le déficit du dépistage précoce de la MRC et de prise en charge des facteurs de risque dans le système de santé traditionnel favorisant la référence tardive et/ou les décès prématurés, ainsi que les limites malheureuses par manque de moyens de la prise en charge de la maladie au stade tardif. Ces études plaident pour la nécessité dun renforcement de la capacité du personnel soignant dans le domaine de détection précoce et de prise en charge des MNT dont la MRC. Elles montrent également quun dépistage annuel de masse de la population de la protéinurie et des facteurs de risque de la MRC est faisable et pourra, nous lespérons, constituer la base dune élaboration dune politique nationale de prévention. Mots-clé : diabète sucré, équation (Cockcroft & Gault, MDRD), hypertension artérielle, maladie rénale chronique, prévalence, protéinurie. SUMMARY Background Chronic kidney disease (CKD) is a worldwide public health problem. Little is known about its burden in Africa. Despite the advances in identification and prevention of CKD and management of end stage renal disease (ESRD), sub-Saharan Africa has been left far behind regarding these advances. This is because of the scarcity of necessary resources. Objective This work was designed to ascertain the epidemiologic knowledge of CKD in Kinshasa in order to define suitable baseline preventive strategies. It would aims ultimately, to reduce the morbidity and mortality from renal disease and related cardiovascular events. Methods: This current work summarises results of 4 studies undertaken in Kinshasa: A retrospective cross sectional study of 412 cases which was done in the Academic hospital of Kinshasa (AHK), from January 2001 to December 2004 to identify the epidemiologic and clinical profile of patients with CKD. The results of this study motivated us to investigate the extent of the burden of CKD in the population and the existing structures of healthcare. Thus, three further studies were carried out; In an epidemiologic cross sectional study, 503 adult residents in 10 of the 35 health zones of Kinshasa, the capital of the DRC were studied in a randomly selected sample; In a second study of higher risk subjects, 527 people in primary and secondary health care areas in the city of Kinshasa were studied from a random sample of at-risk out-patients with hypertension, diabetes, obesity, or who were infected by HIV; Finally, a mass screening for proteinuria and CKD risk factors was conducted in Kinshasa which involved 3,018 subjects. Results: The analysis of the data recorded in health care had showed: An overwhelmingly annual increasing proportion of CKD (60.6%, 65.9%, 67.4% and 70.5%) in AHK, unfortunately for the majority at stage 5, in other words at ESRD. Tragically enough, only 11% of them could be treated by peritoneal dialysis depending on their financial resources. The majority of the patients are young males (sex ratio 2.2/1) undergoing premature death (45.8±14.5). The probable causes of CKD in these subjects were chronic glomerulonephritis (37%), hypertension (27%) and diabetes mellitus (26%). The cross-sectional studies in the general population and the traditional structures of health care (HC) of the city of Kinshasa highlighted the following: The overall prevalence of CKD is 12% in the general population, but only 3% of those with CKD were aware of their condition. The prevalence of chronic renal failure (CRF) (eGFR < 60 ml/min/1.73 m ²) is 8%; CKD affects particularly young adults (52±15 years); Risk factors for CKD considered in this study, including hypertension (28%), diabetes (12%) and obesity (15%), are increasing compared to the former studies. In HC, the overall prevalence of undiagnosed CKD among at-the risk subjects is three times higher the prevalence of CKD in the general population of the same city. In those with the at-risk conditions, the % of CKD was: 44% in the hypertensive, 39% in the diabetics; 16% in the obese and 12% in those who were infected by the human immunodeficiency virus (HIV). 82% of those with history of diabetes had fasting serum glucose levels (> 126 mg/dl), and 78% of those with a history of hypertension did not have blood pressure controlled to less than 140/90 mmHg. The strongest determinants of CRF or CKD 3+ were: hypertension (adjusted OR 3.3), diabetes (OR 2) and proteinuria (OR 2.9). The principal determinants of eGFR < 60 ml/min/1.73 m² in the diabetic patients were age and the duration of diabetes. The results of the campaign of early detection for proteinuria and CKD risk factors revealed that: The prevalence of proteinuria was 17%. The other CKD risk factors identified were: hypertension (37%), diabetes (9%), obesity (11%) and metabolic syndrome (5%). To identify 1 case of proteinuria, one would need to screen 4 persons with diabetes, 5 persons with hypertension, 4 subjects having metabolic syndrome, 5 subjects aged ≥ 50 years and 9 people without any of the conditions mentioned above. The strongest determinants of proteinuria were age > 50 years (adjusted OR 1.4), diabetes (OR 1.3) and overweight (OR 1.2) and low socioeconomic status (OR 1.4). Conclusion: This work documents for the first time in Africa the high prevalence of CKD and its risk factors mainly hypertension, diabetes, obesity and HIV infection. CKD affects younger people in DRC, in contrast to the United States, where CKD is more prevalent in older. Our work also shows the high prevalence of proteinuria among subjects with neither diabetes nor hypertension, the deficit of the early detection and management of CKD risk factors in the traditional health care system leading to late referral or premature deaths, and the limits of renal replacement treatment. They also show that an annual mass screening of the population for proteinuria and CKD risk factors is feasible and will, it is hoped, provide the basis for building a nationwide prevention strategy. Key words: chronic kidney disease, diabetes mellitus, equation (Cockcroft &Gault, MDRD), arterial hypertension, prevalence, proteinuria.

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Introduction : Partant du constat qu'il est possible de conserver plus longtemps les denrées alimentaires grâce à un conditionnement sous atmosphère modifiée enrichie en gaz nobles, nous avons développé une stratégie innovante de conservation de transplants rénaux. Nous avons évalué l'effet protecteur d'une solution de conservation saturée en gaz nobles pour la préservation des transplants rénaux.Matériels et méthodes : Dans un modèle d'autotransplantation rénale hétérotopique chez le porc, les transplants prélevés ont été rincés et mis en conservation 30h à 4°C dans du Celsior présaturée en gaz (air, azote, argon 100% ou xénon 100%, n=6 dans chaque groupe) avant transplantation. Les porcs ont été surveillés quotidiennement pendant 21 j.Résultats : L'argon a amélioré la survie (83,3% vs 33,3% avec l'Air, p=0,04) et la reprise de fonction du transplant. Une sortie de tubulopathie significativement plus précoce des transplants a été observé avec l'Argon. Tous les porcs xénon et azote sont décédés. A J21 les transplants argon avaient une meilleure préservation de leur intégrité structurelle cellulaire avec moins d'inflammation, de fibrose interstitielle et d'atrophie tubulaire. Les rapports RAA/TBARS, et d'Hsp 27, étaient significativement plus élevés avec l'argon. Les taux de TNF alpha, Il 6 et 8 ont montré une diminution de la réponse inflammatoire avec l'argon.Conclusion : Nous avons démontré l'effet bénéfique de l'argon sur la reprise précoce de fonction de transplant et en limitant les lésions d'ischémie-reperfusion. Bien que le mécanisme d'action de l'Argon ne soit pas élucidé, il semble que Hsp 27 soit un élément central de la renoprotection. / Introduction: Based on prolonged preservation of perishable food products under modified atmosphere, we developed an innovative method to preserve kidneys during transplantation using nobles gases. We evaluated the protective effect of argon and xenon on preserving kidney graft functionality and integrity in a clinically relevant pig model of transplantation. Methods: The left kidneys of pigs (n=6 per group) were removed, flushed and stored for 30 h in Celsior solution saturated with air, nitrogen, 100% argon, or 100% xenon. Next, autotransplantation and controlateral nephrectomy were performed. The survival rate, renal function, Hsp27, thiobarbituric acid (TBARS), reduced ascorbic acid (RAA), and TNF alpha were analyzed. A histological examination was completed.Results: Argon improved survival (83.3% for argon vs 33.3% for air, p=0.04) and transplant function recovery. All pigs in the nitrogen and the xenon group died. Diuresis recovery occurred earlier in the argon group (n= 5) when compared with the air group (n=2), p=0.05. On day 7 argon transplants had lower serum creatinine levels and a large reduction in primary non function than the air group. Argon-treated tissues showed better cell structural integrity with minor signs of inflammation, fibrosis, and tubular atrophy. The argon group showed significantly higher RAA/TBARS ratios and Hsp27 levels.Conclusion: We demonstrated that modified atmosphere preservation packaging with argon in cold-storage solution improved early transplant function recovery and long-term quality by minimizing IRI in a pig model of prolonged cold-ischemia. The renoprotective effect of argon may involve the Hsp27 pathway.

Il termine malattia renale cronica (Chronic Kideny Disease: CKD) si riferisce a differenti condizioni caratterizzate da un progressivo declino della funzione renale. Le linee guida internazionali hanno definito la CKD come una condizione in cui siano presenti marcatori di danno renale e/o la velocità di filtrazione glomerulare stimata (Estimated Glomerular Filtration Rtae: eGFR) sia inferiore a 60 ml/min/1.73 m2 per almeno 3 mesi. L’insufficienza renale in stadio terminale è associata ad un alto rischio di malattia cardiovascolare (Cardiovascular Disease: CVD), la più frequente causa di morte in questi pazienti. Fattori di rischio “non-tradizionali” come: infiammazione cronica, stress ossidativo, deplezione proteico-energetica, disordini del metabolismo minerale e deficit di inibitori della calcificazione, partecipano alla patogenesi della CVD. L’infiammazione gioca un ruolo cruciale nella risposta fisiologica all’infezione e al danno renale e partecipa anche nell’evoluzione del danno renale irreversibile con la produzione di diverse molecole infiammatorie a partire da acidi grassi polinsaturi a lunga catena (Long Chain PolyuUsaturated Fatty Acids: LCPUFA) della serie Omega-6. La supplementazione di Omega-3, con effetto antinfiammatorio, nei pazienti affetti da CKD è stata ed è oggetto di molti studi, nonostante ciò, l’effetto sul danno renale è ancora poco chiaro. Comunque, è ampiamente riconosciuto che un alterato profilo lipidico possa determinare la progressione della patologia, inducendo lo stato infiammatorio. Inoltre, elevati/normali livelli di Omega-3 potrebbero essere associati al miglioramento della funzionalità renale, diminuendo quindi il rischio di peggioramento della malattia. Le concentrazioni e il rapporto di Omega-3 e Omega-6 sono strettamente associati alla salute del rene, poiché svolgono ruoli importanti in differenti vie metaboliche. Un altro aspetto, preso poco in considerazione, è l’effetto dei livelli di acidi grassi circolanti e dei loro metaboliti sullo stato infiammatorio e sulla sua modulazione. Il primo scopo di questo studio è stato quello di analizzare il profilo degli acidi grassi in soggetti anziani affetti da CKD. Sono stati arruolati 57 pazienti afferenti agli ambulatori di Nefrologia dell’Ospedale Maggiore Policlinico di Milano e sono stati raccolti campioni di sangue su cui è stata effettuata l’analisi del profilo lipidico. Negli ultimi anni, diversi studi hanno sottolineato la stretta associazione tra infiammazione a livello intestinale e peggioramento del quadro in pazienti con CKD. Il mantenimento di un ottimo stato del tratto gastrointestinale è fondamentale per assicurare lo stato di salute dell’ospite, contribuendo ai processi metabolici, fisiologici e immunologici. Le comunità batteriche instaurano un rapporto mutualistico con l’individuo che colonizzano, giocando un ruolo importante negli stati di salute e malattia. Un’anomala colonizzazione o cambiamenti nella composizione del microbiota intestinale, determina disbiosi, uno squilibrio associato a diverse condizioni patologiche come obesità, diabete di tipo II, malattia intestinale cronica, CVD e anche CKD. Il rapporto tra intestino e rene è bidirezionale, nei pazienti affetti da malattia renale cronica, la composizione del microbiota intestinale risulta essere modificata rispetto a quella del soggetto sano. Alti livelli di urea che si riversano facilmente nel tratto intestinale modificano il microambiente chimico con conseguente innalzamento del pH del colon che esercita una pressione selettiva a favore di specie ureasi-positive, responsabili della conversione dell’urea in ammoniaca. Lo strato protettivo di muco viene degradato e la permeabilità della barriera intestinale viene compromessa. In conseguenza di ciò si ha il passaggio di materiale batterico attraverso la mucosa e l’attivazione di un meccanismo infiammatorio. Nei pazienti con funzionalità renale compromessa, il rene perde progressivamente la capacità di eliminare sia le sostanze provenienti dal metabolismo umano, sia quelle della comunità microbica intestinale. Alcune di queste sostanze sono rappresentate dalle tossine uremiche, tra quelle di derivazione intestinale le principali e più studiate sono p-cresil solfato (PCS) e indossile solfato (IS). IS e p-CS, strettamente legate all’albumina sierica (Human Serum Albumin: HSA), non vengono eliminate facilmente ma rimangono nel torrente ematico. HSA è la più abbondante proteina sierica ed è la principale trasportatrice di composti esogeni ed endogeni, inclusi gli acidi grassi che sembrano rappresentare il maggior ligando endogeno della proteina. Multipli siti di legame vengono utilizzati per gli acidi grassi monoinsaturi (MonoUnsaturated Fatty Acids: MUFA) e PUFA. Acidi grassi e tossine uremiche competono quindi per gli stessi siti di legame sulla proteina. Il potenziale ruolo degli acidi grassi nel contrastare l’accumulo di tossine uremiche derivate dalla comunità batterica intestinale ne giustifica l’importanza della valutazione dei loro livelli ematici. Secondo scopo di questa tesi di dottorato è stato quello di valutare la possibile correlazione tra i livelli di acidi grassi circolanti e la composizione del microbiota intestinale in soggetti affetti da CKD. Sono stati arruolati nello studio 64 pazienti anziani con CKD non dializzati e 15 soggetti anziani con normale funzionalità renale. La composizione del microbiota intestinale è stata precedentemente caratterizzata attraverso l’impiego delle tecniche di elezione: PCR-DGGE e la PCR quantitativa (qPCR). In accordo con la letteratura scientifica, è stata evidenziata una riduzione di batteri saccarolitici e produttori di butirrato nei pazienti con CKD rispetto al gruppo di controllo. Il butirrato sembra giocare un ruolo cruciale nel mantenimento delle ottimali condizioni della barriera intestinale. Tenendo ciò in considerazione è stato deciso di approfondire lo studio e valutare l’associazione tra la comunità microbica intestinale e i livelli di acidi grassi basali in tali pazienti. Come risultato più importante ottenuto, è stata osservata una correlazione positiva statisticamente significativa tra la specie batterica Faecalibacterium Prausnitzii e i livelli totali di Omega-3 entrambi associati a proprietà antinfiammatorie. La presente tesi di dottorato evidenzia la necessità di sostenere ulteriori ricerche per supportare i risultati qui presentati. Studi futuri potrebbero essere utili per migliorare la comprensione del ruolo degli acidi grassi circolanti e i loro metaboliti sulla composizione del microbiota intestinale, sullo stato infiammatorio e sulla malattia renale cronica. / The aim of this thesis was to explore the possible associations between fatty acids (FA) profile and gut microbiota (gMb) with several conditions throughout the lifespan, from infancy to old age. In particular, we focused our attention on elderly subjects with Chronic Kidney Disease (CKD) and children with Acute Otitis Media (AOM). The terms “Chronic Kidney Disease” refers to several disorders with a progressive kidney function decline. International guidelines approved the definition of CKD as a condition with the presence of markers of kidney damage or with the estimated glomerular filtration rate (eGFR) less than 60 ml/min/1.73 m2 or both, for at least three months. End-stage renal disease is associated with a high cardiovascular disease (CVD) risk, the major cause of death in these patients. Chronic inflammation, oxidative stress, protein-energy wasting, disordered mineral metabolism, and deficiency of endogenous calcification inhibitors, known as non-traditional risks factor, take part in cardiovascular pathology in CKD. Inflammatory processes influence the physiological response to renal infection and injury but also participate in the development of potentially irreversible kidney damage with the production of various inflammatory molecular species, among whom eicosanoids and cytokines, from parental omega-6 long-chain polyunsaturated fatty acids (LCPUFA). Several studies focused their attention on the potential role of omega-3 (n-3) LCPUFA supplementation in subjects with CKD. Despite this, their effect on kidney damage is still not clear. However, it is widely agreed that a modified FA profile in CKD can determine a progression of the disease, inducing the inflammatory state. Moreover, high/normal n-3 LCPUFA levels decrease the risk of a decline of the disease. Omega-3 and omega-6 (n-6) LCPUFA concentrations and their ratios are tightly associated with renal health, because of their important roles in different pathways. Another aspect not very considered in the field of CKD is the role of circulating FA levels and their metabolites on the modulation of inflammation. The first aim of this study is to analyze the FA profile in elderly subjects with CKD. Blood samples have been collected from 57 subjects enrolled in the study, and FA analysis has been performed. During the last years, several studies underlined the strong relationship between intestinal inflammation and adverse outcomes in CKD. The health of gastrointestinal tract is fundamental to ensure the well being of the host contributing to its nutrition, metabolism, physiology, and immune function. The bacterial communities colonizing humans have been seen in terms of mutualistic symbiosis with their hosts, a mutually beneficial coexistence, playing an important role in health and disease. Abnormal colonization or changes in the gut microbial composition determine dysbiosis, a state associated with different illnesses, such as obesity, type 2 diabetes, inflammatory bowel disease, cardiovascular disease, and also chronic kidney disease. The relationship between gut and kidney is a bi-directional relation with a mutual influence. Chronic kidney disease influences gMB characteristics, especially through high levels of urea that easily spread in the intestinal fluid where bacterial urease enzymes degrade it, then it is hydrolyzed in ammonium hydroxide that increases fecal pH with a consequent alteration of intestinal cellular junctions. Besides, high levels of urea change intestinal microbiota composition damaging permeability of intestinal barrier and promoting proteolysis with production and absorption of uremic toxins, such as indoxyl sulfate (IS) and p-cresol sulfate (p-CS). These toxins induce an inflammatory process associated with CKD. Under physiologic conditions, the kidney through the urine eliminates these compounds, but CKD patients have a compromised renal clearance. Therefore, these solutes tend to accumulate in the organs. IS and p-CS are tightly bound to human serum albumin (HSA), the most abundant plasma protein in the bloodstream. HSA is recognized as the main means of transport for endogenous and exogenous compounds, including fatty acids that seem to be the main endogenous ligand of HSA, multiple binding sites are used for monounsaturated fatty acids (MUFA) and PUFA. Thus, free fatty acids and uremic toxins compete for the same binding sites on HSA. It is important to assess fatty acid (FA) levels in patients with CKD because of the potential role to contrast the accumulation of uremic toxins derived from the intestinal bacterial community. As a consequence of this bi-directional relation between gut and kidney and the possible involvement of some compounds as metabolites of FA in the inflammatory response, we investigate the correlation between circulating FA levels and the gMB composition in the same subjects with CKD, as the second aim of this thesis. 64 old CKD non-dialysis patients (eGFR 15-45 ml/min/1.73 m2) and 15 elderly subjects (>65 years) with normal renal function (eGFR >60 ml/min/1.73 m2, CKD-EPI) are enrolled. Bacterial composition was studied in a previous observational study by denaturating gel gradient electrophoresis (DGGE), high-throughput sequencing (16S ribosomal RNA), and quantitative real-time PCR (qPCR). This study described an increased abundance of some bacteria associated with pathological conditions. In agreement with the literature, the author found a reduced abundance of saccharolytic and butyrate-producing bacteria (Prevotella, Faecalibacterium prausnitzii, Roseburia) in CKD patients respect to the control group. Butyrate plays a crucial role in the maintenance of the gut barrier function. Taking that into account, we decided to investigate the correlation between gMB composition and FA profile in these subjects. The main result of the study was the significant positive correlation between Faecalibacterium prausnitzii and total n-3 levels, both associated with the antiinflammatory action. The present doctoral thesis underlines the need to perform further investigations in order to support evidence presented. Future studies may be useful to improve understanding of the effect of circulating fatty acids levels and their metabolites on gut microbial composition, inflammation process, and pathological conditions such as kidney disease. Our results showed that CKD patients with previous cardiovascular events had lower total and specific n-3 levels comparing with patients without them. Moreover, higher docosahexaenoic acid (DHA) levels and having had previous cardiovascular events seemed to have protective effects against further cardiovascular events. Moreover, we observed a significant reduction of the genera Roseburia and Faecalibacterium in CKD patients compared to C group and a significant lower abundance of F. prausnitzii and Roseburia spp. in CKD patients. Thus, our results seem in accordance with anti-inflammatory actions of total n-3, DHA, and saccharolytic and butyrateproducing bacteria. Many gMB changes seem to be related both to CKD and CVD. If the different gMB composition might play a causal role in cardiovascular events by an unbalanced production of some toxic substances, or if the gMB changes are merely a consequence of different dietary and lifestyle behaviours of these patients, it cannot be explained by the present study and all the yet available data. Further studies, possibly utilizing new high-throughput tools, will be required to understand the potential correlations between the gMB composition and other inflammation and oxidative stress markers in these patients. Other two studies have been performed during the doctoral course, to reach a better comprehension of fatty acids, gut microbial community and inflammatory states. A prospective pilot clinical study has been performed to to explore possible changes of gMB composition in children with AOM treated with amoxicillin with or without clavulanic acid. AOM is one of the most common bacterial infections in children and is normally treated with antibiotic therapies that lead to increasing antimicrobial resistance rates among otopathogens and may impair the correct development of the microbiota in early life. No significant differences were shown in the gMB composition of the overall cohort at different time intervals of the samples collection and in subjects treated with amoxicillin with or without clavulanic acid at different time intervals (T0, T1 and T2). A literature revision on lipids in infant formulae has been performed to better understanding quality and quality of dietary lipids because of their significant impact on health outcomes, especially when fat storing and/or absorption are limited (e.g., preterm birth and short bowel disease) or when fat byproducts may help to prevent some pathologies. The lipid composition of infant formulae varies according to the different fat sources used, and the potential biological effects are related to the variety of saturated and unsaturated FAs. Instead, ruminant-derived trans FAs and metabolites of n-3 LCPUFA with their anti-inflammatory properties can modulate immune function. Furthermore, dietary fats may influence the nutrient profile of formulae, improving the acceptance of these products and the compliance with dietary schedules. During the doctoral course, I spent a period abroad at Dell Pediatric Research Institute (DPRI), The University of Texas at Austin. In particular, I attended the laboratory of Doctor Brenna. I focused my research activity on a specific regulatory insertion-deletion polymorphism in the FADS gene cluster for better understanding its influence on PUFA and lipid profile.

Background: Renal denervation (RDN) can reduce cardiac sympathetic activity maintained by arterial hypertension (aHT). Its potential antiarrhythmic effect on rhythm outcome in patients with multi-drug resistant aHT undergoing catheter ablation for atrial fibrillation (AF) is unclear. Methods: The RDN+AF study was a prospective, randomized, two-center trial. Patients with paroxysmal or persistent AF and uncontrolled aHT (mean systolic 24-h ambulatory BP > 135 mmHg) despite taking at least three antihypertensive drugs were enrolled. Patients were 1:2 randomized to either RDN+AF ablation or AF-only ablation. Primary endpoint was freedom from any AF episode > 2 min at 12 months assessed by implantable loop recorder (ILR) or 7d-holter electrocardiogram. Secondary endpoints included rhythm outcome at 24 months, blood pressure control, periprocedural complications, and renovascular safety. Results: The study randomized 61 patients (mean age 65 ± 9 years, 53% men). At 12 months, RDN+AF patients tended to have a greater decrease in ambulatory BPs but did not reach statistical significance. No differences in rhythm outcome were observed. Freedom from AF recurrence in the RDN+AF and AF-only group measured 61% versus 53% p = .622 at 12 months and 39% versus 47% p = .927 at 24 months, respectively. Periprocedural complications occurred in 9/61 patients (15%). No patient died. Conclusion: Among patients with multidrug-resistant aHT and paroxysmal or persistent AF, concomitant RDN+AF ablation was not associated with better blood pressure control or rhythm outcome in comparison to AF-only ablation and medical therapy.

Diese Studie untersucht die Bedeutung von Angiotensin II- und Endothelin-1-vermittelten Mechanismen, die im Rahmen von hämodynamischen, hormonellen und renalen Reaktionen bei einen akuten Blutverlust einsetzen. Es wurden wache Hunde mit und ohne Vorbehandlung mit Angiotensin II Typ 1 (AT1) und/oder Endothelin-A (ETA) Rezeptorblockern untersucht. Protokoll 1: Nach einer 60-minütigen Kontrollstunde wurde den Hunden 25% ihres Blutes zügig entzogen. Nach einer Stunde wurde das Blut retransfundiert und die Datenaufzeichnung für eine weitere Stunde fortgesetzt. Protokoll 2: Wie Protokoll 1, aber mit AT1 Blockade durch Losartan i.v. Protokoll 3: Wie Protokoll 1, aber mit ETA Blockade durch ABT-627 i.v. Protokoll 4: Wie Protokoll 1, aber mit kombinierter AT1 plus ETA Blockade. In der Kontrolle sinkt der arterielle Mitteldruck (MAP) nach dem Blutentzug um ~25%, das Herzzeitvolumen (HZV) um ~40%, das Urinvolumen um ~60%, während die Plasmakonzentrationen von Angiotensin II (3.1-fach), Endothelin-1 (1.13-fach), Vasopressin (116-fach) und Adrenalin (3.2-fach) ansteigen. Unter AT1 Blockade kommt es zu einem überproportionalen Abfall des arteriellen Mitteldrucks und die glomeruläre Filtrationsrate (GFR) sinkt. Beim Blutentzug unter ETA Blockade steigt Noradrenalin und nicht Adrenalin an, und der Wiederanstieg des MAP infolge Retransfusion ist unvollständig. In allen Protokollen sinkt das HZV um den gleichen Betrag. Schlussfolgerungen: Für die kurzfristige Regulation des Blutdrucks und die renale Autoregulation der GFR nach Blutverlust spielt Angiotensin II eine wichtigere Rolle als Endothelin-1. Andererseits ist ein intaktes Endothelinsystem eine wichtige Voraussetzung für die vollständige Restitution des arteriellen Mitteldrucks in der Retransfusionsphase. Darüber hinaus scheint Endothelin-1 nach dem Blutentzug die Freisetzung von Adrenalin zu erleichtern, die Freisetzung von Noradrenalin jedoch zu mildern. Die bei einem akuten Blutverlust einsetzenden Kompensationsmechanismen scheinen den Blutfluss (HZV) viel effektiver aufrecht zu erhalten als den Blutdruck (MAP), denn das HZV, nicht aber der arterielle Mitteldruck, sank in allen Protokollen um den gleichen Betrag. / This study investigates angiotensin II and endothelin-1 mediated mechanisms involved in the hemodynamic, hormonal, and renal response towards acute hypotensive hemorrhage. Conscious dogs were pretreated with angiotensin II type 1 (AT1) and/or endothelin-A (ETA) receptor blockers or not. Protocol 1. After a 60 min baseline period, 25% of the dog's blood was rapidly withdrawn. The blood was retransfused 60 min later and data recorded for another hour. Protocol 2. Likewise, but preceded by AT1 blockade with i.v. Losartan. Protocol 3. Likewise, but preceded by ETA blockade with i.v. ABT-627. Protocol 4. Likewise, but with combined AT1 plus ETA blockade. In Controls, hemorrhage decreased mean arterial pressure (MAP) by ~25%, cardiac output by ~40%, and urine volume by ~60%, increased angiotensin II (3.1-fold), endothelin-1 (1.13-fold), vasopressin (116-fold), and adrenaline concentrations (3.2-fold). Glomerular filtration rate and noradrenaline concentrations remained unchanged. During AT1 blockade, the MAP decrease was exaggerated (-40%) and glomerular filtration rate fell. During ETA blockade, noradrenaline increased after hemorrhage instead of adrenaline, and the MAP recovery after retransfusion was blunted. The decrease in cardiac output was similar in all protocols. Conclusions: Angiotensin II is more important than endothelin-1 for the short-term regulation of MAP and glomerular filtration rate after hemorrhage, whereas endothelin-1 seems necessary for complete MAP recovery after retransfusion. After hemorrhage, endothelin-1 seems to facilitate adrenaline release and to blunt noradrenaline release. Hemorrhage-induced compensatory mechanisms maintain blood flow more effectively than blood pressure, since the decrease in cardiac output - but not MAP - was similar in all protocols.

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