A nefropatia diabética (ND) é uma das complicações microvasculares do diabetes e consiste no dano ao parênquima renal por consequência de uma série de fatores hemodinâmicos e moleculares. A ocorrência de ND e de outras complicações mesmo em indivíduos sob adequado controle glicêmico tem sido associada a um fenômeno conhecido como memória metabólica. Neste trabalho foram investigadas vias bioquímicas e moleculares persistentemente alteradas no rim de animais diabéticos tratados após um período inicial de hiperglicemia, com o propósito de entender os mecanismos envolvidos na memória metabólica. Para tanto, ratos com diabetes induzida por estreptozotocina foram mantidos hiperglicêmicos durante 4 semanas (período curto) ou 12 semanas (período longo) e posteriormente tratados com insulina isoladamente ou combinada com metformina (100mg/kg/dia) durante as 4 (período curto) ou 12 (período longo) semanas seguintes. Todos os animais tratados tiveram os seus níveis glicêmicos e função renal normalizados. Os tratamentos também foram capazes de normalizar os níveis elevados de malonaldeído no rim, bem como a excreção aumentada dos adutos de DNA 8-oxo-2\'-desoxiguanosina (8-oxodG) e N2-carboxietil-2\'- desoxiguanosina (CEdG) na urina observados nos animais diabéticos. Níveis aumentados de 8-oxodG foram detectados em DNA mitocondrial (mtDNA), mas não em DNA nuclear, de animais diabéticos apenas no período curto de estudo e também foram normalizados após o controle glicêmico. Nós identificamos uma via gradualmente alterada durante o curso do diabetes que permanece persistentemente alterada após o controle glicêmico tardio. Essa via compreende um declínio precoce do clearance de ácido úrico e expressão da pAMPK, seguida pelo acúmulo de fumarato, expressão aumentada de TGF-β, expressão reduzida de PGC-1α e redução da metilação e hidroximetilação do mtDNA. A redução persistente do clearance de ácido úrico em animais diabéticos tratados pode sustentar as alterações bioquímicas renais prolongadas observadas após o controle glicêmico, e essa regulação é provavelmente mediada pela redução sustentada da expressão de pAMPK e pela indução de inflamação. Este trabalho propõe a primeira consideração do possível papel da hiperuricemia e das alterações bioquímicas subjacentes como parte da memória metabólica na nefropatia diabética. / Diabetic nephropathy is one of the diabetes microvascular complications, and it consists on the damage to the renal parenchyma due to several hemodynamic and molecular factors. The occurrence of diabetic nephropathy and other complications even in those individuals under tight glycemic control has been associated to a phenomenon known as metabolic memory. Here we investigated biochemical and molecular pathways persistently altered in the kidney of diabetic animals treated after a previous period of hyperglycemia, aiming to understand underlying mechanisms in metabolic memory. Streptozotocin-induced diabetic rats were maintained hyperglycemic during 4 (short period) or 12 weeks (long period), and then they were treated with insulin alone or combined with metformin (100 mg/kg/day) for the following 4 or 12 weeks, respectively. All the treated animals had them glycemic levels and renal function normalized. The treatments were also able to control enhanced kidney malondialdehyde levels, as well as the increased urine excretion of the DNA adducts 8-oxo-2\'- deoxyguanosine (8-oxodG) and N2-carboxyethyl-2\'-deoxyguanosine seen in diabetic animals. Increased levels of 8-oxodG were detected in mitochondrial DNA, but not in nuclear DNA of diabetic animals in the short period, and were also recovered after glycemic control. We have identified a kidney pathway that is gradually altered during the course of diabetes and remains persistently changed after late glycemic control. This pathway comprises an early decline of uric acid clearance and pAMPK expression followed by fumarate accumulation, increased TGF-β expression, reduced PGC-1α expression, and downregulation of methylation and hydroxymethylation of mitochondrial DNA. The sustained decrease of uric acid clearance in treated diabetes may support the prolonged kidney biochemical alterations observed after tight glycemic control, and this regulation is likely mediated by the sustained decrease of AMPK activity and the induction of inflammation. This work proposes the first consideration of the possible role of hyperuricemia and the underlying biochemical changes as part of metabolic memory in diabetic nephropathy.

Diabetic nephropathy is the main cause for initiation of renal replacement therapy and early symptoms in patients include increased glomerular filtration rate (GFR), decreased oxygen tension and albuminuria, followed by a progressive decline in GFR and loss of kidney function. Experimental models of diabetes display increased GFR, decreased tissue oxygenation and nitric oxide bioavailability. These findings are likely to be intertwined in a mechanistic pathway to kidney damage and this thesis investigated their roles in the development of diabetic nephropathy. In vivo, diabetes-induced oxidative stress stimulates renal tubular Na+ transport and in vitro, proximal tubular cells from diabetic rats display increased transport-dependent oxygen consumption, demonstrating mechanisms contributing to decreased kidney oxygenation. In control animals, endogenous adenosine reduces vascular resistance of the efferent arteriole via adenosine A2-receptors resulting in reduced filtration fraction. However, in diabetes, adenosine A2-signalling is dysfunctional resulting in increased GFR via increased filtration fraction. This is caused by reduced adenosine A2a receptor-mediated vasodilation of efferent arterioles. The lack of adenosine-signaling in diabetes is likely due to reduced local adenosine concentration since adenosine A2a receptor activation reduced GFR only in diabetic animals by efferent arteriolar vasodilation. Furthermore, sub-optimal insulin treatment also alleviates increased filtration pressure in diabetes. However, this does not affect GFR due to a simultaneously induction of renal-blood flow dependent regulation of GFR by increasing the filtration coefficient. In diabetes, there is decreased bioavailability of nitric oxide, resulting in alterations that may contribute to diabetes-induced hyperfiltration and decreased oxygenation. Interestingly, increased plasma concentration of l-arginine, the substrate for nitric oxide production, prevents the development of increased GFR and proteinuria, but not increased oxygen consumption leading to sustained intra-renal hypoxia in diabetes. This thesis concludes that antioxidant treatment directed towards the NADPH oxidase as well maneuvers to promote nitric oxide production is beneficial in diabetic kidneys but is targeting different pathways i.e. transport-dependent oxygen consumption in the proximal tubule by NADPH oxidase inhibition and intra-renal hemodynamics after increased plasma l-arginine. Also, the involvement and importance of efferent arteriolar resistance in the development of diabetes-induced hyperfiltration via reduced adenosine A2a signaling is highlighted.

Outre les facteurs métaboliques et hémodynamiques, l’inflammation est actuellement considérée comme un facteur pathogénique potentiel de la néphropathie diabétique (ND), pouvant contribuer à l’initiation et à la progression de la maladie. Les mécanismes menant au développement de l’inflammation rénale dans la ND sont encore peu connus, bien qu’une augmentation d’activité des systèmes rénine angiotensine (RAS) et de l’endothéline (ET) semble y contribuer. L’objectif général de cette étude mono-centre, à double aveugle, randomisée et incluant un groupe placebo était de démontrer que l’inhibition simultanée du RAS et du système de l’ET chez des patients avec ND induisait des effets rénoprotecteurs et anti-inflammatoires supérieurs à ceux observés par blocage du RAS seul. L’objectif spécifique de notre étude était d’évaluer la possibilité que l’administration d’un bloqueur des récepteurs de l’ET-1, le bosentan, à des patients atteints de ND et traités par bloqueurs des récepteurs de l’angiotensine II (BRA), réduisait, chez ces derniers, la protéinurie et les marqueurs inflammatoires systémiques et rénaux. Ce travail constitue un rapport d’un cas clinique et illustre les résultats obtenus suite à l’administration pendant 16 semaines du bosentan chez un patient diabétique de type 2 avec néphropathie clinique traité au long cours par BRA. Le protocole de recherche comprenait 6 visites médicales à 4 semaines d’intervalle, la première visite (V1) correspondant au recrutement du patient, la deuxième visite (V2) constituant le temps 0 de l’étude et la dernière visite (V6) représentant la fin de l’étude. Des échantillons de sang et d’urine étaient prélevés à 3 reprises soit à V2, V4 c’est-à-dire 8 semaines après le début du traitement et à V6 soit 16 semaines après le début du traitement pour mesure des taux sériques et urinaires de divers facteurs pro-inflammatoires incluant l’ET-1, le facteur de nécrose tumorale alpha (TNF-α), l’interleukine-6 (IL-6), le facteur chémoattractant des monocytes-1 (MCP-1), la molécule d’adhésion intracellulaire-1 (ICAM-1), la molécule d’adhésion vasculaire-1 (VCAM-1) et la protéine C-réactive (CRP). Un profil lipidique était aussi déterminé au début et à la fin de l’étude. La fonction rénale était mesurée aux visites V1, V2, V4 et V6 par détermination du taux de filtration glomérulaire (TFG) et de l’excrétion urinaire d’albumine (UAE). Des tests biochimiques de routine étaient aussi faits à chaque visite. La corrélation entre les paramètres inflammatoires et rénaux sous étude et la filtration glomérulaire était enfin déterminée. Nos résultats chez ce sujet ont démontré que le bosentan réduisait l’UAE de 32 % et 35% aux semaines 8 et 16, et ce, sans affecter la pression artérielle ou la filtration glomérulaire. L'effet anti-protéinurique du bosentan était associé à une réduction des concentrations urinaires de VCAM-1, ICAM-1, IL-6, TNF-α et d’ET-1 ainsi qu’à une diminution des concentrations sériques de TNF-α. Le changement dans la protéinurie était corrélé de manière positive avec les changements des niveaux urinaires de VCAM-1 (r=0.86), ICAM-1 (r=0.88), ET-1 (r=0.94), et du TNF-α (r=0.96) ainsi qu’avec les changements des niveaux sériques de TNF-α (r=0.98). Ces données suggèrent que l’inhibition du système de l’ET induit dans la ND des effets rénoprotecteurs additifs à ceux observés par blocage du RAS seul. Ils supportent le concept que l’activation du système de l’ET au niveau rénal, par ses effets inflammatoires, puisse jouer un rôle important dans la pathogenèse de la ND. L’effet anti-inflammatoire et anti-protéinurique du bosentan constitue une découverte intéressante susceptible d’engendrer dans le futur une alternative thérapeutique et préventive dans la prise en charge de la ND. / Apart from metabolic and hemodynamic factors, inflammation has recently been introduced as a potential key pathogenic mechanism involved in the development and progression of diabetic nephropathy (DN). The mechanisms by which renal inflammation occurs in DN are still poorly understood, yet increased renal activity of the renin-angiotensin system (RAS) and endothelin (ET) system may play a key role. The main objective of this mono-centre, double blind, randomized, placebo-controlled study was to demonstrate that concomitant blockade of the RAS and ET system in patients with DN produces greater renal protective effects and exerts greater anti-inflammatory changes than those seen with blockade of the RAS system alone. The specific aim of the study was to evaluate whether administration of bosentan to patients with DN on angiotensin II receptor blockers (ARB) reduces systemic and renal inflammation and improves glomerular filtration. The work presented herein illustrates the results obtained in one type 2 diabetic patient with clinical DN and treated with ARB following the administration of bosentan for 16 weeks. The study protocol included 6 medical visits at 4 weeks interval, with the first visit (V1) being the screening visit and the second visit (V2) being the baseline and randomization visit. Blood and urine samples were taken at V2, after 8 weeks of treatment (V4), and at the end of the study (V6) for determination of serum and urinary inflammatory markers including ET-1, tumour necrosis factor alpha (TNF-α), interleukin-6 (IL-6), monocyte chemotactic protein-1 (MCP-1), intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1) and C-reactive protein (CRP). Lipid profile was done at the beginning and end of the study. Renal function was assessed at V1, V2, V4 and V6 by determination of glomerular filtration rate and urinary albumin excretion (UAE). Routine biochemical analyses were done at each visit. Correlation between serum and urinary inflammatory markers and UAE was determined. Our results demonstrated that bosentan administration to this patient reduced UAE by 32% and 35% at weeks 8 and 16, respectively, without affecting blood pressure and glomerular filtration. The anti-proteinuric effect of bosentan was associated with a reduction in urinary levels of VCAM-1, ICAM-1, IL-6, TNF- and ET-1 and a reduction in serum TNF- levels. Change in UAE was positively correlated with changes in urinary levels of VCAM-1 (r=0.86), ICAM-1 (r=0.88), ET-1 (r=0.94), and TNF- (r=0.96) and with change in serum TNF- levels (r=0.98). Our data suggest that blockade of the ET system in top of RAS inhibition exerts additive renoprotective effects in DN. They support the notion that activation of the ET system, by promoting renal inflammation, may play a role in the pathogenesis of DN. The anti-inflammatory and anti-proteinuric effect of bosentan represents an interesting finding which may leads in the future to an alternate therapeutic and preventive for the treatment of DN.

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Made available in DSpace on 2014-12-17T14:16:26Z (GMT). No. of bitstreams: 1 MarcelaAGU_DISSERT.pdf: 6376152 bytes, checksum: 6d7d86fec335062b8c283cdea3878878 (MD5) Previous issue date: 2009-03-30 / Conselho Nacional de Desenvolvimento Cient?fico e Tecnol?gico / Inflammation has been pointed out as an important factor in development of chronic diseases, as diabetes. Hyperglycemia condition would be responsible by toll-like receptors, TLR2 and TLR4, and, consequently by local and systemic inflammation induction. Thus, the objective of present study was to evaluate type 1 Diabetes mellitus (T1DM) pro-inflammatory state through mRNA expression of TLRs 2 and 4 and proinflammatory cytokines IL-1?, IL-6 and TNF-? correlating to diabetic nephropathy. In order to achieve this objective, 76 T1DM patients and 100 normoglycemic (NG) subjects aged between 6 and 20 years were evaluated. T1DM subjects were evaluated as a total group DM1, and considering glycemic control (good glycemic control DM1G, and poor glycemic control DM1P) and considering time of diagnosis (before achieving 5 years of diagnosis DM1< 5yrs, and after achieving 5 years of diagnosis DM1 <5yrs). Metabolic control was evaluated by glucose and glycated hemoglobin concentrations; to assess renal function serum urea, creatinine, albumin, total protein and urinary albumin-to-creatinine ratio were determined and to evaluate hepatic function, AST and ALT serum activities were measured. Pro-inflammatory status was assessed by mRNA expression of TLRs 2 and 4 and the inflammatory cytokines IL-1?, IL-6 and TNF-?. Except for DM1G group (18.4%), DM1NC patients (81.6%) showed a poor glycemic control, with glycated hemoglobin (11,2%) and serum glucose (225,5 md/dL) concentrations significantly increased in relation to NG group (glucose: 76,5mg/dL and glycated hemoglobin: 6,9%). Significantly enhanced values of urea (20%) and ACR (20,8%) and diminished concentrations of albumin (5,7%) and total protein (13,6%) were found in T1DM patients, mainly associated to a poor glycemic control (DM1P increased values of urea: 20% and ACR:49%, and diminished of albumin: 13,6% and total protein:13,6%) and longer disease duration (DM1 <5yrs - increased values of urea: 20% and ACR:20,8%, and diminished of albumin: 14,3% and total protein:13,6%). As regarding pro-inflammatory status evaluation, significantly increased mRNA expressions were presented for TLR2 (37,5%), IL-1? (43%), IL-6 (44,4%) and TNF-? (15,6%) in T1DM patients in comparison to NG, mainly associated to DM1P (poor glycemic control TLR2: 82%, IL-1?: 36,8% increase) and DM1 <5yrs (longer time of diagnosis TLR2: 85,4%, IL-1?: 46,5% increased) groups. Results support the existence of an inflammatory state mediated by an increased expression of TLR2 and pro-inflammatory cytokines IL-1?, IL-6 and TNF-? in T1DM / A inflama??o tem sido descrita como um fator importante no desenvolvimento de doen?as cr?nicas como o diabetes, e a condi??o da hiperglicemia seria a respons?vel pela ativa??o dos receptores toll-like (TLRs), TLR2 e TLR4, e, conseq?entemente, pela indu??o da inflama??o local e sist?mica. Nesse sentido, o presente estudo teve como objetivo de avaliar o estado pr?-inflamat?rio do Diabetes mellitus tipo 1 (DM1) atrav?s da express?o g?nica de TLRs 2 e 4 e das citocinas pr?-inflamat?rias IL-1?, IL-6 e TNF- ?, e correlacionar com o desenvolvimento da nefropatia diab?tica. Foram estudados 76 pacientes diab?ticos tipo 1 e 100 indiv?duos normoglic?micos NG, na faixa et?ria de 6 a 20 anos. Os indiv?duos diab?ticos foram avaliados como um grupo total DM1, e subdivididos em fun??o do controle glic?mico (diab?ticos compensados DM1C, e n?o-compensados DM1NC) e em fun??o do tempo de diagn?stico (diab?ticos com menos de 5 anos de diagn?stico DM1< 5anos, e a partir de 5 anos de diagn?stico DM1 <5 anos). Para a avalia??o do controle metab?lico foram determinadas as concentra??es de glicose e de hemoglobina glicada; para avaliar a fun??o renal as concentra??es s?ricas de ur?ia, creatinina, albumina, prote?na total e a rela??o albumina/creatinina (RAC) urin?ria; e para fun??o hep?tica a atividade s?rica de AST e ALT. O estado pr?-inflamat?rio foi avaliado a partir da express?o do mRNA dos TLRs 2 e 4, e das citocinas IL-1?, IL-6 e TNF-?. Com exce??o do grupo DM1C (18,4%), os pacientes DM1NC (81,6%) apresentaram um controle glic?mico insatisfat?rio, com valores de mediana para glicose (225,5mg/dL) e hemoglobina glicada (11,2%) significativamente superiores ao grupo NG (glicose: 76,5mg/dL e hemoglobina glicada: 6,9%). Foram obtidos valores aumentados para a ur?ia s?rica (20%) e RAC urin?ria (20,8%); e diminu?dos para albumina (5,7%) e prote?na total (13,6%) nos indiv?duos diab?ticos; e associados a um controle glic?mico insatisfat?rio (DM1NC aumento de 20% para ur?ia e 49% para RAC; e diminui??o de 8,6% para albumina e 12,1% para prote?na total) e a um maior tempo de diagn?stico (DM1 <5anos aumento de 20% para ur?ia e 20,8% para RAC; e diminui??o de 14,3% para albumina e 13,6% para prote?na total). No tocante ? avalia??o do estado pr?-inflamat?rio, as express?es de mRNA se apresentaram elevadas para TLR2 (37,5%), IL-1? (43%), IL-6 (44,4%) e TNF-? (15,6%) nos indiv?duos diab?ticos em rela??o aos NG, sendo principalmente associadas aos grupos DM1NC (controle glic?mico insatisfat?rio TLR2: 82%, IL-1?: 43% de aumento) e DM1 <5 anos (maior tempo de diagn?stico TLR2: 85,4%, IL-1?: 46,5% de aumento). O conjunto de resultados suporta a exist?ncia de um quadro inflamat?rio mediado pelo aumento da express?o do TLR2 e das citocinas pr?-inflamat?rias IL-1?, IL-6 e TNF-? no diabetes tipo 1

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Nous avons précédemment rapporté que le diabète maternel altère la néphrogenèse chez la progéniture diabétique de notre modèle murin de diabète maternel. Notre analyse par puce à ADN réalisée sur les reins néonatals de ces descendants de grossesse diabétique a révélé que l'expression de la protéine interagissant avec hedgehog (Hhip) était significativement surexprimée par rapport au groupe contrôle. En tant qu'antagoniste de Sonic hedgehog (Shh) qui joue un rôle essentiel dans la différenciation cellulaire, la croissance et le modelage tissulaire pendant le développement des reins et la fibrose rénale, Hhip se lie à Shh et atténue sa bioactivité. Il a été rapporté que la protéine Hhip est principalement exprimée dans les cellules endothéliales vasculaires et les cellules adjacentes à celles exprimant Shh. Cependant, le rôle fonctionnel de l'expression de Hhip dans les reins en développement et matures est à peine connu. Nous avons tout d'abord établi le profil d'expression de Hhip dans les reins embryonnaires. Dans notre modèle murin de diabète maternel, nous avons observé que l'expression du gène Hhip est différemment surexprimée dans le mésenchyme métanéphrique différencié et l'épithélium du bourgeon urétérique de la progéniture. En utilisant des cellules mésenchymateuses métanéphriques en culture (cellules MK4), nous avons démontré que le D-glucose élevé (25 mM D-glucose) stimulait spécifiquement l'expression de Hhip de façon dépendante du temps, puis ciblait la signalisation TGFβ1. De plus, la surexpression de Hhip augmentait l'expression des gènes pro-apoptotiques [NF-kB (P50/p65) et p53] et inhibait l'expression des gènes prolifératifs (i.e. Shh, Pax2, N-myc et p27Kip1). Finalement, nous avons démontré que le diabète maternel qui altère le développement des reins pourrait jouer un rôle dans les interactions de la voie Hhip et TGFβ1-SMAD (Diabetologia 2014). Après la naissance, dans le rein normal non-diabétique, l'expression de Hhip est quiescente; une expression basale et limitée de Hhip (ARNm et protéine) est détectable dans les cellules endothéliales glomérulaires matures, les podocytes et les cellules épithéliales tubulaires rénales. Alors que les lésions des cellules épithéliales glomérulaires (GECs) et des podocytes sont la caractéristique de la lésion rénale précoce de la néphropathie diabétique (DN), nous avons examiné plus en détail le rôle de l'expression rénale de Hhip dans certains modèles de diabète murin—e.g. souris Akita, souris db/db et souris Hhip hétérozygote (Hhip +/−) rendus diabétiques avec de faibles doses de streptozotocine (LDSTZ). Nos données révèlent que l'hyperglycémie (ou un taux élevé de glucose in vitro) active l'expression du gène Hhip dans les cellules endothéliales glomérulaires et les podocytes. En particulier, l'hétérozygosité de Hhip protège l'intégrité glomérulaire avec moins de pertes de podocytes et améliore les paramètres rénaux [c.-à-d., une diminution du ratio albumine/créatinine urinaire (ACR, une caractéristique de l'apparition de la maladie rénale) et les caractéristiques de la DN (hypertrophie rénale, augmentation du taux de filtration glomérulaire, glomérulosclérose et fibrose)]. Prises dans leur ensemble, nos données suggèrent que l’augmentation de l'expression rénale de Hhip due aux concentrations élevées de glucose peut déclencher directement le processus d'apoptose et de fibrose des cellules endothéliales glomérulaires observé dans le diabète et qu’elle joue un rôle clé dans le développement et la progression de la DN (Sci. Report 2018). Mots-clés : Expression des gènes de Hhip, diabète maternel, néphrogenèse, néphropathie diabétique, dérivé réactif de l'oxygène / We previously reported that maternal diabetes impairs nephrogenesis in diabetic progeny in our maternal diabetes murine model. Our gene-array performed in neonatal kidneys of those affected offspring revealed that hedgehog interacting protein (Hhip) expression was significantly upregulated, as compared to the control group. As an antagonist of sonic hedgehog (Shh) which plays an essential role in cell differentiation, growth and tissue patterning during kidney development and kidney fibrosis, Hhip binds to Shh and attenuates its bioactivity. It is reported that Hhip is predominantly expressed in vascular endothelial cells and cells adjacent to those expressing Shh. However, the functional role of Hhip expression in the developing and mature kidney is barely known. We first established the expression pattern of Hhip in embryonic kidneys. We observed in our murine model of maternal diabetes that Hhip gene expression is differentially up-regulated in differentiated metanephric mesenchyme and ureteric bud epithelium of the offspring. Using cultured metanephric mesenchymal cells (MK4 cells), we demonstrated that high D-glucose (25 mM D-Glucose) specifically stimulated Hhip expression in a time-/dose-dependent manner, and then targeted TGFβ1 signaling. In addition, overexpression of Hhip increased the expression of pro-apoptotic genes [NF-kB (p50/p65) and p53] and inhibited the expression of proliferative genes (i.e., Shh, Pax2, N-myc, and p27kip1). Finally, we demonstrated that maternal diabetes impairing kidney development might mediate the interactions of Hhip and TGFβ1-Smad pathway (Diabetologia 2014). After birth, in normal non-diabetic state, Hhip expression is quiescent; a limited basal Hhip expression (mRNA and protein) is detectable in mature glomerular endothelial cells, podocytes and renal tubular epithelial cells. While glomerular epithelial cells (GECs) and podocyte injury is the hallmark of early renal injury in diabetic nephropathy (DN), we further examined the role of renal Hhip expression in murine diabetes models--e.g., Akita mice, db/db mice and heterozygous Hhip deficiency (Hhip+/−) mice rendered diabetic with low-dose streptozotocin (LDSTZ). Our data revealed that hyperglycemia (or high glucose in vitro) activates Hhip gene expression in glomerular endothelial cells and podocytes. In particular, Hhip heterozygosity protects glomerular integrity with less podocyte loss and improved renal outcomes [i.e., decreased urinary albumin/Creatinine (Cre) ratio (ACR, a hallmark of onset of kidney disease) and DN-features (renal hypertrophy, increased glomerular filtration rate, glomerulosclerosis and fibrosis)]. Taken together, our data suggest that high glucose-elevated renal Hhip expression may directly trigger the process of apoptosis and fibrosis in glomerular endothelial cells in diabetes and it plays a key role in the development and progression of DN (Sci. Report 2018). Keywords: Hhip gene expression, maternal diabetes, nephrogenesis, diabetic nephropathy, reactive oxygen species

La néphropathie diabétique (DN) est l’une des premières causes de maladie rénale en phase terminale (ESKD). L’ESKD est un important facteur de risque d'insuffisance cardiaque et d'accidents vasculaires cérébraux. La dysfonction du système rénine-angiotensine intrarénal (iRAS) est considérée comme étant l'une des principales causes du développement de la DN. Tous les composants du iRAS sont identifiés dans les cellules épithéliales des tubules rénaux proximaux (RPTCs), y compris l'angiotensinogène (Agt), le seul précurseur de toutes les angiotensines. Notre laboratoire a rapporté précédemment que la surexpression spécifique de l’Agt dans les RPTCs provoque l’hypertension, la protéinurie, la fibrose rénale, l’apoptose et des lésions rénales. Nrf2 (Nuclear factor erythroid 2-related factor 2) est un facteur de transcription qui est exprimé de façon abondante dans les RPTCs et a été considéré comme étant un régulateur central de l'équilibre redox dans les réponses cytoprotectrices cellulaires. Le rôle de l’activation du Nrf2 dans la DN, toutefois, est controversé. L’objectif général de cette thèse est de comprendre le rôle physiologique du Nrf2 dans la DN et d’étudier le(s) mécanisme(s) moléculaire(s) de l’action de Nrf2. Premièrement, nous avons démontré que la délétion génétique de Nrf2 ou l’inhibition pharmacologique de Nrf2 avec de la trigonelline chez les souris Akita diabétiques de type 1 régule à la hausse la voie Ace2/MasR et supprime l’expression de Agt/ACE dans les RPTCs, ce qui a pour effet d'atténuer l’hypertension systémique et les lésions rénales. Conformément, dans les cellules immoratalisées de tubule proximal de rat (IRPTC) en culture, la transfection de ARNsi ou le traitement à la trigonelline empêche la régulation positive de Agt/ACE induite par le HG, avec une baisse subséquente de l’expression des gènes Ace2/MasR. Ces données identifient un nouveau mécanisme dans lequel l’activation de Nrf2 stimule l’expression et l’activation des gènes du iRAS, menant au développement de l’hypertension et de la néphropathie dans le diabète. Deuxièmement, nous avons généré des souris Nrf2 transgéniques qui surexprime spécifiquement Nrf2 dans les RPTCs (souris Nrf2RPTC Tg), sous le contôle du promoteur KAP (kidney specific androgen-regulated protein). Nous avons ensuite croisé les souris Nrf2RPTC Tg avec les 6 souris Akita Nrf2-/- pour générer des souris Akita Nrf2-/- /Nrf2RPTC Tg. Nous avons trouvé que la surexpression de Nrf2 dans les RPTCs des souris Akita Nrf2-/- augmentait significativement l’expression du gène SGLT2, entraînant une élévation du glucose sanguin, du taux de filtration glomérulaire, du rapport albumine/créatinine urinaire et de la fibrose tubulo-interstitielle. Dans les cellules tubulaires proximales humaines immortalisées (HK2), le traitement à l’oltipraz ou la transfection de l’ADNc du NRF2 stimule l’expression de l’ARNm du SGLT2 et l’activité de son promoteur. De plus, des tests de retard sur gel et d’immunoprécipitation de chromatine ont montrés que NRF2 se lie au NRF2-RE du promoteur du SGLT2. En outre, une expression plus élevée de NRF2 et SGLT2 est observée dans les RPTCs de reins de patients diabétiques que dans les reins de patients non diabétiques. Ces données ont établi un nouveau mécanisme de la régulation du NRF2 sur l’expression et l’activation du gène SGLT2, menant à une exacerbation du glucose sanguin, de l’hyperfiltration et des lésions rénales dans le diabète. En somme, cette thèse a démontré que le stress oxidatif (hyperglycémie) induisait l’activation du Nrf2 qui stimulait le iRAS et l’expression de SGLT2, contribuant ainsi à la progression de la DN. Ces études suggèrent que le Nrf2 pourrait être une cible thérapeutique potentielle dans le traitement de la DN et pourront fournir de valabless données pré-cliniques pour les essais cliniques en cours avec le bardoxolone méthyle (un activateur de Nrf2). / Diabetic nephropathy (DN) is one of the leading causes of end-stage kidney disease (ESKD). ESKD is a major risk factor for heart failure and stroke. Dysfunction of intrarenal renin angiotensin system (iRAS) is considered as one of the main reasons that caused the DN. All components of the iRAS are identified in the renal proximal tubule cells (RPTCs), including angiotensinogen (Agt), the sole precursor of all angiotensins. Our lab has previously reported that specific overexpression of Agt in RPTCs induces hypertension, proteinuria, kidney fibrosis, apoptosis and kidney injury. Nuclear factor erythroid 2-related factor 2 (Nrf2) is a transcription factor that abundantly expresses in RPTCs and has been considered as a master regulator of redox balance in cellular cytoprotective responses. The role of Nrf2 activation in DN, however, is not clear. The overall aim of this study is to understand the physiological role of Nrf2 in DN and investigate the molecular mechanism(s) of Nrf2 action. First, we have demonstrated that genetic deletion of Nrf2 or pharmacological blockade of Nrf2 with trigonelline in type 1 diabetic Akita mice effectively upregulates Ace2/MasR and suppresses Agt/ACE expression in isolated RPTCs, resulting in attenuation of systemic hypertension and kidney injury. Consistently, in cultured IRPTCs, Nrf2 siRNA transfection or trigonelline treatment prevents high glucose-induced upregulation of Agt/ACE with downregulation of Ace2/MasR gene expression. These data identified a novel mechanism in which Nrf2 activation stimulates iRAS gene expression and activation, leading to the development of hypertension and nephropathy in diabetes. Second, we have generated Nrf2 transgenic mice under the kidney specific androgen regulated protein (KAP) promoter which specifically overexpress Nrf2 in RPTCs (Nrf2RPTC Tg mice). We further crossbred the Nrf2RPTC Tg mice with Akita Nrf2-/- mice to generate Akita Nrf2-/- /Nrf2RPTC Tg mice. We have found that overexpression of Nrf2 in RPTCs of Akita Nrf2-/- mice significantly unregulated sodium-glucose transporter-2 (SGLT2) expression, resulting in elevation of blood glucose, glomerular filtration rate, albumin-creatinine ratio and tubulointerstitial fibrosis. In 8 immortalized human proximal tubular cells (HK2), oltipraz treatment or NRF2 cDNA transfection stimulated SGLT2 mRNA expression and its promoter activity. Furthermore, NRF2 bound to NRF2- RE of SGLT2 promoter were identified by gel mobility shift assay and chromatin immunoprecipitation assay. Moreover, human diabetic kidneys exhibited higher expression of NRF2 and SGLT2 in RPTCs than non-diabetic kidneys. These data established a novel mechanism of NRF2’s regulation on SGLT2, leading to exacerbation of blood glucose, hyperfiltration and kidney injury in diabetes. In summary, this study documented that activation of Nrf2 in hyperglycemia contributed to the progression of DN via regulation of iRAS and SGLT2, suggesting that Nrf2 might be a potential therapeutic target in the treatment of DN.