Les microARNs (miARN) sont de petits ARN régulant l’expression des gènes au niveau post-transcriptionnel. Ils sont impliqués dans la régulation de nombreux processus biologiques nécessaires au fonctionnement cellulaire, comme le contrôle de la réponse à l’inflammation. Dans ce travail, nous avons évalué l’implication des miARN dans la réponse tubulaire rénale à l’inflammation. Au cours d’un premier travail nous avons étudié par une approche globale le profil d’expression des miARN dans des cellules tubulaires proximales de la lignée HKJ2 exposées à des cytokines pro-inflammatoires. Nous avons ainsi identifié la forte induction de miR-146a en réponse au stimulus inflammatoire. Nous avons ensuite mis en évidence in vitro que l’induction de miR-146a par l’IL-1β dans les cellules HK-2 est secondaire à l’activation de la voie NF-κB, constitue un rétrocontrôle négatif de cette voie et régule l’expression de CXCL8 en aval. In vivo, l’étude du phénotype des souris invalidées pour miR-146a dans un modèle d’agression tubulaire rénale où l’inflammation joue un rôle significatif a mis en évidence une aggravation des lésions tubulaires, de l’infiltrat inflammatoire et de la fibrose interstitielle en réponse à l’ischémie-reperfusion. Le blocage de la signalisation induite par CXCL8 par la réparixine, un inhibiteur du récepteur de CXCL8 (CXCR1), permet de limiter le développement des lésions induites par l’ischémie-reperfusion chez les souris miR-146a-/-. Dans un second travail nous avons exploré le phénotype rénal des souris invalidées pour miR-146a, connues pour développer une auto-immunité. / Independently of its cause, acute kidney injury leads to the development of tubular injury and interstitial inflammation that need to be controlled to avoid fibrosis development. We hypothesized that microRNAs (miRNAs) are involved in the regulation of the balance between lesions and adaptive repair. Using HK2 human proximal tubular epithelial cells, we studied in vitro the response to pro-inflammatory cytokines and the regulation of miR-146a. We explored its targets in HK2 cells after stimulation by IL-1β. In vivo we explored the effect of unilateral renal ischemia-reperfusion injury (IRI) in wild-type or miR-146a invalidated mice. In pro-inflammatory conditions, we identified miR-146a to be transcriptionally upregulated by ligands of the interleukin-1-toll-like receptor signaling in HK2 cells. IL-1β treatment induced miR-146a expression in a time- and concentration-dependent manner through the activation of NF-κB, as confirmed by siRNA and luciferase reporter vector experiments. MiR-146a acted as a negative feedback regulator of this critical pathway by targeting IRAK1, thus decreasing CXCL8/CXCL1 expression by injured tubular cells. In vivo, miR-146a was found to be induced in response to renal IRI in a mouse model of renal unilateral IRI seven days after the injury. In human, miR-146a was found to be induced in the renal allograft of patients who experienced acute tubular necrosis early after transplantation as compared to patients with normal allograft biopsy results (P<0.05). Mir-146a levels were also increased in urine samples collected ten days after renal transplantation in recipients of a deceased donor kidney as compared to recipients of a living donor kidney (P<0.01). In situ hybridization localized up-regulated miR-146a mostly in tubular cells after IRI. Fourteen days after unilateral IRI, miR-146a-/- mice had greater tubular injury, inflammatory infiltrate and fibrosis compared with wild-type mice. Inhibition of the CXCL8/CXCL1 signaling using reparixin, a CXCR2 inhibitor, prevented the development of tubular injury, inflammation and fibrosis after IRI in miR-146a-/- mice. In conclusion, these results highlight miR-146a as a key mediator of the renal response to injury by limiting the consequences of inflammation, a key process in the development of acute and chronic kidney diseases.

Acute kidney injury (AKI) is a major public health concern, which contributes to serious hospital complications, chronic kidney disease (CKD) and even death. Renal ischemia- reperfusion injury (IRI) remains a leading cause of AKI.IRI combines major cell stress, significant burst of free radicals, and strong inflammatory responses leading to extensive cell injury, necrosis, and late interstitial fibrosis. Moreover, IRI- induced AKI releases pro-inflammatory cytokines (e.g. IL-1β, TNF-α, IL-6) that induce a systemic inflammatory response, resulting in pro-inflammatory cells recruitment and remote organ damage. AKI is associated with poor outcomes, particularly when extrarenal complications or distant organ injuries occur.The stress-responsive enzyme, heme oxygenase-1 (HO-1) mediates protection against renal IRI and may be preventively induced using hemin prior to renal insult. This HO-1 induction pathway called hemin preconditioning is largely known in the literature to be effective.We first confirmed that hemin-induced HO-1 improved renal outcomes after IRI (i.e. fewer renal damage, renal inflammation and oxidative stress). We then demonstrated that this protective pathway mitigated AKI-induced ALI, a major extrarenal complication after renal IRI, through modulation of systemic and lung inflammation.Afterwards, we focused on the specific contribution of myeloid HO-1 to renal IRI, which remains poorly characterized. We therefore investigated the contribution of myeloid HO-1 to renal IRI using mice with myeloid-restricted deletion of HO-1 (HO-1M-KO). We observed that myeloid HO-1 appeared to be a critical regulator of the earliest phases of IRI (i.e. higher plasma creatinine, tubular damage, and renal inflammation/oxidative stress in HO-1M-KO mice).As a link between the severity of renal injury and the risk maladaptive repair leading to CKD has been established, we thereby decided to focus on tubular repair and fibrosis deposition upon IRI. We identified that myeloid HO-1 prevented maladaptive repair and subsequent CKD through modulation of cell-cycle and autophagy regulatory proteins.We then showed that hemin-mediated protection requires specific expression of HO-1 within myeloid cells. We therefore identified CD11b+ F4/80lo macrophages as the main protective myeloid source of HO-1 upon renal IRI. Interestingly, we observed this myeloid cell sub- population in the kidney and spleen, suggesting that protective effects might be provided by both tissue-resident and infiltrating/circulating HO-1+ myeloid cells.Based on its promising cytoprotective effects when giving preemptively, we investigated the use of hemin-induced myeloid HO-1 as a strategy to mitigate established AKI. However, due to its chemical structure and oxidative properties, hemin worsened IRI-induced AKI. We thereby identified that hemin had a dual effect on renal IRI, protective or deleterious, depending on the timing of its administration.Altogether, this work suggests that myeloid HO-1 plays a critical role in the modulation of IRI- induced AKI by improving short- and long-term functional outcomes after renal IRI. We conclude that hemin-induced myeloid HO-1 pathway might be an efficient preventive strategy in many renal IRI situations with predictable AKI such as renal transplantation or partial nephrectomy. / Doctorat en Sciences médicales (Médecine) / info:eu-repo/semantics/nonPublished

Introdução: O transplante renal é amplamente reconhecido como a melhor forma de tratamento para os pacientes que necessitam de terapia de substituição renal. Frequentemente a equipe transplantadora se depara com a difícil questão de determinar se órgãos, provenientes de doadores falecidos e limítrofes ou em parada cardíaca, estão aptos para serem transplantados. É difícil quantificar a intensidade do dano provocado pela isquemia no enxerto a ser utilizado, especialmente se o doador apresentou quadro de instabilidade hemodinâmica com queda significativa da perfusão tecidual e aumento do risco de diminuir a função do enxerto e afetar adversamente sua sobrevida. Desta forma torna-se justificável a utilização da técnica de espectroscopia de fluorescência induzida pelo laser, na tentativa de se avaliar a correlação entre os seus achados e o grau de lesão histológica renal experimental, uma vez que se trata de um método objetivo, não invasivo, rápido e em tempo real que, futuramente, pode ser aplicada nos transplantes renais em humanos. Objetivos: Avaliar a correlação entre os dados da espectroscopia de fluorescência induzida pelo laser e alterações histológicas na isquemia e reperfusão renal em ratos, e se existe diferença significativa na leitura da espectroscopia entre os polos superior, inferior e o terço médio. Materiais e Métodos: Foram utilizados 33 ratos (Rattus norvegicus) machos adultos da linhagem Wistar que, depois de anestesiados, tiveram seus rins esquerdos abordados. Inicialmente os rins foram submetidos à detecção da espectroscopia de fluorescência dos pólos superiores, inferiores e terços médios. As excitações foram geradas por lasers com comprimentos de onda de 408, 442 e 532 nm. Em seguida os pedículos renais esquerdos foram dissecados, isolados e clampeados com auxílio de mini-pinça vascular. Então, os animais foram divididos aleatoriamente em três grupos isquêmicos de 30, 60 e 120 minutos de isquemia quente. Em cada um dos grupos, os rins foram novamente analisados pela espectroscopia de fluorescência, bem como após 5 minutos de reperfusão, utilizando novamente feixes excitatórios com os mesmos comprimentos de onda, nas mesmas regiões renais. Posteriormente os rins esquerdos foram coletados e enviados para estudo histológico. Resultados: O tempo de isquemia mostrou forte influência com a graduação histológica. Com 30 minutos de isquemia, nenhum comprimento de onda (408, 442 e 532 nm) apresentou correlação com a graduação histológica (p = 0,81; p = 0,11; p = 0,21, respectivamente). Com 60 minutos de isquemia, o laser de excitação de 532 nm (na fase de reperfusão) apresentou coeficiente de correlação negativa significativa (r = - 0,61) com a graduação histológica. Na isquemia de 120 minutos, o laser com 442 nm de comprimento de onda (na fase de reperfusão) mostrou o coeficiente de correlação negativa significativa (r = - 0,73) com a graduação histológica. O terço médio renal apresentou média estatística superior à dos polos (p < 0,001) na leitura da espectroscopia de fluorescência. Conclusões: Há correlação entre os dados da espectroscopia de fluorescência induzida pelo laser e as alterações histológicas na isquemia renal em ratos, sendo necessário, durante a investigação, analisar apenas o terço médio renal. / Introduction: Renal transplantation is widely recognized as the best form of treatment for patients who require renal replacement therapy. Often, the transplant team is faced with a difficult question, if organs from deceased marginal donors or non-heart beating donors are able to be transplanted. It is difficult to quantify the intensity of damage caused by ischemia in the graft to be used, especially if the donor had hemodynamic instability with a significant decrease of the tissue perfusion and an increased of the risk of diminishing the graft function which could affect adversely its survival. Thus it is justified to use the technique of laser-induced fluorescence spectroscopy, to assess the correlations between its results and the histological grade in experimental renal injury, since it is an objective, non-invasive, fast and in real-time analysis, which can be applied, in the future, in human kidney transplants. Objectives: To evaluate the correlation between the data of laser-induced fluorescence spectroscopy and histological changes in kidney ischemia and reperfusion in rats, and if there are significant differences of reading between the upper and lower poles and the middle area of such kidneys. Materials and Methods: We used 33 adults male rats (Rattus norvegicus) of Wistar strain, which after anesthetized, had their left kidney addressed. Initially such kidneys were submitted to detection of the fluorescence spectroscopy of the upper pole, lower pole and the middle area. Excitations were generated by lasers having wavelengths of 408, 442 and 532 nm. Then the left renal pedicles were dissected, isolated and clamped. Then the animals were randomized into three ischemic groups of 30, 60 and 120 minutes. In each group, the kidneys were analyzed by fluorescence spectroscopy for the second time, and again after 5 minutes of reperfusion, using excitatory beam with same wavelength, at the poles (upper and lower) and the middle area of the kidneys. Later, the left kidney were collected and sent for histological examination. Results: The ischemia time showed a strong influence on the histological grade. With 30 minutes of ischemia, no wavelength (408, 442 and 532 nm) was correlated with the histological lesions (p = 0.81, p = 0.11, p = 0.21, respectively). With 60 minutes of ischemia, the laser excitation of 532 nm (in the reperfusion phase) showed a significant negative correlation coefficient (r = - 0.61) with the histological grading. In 120 minutes of ischemia, laser with 442 nm wavelength (in the reperfusion phase) showed a significant negative correlation coefficient (r = - 0.73) with the histological grade. The middle area of the kidneys showed a higher average statistically (p< 0,001) than the poles in the reading of fluorescence spectroscopy. Conclusions: There is a strong correlation between the data of laser-induced fluorescence spectroscopy and the histological changes in rats renal ischemia, being necessary, during the investigation, to analyze only the middle area of the kidneys.

Introdução: O transplante renal é amplamente reconhecido como a melhor forma de tratamento para os pacientes que necessitam de terapia de substituição renal. Frequentemente a equipe transplantadora se depara com a difícil questão de determinar se órgãos, provenientes de doadores falecidos e limítrofes ou em parada cardíaca, estão aptos para serem transplantados. É difícil quantificar a intensidade do dano provocado pela isquemia no enxerto a ser utilizado, especialmente se o doador apresentou quadro de instabilidade hemodinâmica com queda significativa da perfusão tecidual e aumento do risco de diminuir a função do enxerto e afetar adversamente sua sobrevida. Desta forma torna-se justificável a utilização da técnica de espectroscopia de fluorescência induzida pelo laser, na tentativa de se avaliar a correlação entre os seus achados e o grau de lesão histológica renal experimental, uma vez que se trata de um método objetivo, não invasivo, rápido e em tempo real que, futuramente, pode ser aplicada nos transplantes renais em humanos. Objetivos: Avaliar a correlação entre os dados da espectroscopia de fluorescência induzida pelo laser e alterações histológicas na isquemia e reperfusão renal em ratos, e se existe diferença significativa na leitura da espectroscopia entre os polos superior, inferior e o terço médio. Materiais e Métodos: Foram utilizados 33 ratos (Rattus norvegicus) machos adultos da linhagem Wistar que, depois de anestesiados, tiveram seus rins esquerdos abordados. Inicialmente os rins foram submetidos à detecção da espectroscopia de fluorescência dos pólos superiores, inferiores e terços médios. As excitações foram geradas por lasers com comprimentos de onda de 408, 442 e 532 nm. Em seguida os pedículos renais esquerdos foram dissecados, isolados e clampeados com auxílio de mini-pinça vascular. Então, os animais foram divididos aleatoriamente em três grupos isquêmicos de 30, 60 e 120 minutos de isquemia quente. Em cada um dos grupos, os rins foram novamente analisados pela espectroscopia de fluorescência, bem como após 5 minutos de reperfusão, utilizando novamente feixes excitatórios com os mesmos comprimentos de onda, nas mesmas regiões renais. Posteriormente os rins esquerdos foram coletados e enviados para estudo histológico. Resultados: O tempo de isquemia mostrou forte influência com a graduação histológica. Com 30 minutos de isquemia, nenhum comprimento de onda (408, 442 e 532 nm) apresentou correlação com a graduação histológica (p = 0,81; p = 0,11; p = 0,21, respectivamente). Com 60 minutos de isquemia, o laser de excitação de 532 nm (na fase de reperfusão) apresentou coeficiente de correlação negativa significativa (r = - 0,61) com a graduação histológica. Na isquemia de 120 minutos, o laser com 442 nm de comprimento de onda (na fase de reperfusão) mostrou o coeficiente de correlação negativa significativa (r = - 0,73) com a graduação histológica. O terço médio renal apresentou média estatística superior à dos polos (p < 0,001) na leitura da espectroscopia de fluorescência. Conclusões: Há correlação entre os dados da espectroscopia de fluorescência induzida pelo laser e as alterações histológicas na isquemia renal em ratos, sendo necessário, durante a investigação, analisar apenas o terço médio renal. / Introduction: Renal transplantation is widely recognized as the best form of treatment for patients who require renal replacement therapy. Often, the transplant team is faced with a difficult question, if organs from deceased marginal donors or non-heart beating donors are able to be transplanted. It is difficult to quantify the intensity of damage caused by ischemia in the graft to be used, especially if the donor had hemodynamic instability with a significant decrease of the tissue perfusion and an increased of the risk of diminishing the graft function which could affect adversely its survival. Thus it is justified to use the technique of laser-induced fluorescence spectroscopy, to assess the correlations between its results and the histological grade in experimental renal injury, since it is an objective, non-invasive, fast and in real-time analysis, which can be applied, in the future, in human kidney transplants. Objectives: To evaluate the correlation between the data of laser-induced fluorescence spectroscopy and histological changes in kidney ischemia and reperfusion in rats, and if there are significant differences of reading between the upper and lower poles and the middle area of such kidneys. Materials and Methods: We used 33 adults male rats (Rattus norvegicus) of Wistar strain, which after anesthetized, had their left kidney addressed. Initially such kidneys were submitted to detection of the fluorescence spectroscopy of the upper pole, lower pole and the middle area. Excitations were generated by lasers having wavelengths of 408, 442 and 532 nm. Then the left renal pedicles were dissected, isolated and clamped. Then the animals were randomized into three ischemic groups of 30, 60 and 120 minutes. In each group, the kidneys were analyzed by fluorescence spectroscopy for the second time, and again after 5 minutes of reperfusion, using excitatory beam with same wavelength, at the poles (upper and lower) and the middle area of the kidneys. Later, the left kidney were collected and sent for histological examination. Results: The ischemia time showed a strong influence on the histological grade. With 30 minutes of ischemia, no wavelength (408, 442 and 532 nm) was correlated with the histological lesions (p = 0.81, p = 0.11, p = 0.21, respectively). With 60 minutes of ischemia, the laser excitation of 532 nm (in the reperfusion phase) showed a significant negative correlation coefficient (r = - 0.61) with the histological grading. In 120 minutes of ischemia, laser with 442 nm wavelength (in the reperfusion phase) showed a significant negative correlation coefficient (r = - 0.73) with the histological grade. The middle area of the kidneys showed a higher average statistically (p< 0,001) than the poles in the reading of fluorescence spectroscopy. Conclusions: There is a strong correlation between the data of laser-induced fluorescence spectroscopy and the histological changes in rats renal ischemia, being necessary, during the investigation, to analyze only the middle area of the kidneys.

Le syndrome d'ischémie-reperfusion (IR), inhérent à la transplantation rénale, est caractérisé par un infiltrat leucocytaire important et des lésions tissulaires graves dont les signaux initiateurs restent à ce jour peu décrits. Postulant que la libération d'alarmines par les cellules en nécrose est décisive dans ce processus, l'objectif principal du présent travail a été d'étudier la contribution de l'alarmine IL-33 dans la genèse des lésions tissulaires dans un modèle murin d'IR rénale. Nos résultats montrent que l'IL-33 est rapidement libérée du rein après IR comme protéine circulante, dès une heure de reperfusion. Les souris IL-33gt/gt, déficientes en IL-33, sont moins sensibles aux lésions induites par l’IR, comme l'attestent le maintien de la fonction rénale et des lésions histologiques atténuées avec un recrutement de polynucléaires neutrophiles (PNN) diminué par rapport aux souris contrôles. Ceci est associé à la perte du recrutement de cellules iNKT productrices d'IFN-γ/IL-17A. Parallèlement, les souris Jα18KO, déficientes en cellules iNKT et protégées contre les lésions d'IR, possèdent également des niveaux élevés d'IL-33 circulante. Nous proposons donc que l'IL-33 endogène contribue aux lésions d'IR en favorisant le recrutement de cellules iNKT, conduisant ainsi à un recrutement amplifié de PNN au niveau du rein lésé. Notre étude, en identifiant l'alarmine IL-33 comme un médiateur précoce de la réponse immunitaire innée induite par l'IR rénale, mettant en jeu les cellules iNKT, contribue à la compréhension des mécanismes impliqués dans la genèse des lésions associées à la greffe rénale et permet de proposer de nouvelles stratégies thérapeutiques. / Ischemia-reperfusion (IR) injury in renal transplantation is characterized by leukocyte infiltration and tissue damage. However, the signals that initiate these events remain poorly understood. Assuming that alarmin release by necrotic cells during IRI is critical, the main objective of the present study was to investigate the role of alarmin IL-33 in kidney injury using a mouse model of renal IR. We observed release of IL-33 shortly after kidney IR concomitantly with an increase in plasma levels of IL-33 within one hour of reperfusion. IL-33 deficient mice (IL-33gt/gt) exhibited reduced renal IR-induced injury, as attested by function preservation, reduced histological change and attenuation of neutrophil recruitment compared to control mice. This was associated with the loss of IFN-γ/IL-17A-producing iNKT cell recruitment. In the meantime, iNKT cell-deficient (Jα18KO) mice, also protected against IRI, have increased levels of circulating IL-33.These findings lead us to propose that endogenous IL-33 contributes to kidney IRI by promoting iNKT cell recruitment and cytokine production, thereby promoting amplified neutrophil recruitment to the injured kidney. The present study identifies the nuclear alarmin interleukin (IL)-33 as an important and early mediator of innate immune response, involving iNKT cells, following experimental kidney ischemia-reperfusion in mice. Our findings contribute to a better understanding of IR-induced injury and may lead to new therapeutic insights into renal-induced injury associated with renal transplantation.