Extracellular DNA contributes to cholesterol crystal embolism-induced clot formation, acute kidney injury, and tissue infarction

Atherosklerose ist weltweit eine der Hauptursachen für Morbidität und Mortalität. Bei fortgeschrittener Atherosklerose ist die Cholesterinkristallembolie (CCE) eine potenziell lebensbedrohliche Komplikation mit einer durchschnittlichen Mortalität von 62,8%. Autopsien oder Gewebebiopsien zeigen Cholesterinkristalle (CC) im arteriellen Lumen, umgeben von einer fibrotischen Matrix, die das Gefäßlumen verschließt. Über die genauen zellulären und molekularen Mechanismen nach CCE ist wenig bekannt, was teilweise auf das Fehlen eines Tiermodells zurückzuführen ist. Wir stellten daher die Hypothese auf, dass die Entwicklung eines reproduzierbaren Mausmodells der CCE zur Nachahmung der morphologischen und funktionellen Eigenschaften der CCE beim Menschen dazu beitragen würde, die molekularen Mechanismen des CC-gesteuerten arteriellen Verschlusses, des Gewebeinfarkts und des Organversagens zu untersuchen. CCE wurden in C57BL/6J-Mäusen durch Injektion von CC über einen minimal- invasiven Eingriff in die linke Nierenarterie induziert. Primärer Endpunkt war die glomeruläre Filtrationsrate (GFR), die am wachen und frei beweglichen Tier gemessen wurde, um den Abfall der exkretorischen Nierenfunktion als Marker eines akuten Nierenversagens zu bestimmten. Die Größe des Niereninfarkts wurde, wie bei Myokardinfarkt oder Schlaganfallmodellen etabliert, per TTC-Färbung von Nierenschnitten und Planimetrie quantifiziert. Injektion von CC verursachte einen dosis-abhängigen Abfall der GFR und Territorialinfarkte der Niere. Ursache waren Verschlüsse präglomerulärer Arterien und Arteriolen. Der Kristallanteil am Gefäßverschluss war gering, stattdessen fanden sich Fibrin+ Thrombusmaterial um die Kristalle, die das arterielle Lumen ausfüllten. Wir nannten diese Strukturen “Kristallthrombosen”. Im Vergleich zum GFR Abfall, war das Ausmaß der Infarktgrößen variabler. 3D Rekonstruktionen von Angio-μCTs zeigte partielle und vollständige arterielle Verschlüsse und Rarefizierung der arteriellen Blutgefäße. Histologisch fand sich nach 24 h ein deutliches perilesionales Neutrophileninfiltrat. Somit imitiert unser Modell periphere CCE mit arteriellen Verschlüssen, die akute territoriale Infarkte, perilesionale Entzündung und fiunktionelles Organversagen. Das Blockieren der Nekroinflammation/Infarzierung in mixed lineage kinase domain-like (Mlkl)-defizienten Mäusen oder mit dem Inhibitor Nec-1s bzw. einem NLRP3-Inhibitor reduzierte signifikant die Infarktgröße und Infiltration von Neutrophilen im Vergleich zu Kontrollen. Keine dieser Interventionen hatte jedoch Auswirkungen auf den durch CCE verursachten GFR-Verlust (=Organversagen), weil keine der Interventionen Einfluss auf die arteriellen Verschlüsse hatte. Da in der Niere die Funktion zu allererst von der glomerulären Perfusion abhängt, verhindert die Hemmung der ischämen Nekrose alleine noch nicht das Nierenversagen. Somit war klar, dass die Kristallthrombosen das entscheidende Therapietarget bei CCE darstellen. Histologisch bestanden die Kristallthrombosen aus Erythrozyten, Plättchen, Neutrophile, Fibrin, und extrazelluläre DNA (ecDNA). Zunächst haben wir Neutrophile mit einem depletierenden Antikörper selektiv entfernt, bzw. die Bildung von neutrophil extracellular traps (NETs) mit einem Inhibitor gehemmt. Beide Interventionen verringerten die Größe des Niereninfarkts im Vergleich zur Kontrollgruppe, dies hatte jedoch keinen signifikanten Einfluss auf die arteriellen Verschlüsse bzw. den GFR-Verlust. Im Gegensatz dazu schützte der Thrombozyten-P2Y12-Rezeptorantagonist Clopidogrel Mäuse vollständig vor Kristallthrombosen, GFR-Abfall und Niereninfarkt. Daher sind Blutplättchen, jedoch nicht neutrophile Granulozyten, von zentraler Bedeutung für CCE-induzierte Kristallthrombosen und ihre Folgen. Als Nächstes testeten wir die Wirkung von Heparin und des Fibrinolytikums Urokinase. Nach 24 Stunden reduzierten sowohl Heparin als auch Urokinase die Anzahl der arteriellen Verschlüsse signifikant. Niereninfarkt, Nierenverletzung, Infiltration von Neutrophilen, Gefäßverletzung sowie tubuläre Nekrose waren nahezu vollständig abwesend. Beide Behandlungen hatten im Vergleich zu mit Vehikel-behandelten Mäusen einen vollständigen Schutz vor GFR-Abfall. Zusammenfassend lässt sich sagen, dass nicht die Kristalle an sich, sondern die Kristallthrombosen arterielle Obstruktion, Gewebeinfarkt und Organversagen verursachen. Um die Bedeutung der ecDNA zu untersuchen, gaben wir rekombinante DNase I, die ecDNA degradiert. Tatsächlich war 24 h nach DNase I Gabe in den Arterien keine ecDNA mehr nachweisbar und, überraschenderweise, traten auch Kristallthrombosen nicht mehr auf. Die Verhinderung von arteriellen Verschlüssen war mit einem vollständigen Schutz vor GFR-Abfall, einer signifikanten Verringerung der Niereninfarktgröße verbunden. Somit ist ecDNA eine weitere nicht-redundante Komponente der CCE-bedingten arteriellen Obstruktion, des Gewebeinfarkts und des Organversagens. Da Herz- oder Aortenoperationen die Verwendung von Antikoagulanzien oder Fibrinolytika ausschließen, betrachteten wir rekombinante DNase I als mögliche Alternative zur Abschwächung der CC-Gerinnselbildung durch Hemmung der Fibrinbildung und der ecDNA-Akkumulation. Zuerst testeten wir das therapeutische Zeitfenster und stellten fest, dass die DNase I-Behandlung, die 3 Stunden nach der CCE verabreicht wurde, immer noch einen Trend zu verbesserten Ergebnissen im Vergleich zu 6 und 12 Stunden zeigte. Um die Ergebnisse bei der Einstellung einer CCE im Zusammenhang mit kardiovaskulären Eingriffen weiter zu optimieren, haben wir ein Regime getestet, das eine präventive Einzeldosis des Nekroptosehemmers Nec-1s mit therapeutischer Gabe von rekombinanter DNase 1 3 Stunden nach Kristallinjektion kombiniert. Hierunter kam es zu einer vollständigen Protektion von Kristallthrombosen Organversagen und Infarkt, was eine neue Behandlungsoption bei elektiven Eingriffen bei Hochrisikopatienten aufzeigen könnte. Mechanistische in vitro Untersuchungen im Organ-Chip Modell, zeigten wie Cholesterin-Kristalle zu Endothelschäden führen, dass ecDNA v.a. aus Endothel und Neutrophilen freigesetzt wird. Thrombozyten setzen nur wenig mitochondriale DNA frei. DNase I inhibiert die CC-induzierte Thrombozytenaktivierung, möglicherweise durch Abbau von ADP. Zusammengenommen präsentieren wir erstmals ein Mausmodell einer CCE mit Organversagen und Infarkt, das pathophysiologische Studien gestattet. Nicht der Infarkt an sich, sondern die Kristallthrombosen sind für das Organversagen entscheidend. Endothelschädigung mit Freisetzung von ecDNA und Plättchenaktivierung führen zur Bildung der Kristallthrombosen und bieten alte und neue Therapietargets. Eine prophylaktische Einmalgabe eines Zelltodinhibitors und die Gabe von DNase I im postinterventionellen Zeitfenster von 3 h (bei der Maus) könnten helfen, die Prognose von Patienten mit Prozedur-assoziierter CCE zu verbessern.Atherosclerosis is a leading cause of global morbidity and mortality. In advanced atherosclerosis, cholesterol crystal (CC) embolism (CCE) is a potentially life-threatening complication with an average mortality of 62.8 %. Autopsies or tissue biopsies reveal CC inside the arterial lumen surrounded by an undefined biological matrix obstructing the vessel lumen. Little is known about the precise cellular and molecular mechanisms following CCE, in part due to the lack of animal models. Therefore, I hypothesized that developing a reproducible mouse model of CCE to mimic the morphological and functional characteristics of CCE in humans would be instrumental to dissect the molecular mechanisms of CC-driven arterial occlusion, tissue infarction, and organ failure. To induce CCE, different doses of CC were injected into the left kidney artery of C57BL/6J mice. Acute kidney failure was evaluated by kidney function (i.e. GFR), kidney infarction was quantified using the TTC method. CC caused crystal clots occluding intrarenal arteries and a dose-dependent drop in GFR. In contrast, the extent of kidney infarction was more variable. 3D μCT showed partial and complete arterial occlusions, blood vessel rarefaction, and volume change. The macroscopic analysis revealed kidney swelling and territorial infarctions, tubular necrosis, interstitial edema, neutrophil infiltrates, and loss of CD31. Thus, intraarterial CC injection induces arterial occlusions causing acute territorial infarctions, perilesional inflammation, and organ failure. Blocking necroptosis with Mlkl-/- mice or Nec-1s, and the NLRP3 inhibitor significantly reduced infarct size, kidney injury, and neutrophil infiltration at 24 h compared to WT controls. However, none of these interventions affected CCE-related GFR loss. Consistently, necroinflammation is involved in kidney infarction but not in arterial occlusions as an upstream event. Thus, as nephron perfusion is ultimately required for kidney function, inhibiting infarction alone does not prevent acute kidney failure. Immunostaining revealed that crystal clots involved platelets, neutrophils, fibrin, and extracellular DNA (ecDNA). Therefore, I depleted neutrophils or inhibited NET formation before CC injection. Neutrophil depletion or NET inhibition significantly decreased kidney infarction compared to the control groups, but this had no significant effect on arterial obstructions or GFR loss, maybe because mononuclear cells had partially replaced neutrophils inside crystal clots as a source of ecDNA. In contrast, the platelet P2Y12 receptor antagonist clopidogrel completely protected mice from intravascular obstructions, GFR loss, kidney infarction, and perilesional neutrophil infiltrate. Therefore, CC occlude arteries by forming crystal clots consisting of fibrin, platelets, and neutrophils. Thus, platelets but not neutrophils are central for CCE-related arterial occlusion, organ failure, and tissue infarction. Next, I tested the effects of the anticoagulant heparin and the fibrinolytic agent urokinase. At 24h both heparin and urokinase significantly reduced the arterial occlusions number, kidney infarction, kidney injury, neutrophils infiltration, vascular injury as well as tubular necrosis. Both treatments had complete protection from GFR loss compared to vehicle-treated mice. Conclusively, not the crystals per se but rather crystal clots cause arterial obstruction, tissue infarction, and organ failure. In the DNase I treatment group, I noticed a significant reduction in the percentage of CC clots with ecDNA. After 24h of DNase I treatment intraarterial ecDNA had disappeared and the number of arterial occlusions was significantly reduced. Preventing arterial occlusions was associated with complete protection from GFR loss, a significant reduction in kidney infarct size as well as kidney cell death, neutrophil infiltrates, and vascular rarefaction. Thus, ecDNA is another non-redundant component of CCE-related arterial obstruction, tissue infarction, and organ failure. As cardiac or aorta surgeries preclude the use of anticoagulants or fibrinolytic agents, I considered recombinant DNase I as a possible alternative to attenuate CC clot formation by inhibiting fibrin formation and ecDNA accumulation. First, I tested the therapeutic window-of-opportunity and found that DNase I treatment given 3 h after CCE showed trends towards improved outcomes compared to 6 h and 12 h. To further optimize outcomes in the setting of a cardiovascular procedure-related CCE I tested a regimen combining a pre-emptive single dose of the necroptosis inhibitor Nec-1s with therapeutic recombinant DNase I gave 3 h after intraarterial CC injection. This approach could be feasible as prophylaxis given to all patients at risk, while DNase I would be only given to those with signs of CCE into the kidney, e.g. an early decline of urinary output. This dual strategy resulted in significant protection from GFR loss and kidney infarction in almost all animals together with a significant reduction in vascular occlusions by crystal clots. In my in vitro studies, platelets were exposed to thrombin with or without CC and found that CC enhances fibrinogen release from platelet alpha (α)-granules, which further promotes fibrin clot formation. CC exposure also induced ATP secretion from dense (δ)-granules, but co-incubation with DNase I strongly reduced these extracellular ATP releases. Next, I stimulated platelets with thrombin and collagen-related peptide and indicated DNase I can inhibit fibrinogen and ATP secretion and subsequent fibrin formation and P2Y12 receptor signaling, respectively. To model this process in vitro, I tested collagen-driven platelet aggregation with or without CC. Indeed, collagen I triggered massive platelet aggregation within 5 min with CC, DNase I treatment normalized this accelerated aggregation response. Endothelial cells and neutrophils studies showed that CC did not directly induce plasmatic coagulation but induced NET formation and DNA release mainly from kidney endothelial cells, neutrophils, and few from platelets. Thus, the in vitro studies support that CC and platelet dependent ecDNA release from neutrophils and endothelial cells, and DNase I can attenuate CC-induced platelet activation, aggregation, and fibrin clot formation. In summary, not CC by itself but the fibrin clots forming around CC obstruct peripheral arteries causing tissue infarction and organ failure. Hence, crystal clots represent the primary target for therapeutic interventions. Among the possible molecular targets in thrombosis and haemostasis, especially enhancing fibrinolysis or inhibiting platelet purinergic signaling could reduce arterial occlusions, infarction, and organ failure albeit with a relatively short window-of-opportunity up to 3 h. My results suggest that prophylactic necroptosis inhibition with a combination of DNase I therapy could have a synergistic effect on CC induced clot formation in mice and might be a feasible two-step prophylactic/therapeutic approach in human patients with a risk for procedure-related CCE

Hemodynamics

Hemodynamics is study of the mechanical and physiologic properties controlling blood pressure and flow through the body. The factors influencing hemodynamics are complex and extensive. In addition to systemic hemodynamic alterations, microvascular alterations are frequently observed in critically ill patients. The book "Hemodynamics: New Diagnostic and Therapeuric Approaches" is formed to present the up-to-date research under the scope of hemodynamics by scientists from different backgrounds

Multimodality ImAging of Cardiovascular Dysfunction : risk factors, diagnostics and treatment options

Cardiovascular diseases are a major cause of mortality worldwide, and includes all diseases of the heart and circulation. Despite improvements in knowledge and treatment options over the last decades, it remains one of the leading causes of disability and death. The underlying mechanisms vary depending on the disease in question. Some of these risk factors can be avoided, controlled, treated or modified such as high cholesterol, high blood pressure and obesity. While others, such as family history and gender, cannot be avoided and their emphasis lies on monitoring and treatment. In this thesis, the role of DNA damage, atherosclerosis and the renin angiotensin system, factors that modulate cardiovascular damage and disease, are investigated and discussed. Additionally, the effect of nutritional and therapeutic interventions is explored

Transglutaminase 2 Conformation as a New Target to Treat Vascular Dysfunction in Aging and Diabetes

Endothelial dysfunction is an independent risk factor for cardiovascular complications associated with aging and diabetes. Transglutaminase 2 (TG2) is a ubiquitously expressed enzyme with calcium-dependent transamidase activity; it is overactive in aging and diabetes and has been linked with fibrotic processes in the cardiovascular and renal systems (e.g. stiffening, calcification) as well as with endothelial dysfunction in diabetes and inward remodeling in hypertension. These effects have been ascribed to the open TG2 conformation. On the other hand, the closed conformation of TG2 has GTP-binding activity, acting as a G protein in transmembrane signaling, opening potassium channels in vascular smooth muscle cells (VSMCs) and participating in cell survival. This led to the hypothesis that induction of the closed conformation by pharmacological means would present blood-pressure lowering effects and prevent endothelial dysfunction..

Studies on Atrial Natriuretic Peptide

Atrial Natriuretic Peptide (ANP) is a hormone secreted by the heart with a unique spectrum of actions: natriuresis, diuresis, vasorelaxation and suppression of the renin-angiotensin system. This thesis is divided into four sections each of which examines a different aspect of ANP. The common theme is the question of how the actions of ANP may be "harnessed" therapeutically, either directly using novel inhibitors of the enzyme Neutral Endopeptidase (NEP; EC 3.4.24.11) which degrades ANP, or indirectly, by investigating potential interactions with available therapeutic agents including Angiotensin Converting Enzyme (ACE) inhibitors, and calcium channel blockers

Le diabète maternel influence la morphogenèse rénale et la programmation périnatale

Le diabète maternel est un facteur de risque majeur pour le développement de malformations congénitales. Dans le syndrome de l’embryopathie diabétique, l’exposition prolongée du fœtus à de hautes concentrations ambientes de glucose induit des dommages qui peuvent affecter plusieurs organes, dont les reins. Les malformations rénales sont la cause de près de 40 pourcent des cas d’insuffisance rénale infantile. L’hyperglycémie constitue un environnement utérin adverse qui nuit à la néphrogenèse et peut causer l’agenèse, la dysplasie (aplasie) ou l’hypoplasie rénale. Les mécanismes moléculaires par lesquels les hautes concentrations ambientes de glucose mènent à la dysmorphogenèse et aux malformations demeurent toutefois mal définis. Le diabète maternel prédispose aussi la progéniture au développement d’autres problèmes à l’âge adulte, tels l’hypertension, l’obésité et le diabète de type 2. Ce phénomène appelé ‘programmation périnatale’ a suscité l’intérêt au cours des dernières décennies, mais les mécanismes responsables demeurent mal compris. Mes études doctorales visaient à élucider les mécanismes moléculaires par lesquels le diabète maternel ou un environnement in utero hyperglycémique affecte la néphrogenèse et programme par la suite la progéniture a développer de l’hypertension par des observations in vitro, ex vivo et in vivo. Nous avons utilisé les cellules MK4, des cellules embryonnaires du mésenchyme métanéphrique de souris, pour nos études in vitro et deux lignées de souris transgéniques (Tg) pour nos études ex vivo et in vivo, soient les souris HoxB7-GFP-Tg et Nephrin-CFP-Tg. Les souris HoxB7-GFP-Tg expriment la protéine fluorescente verte (GFP) dans le bourgeon urétérique (UB), sous le contrôle du promoteur HoxB7. Les souris Nephrin-CFP expriment la protéine fluorescente cyan (CFP) dans les glomérules, sous le contrôle du promoteur nephrin spécifique aux podocytes. Nos études in vitro visaient à déterminer si les hautes concentrations de glucose modulent l’expression du gène Pax2 dans les cellules MK4. Les cellules MK4 ont été traitées pendant 24h avec du milieu contenant soit 5mM D-glucose et 20mM D-mannitol ou 25mM D-glucose et avec ou sans antioxydants ou inhibiteurs de p38 MAPK, p44/42 MAPK, PKC et NF-kB. Nos résultats ont démontré que le D-glucose élevé (25mM) augmente la génération des espèces réactives de l’oxygène (ROS) dans les cellules MK4 et induit spécifiquement l’expression du gène Pax2. Des analogues du glucose tels le D-mannitol, L-glucose ou le 2-Deoxy-D-glucose n’induisent pas cette augmentation dans les cellules MK4. La stimulation de l’expression du gène Pax2 par le D-glucose dans les cellules MK4 peut être bloquée par des inhibiteurs des ROS et de NF-kB, mais pas par des inhibiteurs de p38 MAPK, p44/42 MAPK ou PKC. Ces résultats indiquent que la stimulation de l’expression du gène Pax2 par les concentrations élevées de glucose est due, au moins en partie, à la génération des ROS et l’activation de la voie de signalisation NF-kB, et non pas via les voies PKC, p38 MAPK et p44/42 MAPK. Nos études ex vivo s’intéressaient aux effets d’un milieu hyperglycémique sur la morphogenèse de la ramification du bourgeon urétérique (UB). Des explants de reins embryonnaires (E12 à E18) ont été prélevés par micro-dissection de femelles HoxB7-GFP gestantes. Les explants ont ensuite été cultivés dans un milieu contenant soit 5mM D-glucose et 20mM D-mannitol ou 25mM D-glucose et avec ou sans antioxydants, catalase ou inhibiteur de PI3K/AKT pour diverses durées. Nos résultats ont démontré que le D-glucose stimule la ramification du UB de manière spécifique, et ce via l’expression du gène Pax2. Cette augmentation de la ramification et de l’expression du gène Pax2 peut être bloquée par des inhibiteurs des ROS et de PI3K/AKT. Ces études ont démontré que les hautes concentrations de glucose altèrent la morphogenèse de la ramification du UB via l’expression de Pax2. L’effet stimulant du glucose semble s’effectuer via la génération des ROS et l’activation de la voie de signalisation Akt. Nos études in vivo visaient à déterminer le rôle fondamental du diabète maternel sur les défauts de morphogenèse rénale chez la progéniture. Dans notre modèle animal, le diabète maternel est induit par le streptozotocin (STZ) chez des femelles HoxB7-GFP gestantes (E13). Les souriceaux ont été étudiés à différents âges (naissants et âgés de une, deux ou trois semaines). Nous avons examiné leurs morphologie rénale, nombre de néphrons, expression génique et les événements apoptotiques lors de cette étude à court terme. La progéniture des mères diabétiques avait un plus faible poids, taille et poids des reins, et possédait des glomérules plus petits et moins de néphrons par rapport à la progéniture des mères contrôles. La dysmorphogenèse rénale observée est peut-être causée par l’augmentation de l’apoptose des cellules dans la région du glomérule. Nos résultats ont montré que les souriceaux nés de mères diabétiques possèdent plus de podocytes apoptotiques et plus de marquage contre la caspase-3 active dans leurs tubules rénaux que la progéniture des mères contrôles. Les souriceaux des mères diabétiques montrent une augmentation de l’expression des composants du système rénine angiotensine (RAS) intrarénal comme l’angiotensinogène et la rénine, ainsi qu’une augmentation des isoformes p50 et p65 de NF-kB. Ces résultats indiquent que le diabète maternel active le RAS intrarénal et induit l’apoptose des glomérules, menant à une altération de la morphogenèse rénale de la progéniture. En conclusion, nos études ont permis de démontrer que le glucose élevé ou l’environnement in utero diabétique altère la morphogenèse du UB, qui résulte en un retard dans la néphrogenèse et produit des reins plus petits. Cet effet est dû, au moins en partie, à la génération des ROS, à l’activation du RAS intrarénal et à la voie NF-kB. Nos études futures se concentreront sur les mécanismes par lesquels le diabète maternel induit la programmation périnatale de l’hypertension chez la progéniture adulte. Cette étude à long terme porte sur trois types de progénitures : adultes nés de mères contrôles, diabétiques ou diabétiques traitées avec insuline pendant la gestation. Nous observerons la pression systolique, la morphologie rénale et l’expression de divers gènes et protéines. Nous voulons de plus déterminer si la présence d’un système antioxydant (catalase) peut protéger la progéniture des effets néfastes des ROS causés par l’environnement in utero hyperglycémique. Les souris Catalase-Tg expriment la catalase spécifiquement dans les tubules proximaux et nous permettrons d’explorer notre hypothèse sur le rôle des ROS dans notre modèle expérimental de diabète maternel.Maternal diabetes is a major risk factor for congenital malformations. When the fetus is exposed to high, sustained, ambient glucose levels, widespread fetal damage may affect multiple organs, including the kidneys, evoking diabetic embryopathy syndrome. Renal malformations account for up to 40% of childhood renal failure cases. Hyperglycemia constitutes an adverse in utero environment that dynamically impairs nephrogenesis, resulting in renal agenesis, dysplasia, aplasia or hypoplasia. However, the molecular mechanisms by which high, ambient glucose levels lead to renal dysmorphogenesis and birth defects have not yet been delineated. Maternal diabetes also programs the offspring to develop other problems later in life, such as hypertension, obesity and type 2 diabetes. This phenomenon, called ‘perinatal programming’, has attracted worldwide attention in recent decades, yet the mechanisms by which it occurs are incompletely understood. My PhD studies are designed to elucidate the underlying molecular pathways by which maternal diabetes or hyperglycemic environments in utero impair nephrogenesis and subsequently make the offspring develop perinatal programming of hypertension in vitro, ex vivo and in vivo. We employed mouse embryonic metanephric mesenchyme cells, namely MK4 cells, for our in vitro experiments, and 2 transgenic (Tg) mouse lines, Hoxb7-GFP-Tg and Nephrin-CFP-Tg mice, for ex vivo and in vivo investigations. Hoxb7-GFP-Tg mice specifically express green fluorescent protein (GFP) in ureteric buds (UB), driven by the Hoxb7 promoter. Nephrin-CFP-Tg mice express cyan fluorescent protein (CFP) in glomeruli, driven by the podocyte-specific nephrin promoter. In our in vitro studies, we examined whether high glucose alters Pax2 gene expresson in MK4 cells. The cells were treated with either 5 mM D-glucose plus 20 mM D-mannitol or 25 mM D-glucose media with or without reactive oxygen species (ROS) blockers (DPI, rotenone), and inhibitors of p38 mitogen-activated protein kinase (MAPK) (SB203580), p44/22 MAPK (PD98059), protein kinase C (PKC) (GF109203X), or nuclear factor kappa B (NK-kB) (PDTC) for 24-hr incubation. Our data showed that high D-glucose (25 mM) increased ROS generation and specifically induced Pax2 gene expression, but not other glucose analogs such as D-mannitol, L-glucose or 2-deoxy-D-glucose in MK4 cells. The stimulatory effect of high D-glucose on Pax2 gene expression could be blocked by ROS and NF-kB inhibitors in MK4 cells but not by inhibitors of p38 MAPK (SB203580), p44/22 MAPK (PD98059), and PKC (GFX) in MK4 cells. These data indicated that the stimulatory effect of high glucose on Pax2 gene expression is mediated, at least in part, via ROS generation and activation of NF-κB, but not via the PKC, p38 MAPK and p44/42 MAPK signalling pathways. In our ex vivo studies, we investigated the influence of a high-glucose milieu on UB branching morphogenesis. Kidney explants (E12 to E18) were microdissected from timed-pregnant Hoxb7-GFP mice and cultured with either 5 mM D-glucose plus 20 mM D-mannitol or 25 mM D-glucose media with or without ROS blockers (DPI, rotenone), catalase and phosphoinositide-3-kinase (PI3K)/AKT inhibitor at different time points, depending on the experiment. We found that high D-glucose specifically stimulated UB branching in a time-dependent manner. High D-glucose stimulation of UB branching morphogenesis was mediated via Pax2 gene expression. High D-glucose-induced UB branching and Pax2 gene expression could be blocked by ROS and PI3K/AKT inhibitors. These studies demonstrated that high glucose alters UB branching morphogenesis via Pax2 gene and protein expression. The stimulatory effect of high glucose seems to be mediated via ROS generation and activation of the AKT signalling pathway. In our in vivo studies, we explored the fundamental role of maternal diabetes on renal morphogenesis impairment in offspring. In our experimental model, maternal diabetes was induced by streptozotocin in pregnant Hoxb7-GFP mice at embryonic day 13. The offspring were examined at several time points after birth (neonatal, 1 week, 2 weeks, and 3 weeks) with follow-up of kidney morphology, nephron number, gene expression, and apoptotic events in this short-term postnatal experiment. We observed that the offspring of diabetic mice had lower body weight, body size, kidney weight, small volume of glomeruli and a reduced number of nephrons in comparison to non-diabetic control offspring. Renal dysmorphogenesis may have been the result of increased cell apoptosis in glomeruli. Our findings showed that the offspring of diabetic mice displayed significantly more apoptotic podocytes as well as augmented active caspase-3 immunostaining in renal tubules compared to control mice offspring. Diabetic mice offspring presented heightened expression of intrarenal renin-angiotensin system (RAS) components, such as angiotensinogen and renin, with upregulation of p50 and p65 NF-kB isoforms. These data indicated that maternal diabetes activates the intrarenal RAS and induces glomerular apoptosis, resulting in impairment of renal morphogenesis in diabetic offspring. In conclusion, our findings indicated that a high-glucose milieu in utero or maternal diabetic alters UB morphogenesis, culminating in retardation of nephrogenesis with smaller kidney size. The underlying mechanism(s) is mediated, at least in part, via ROS generation and activation of the intrarenal RAS and NF-kB pathways. In the future, we aim to investigate the underlying mechanism(s) of how maternal diabetes induces perinatal programming of adult hypertension in offspring in vivo. This long-term postnatal study will be undertaken in 3 groups: adult offspring (20 weeks) of control mice, adult offspring of diabetic pregnant mice, and adult offspring of insulin-treated, diabetic, pregnant mice. We will follow-up by tracking hypertension, kidney morphology, and gene expression. Furthermore, we also plan to determine whether an antioxidant system (catalase) can protect against an hyperglycemic environment in utero that affects embryonic organogenesis via an increase in ROS generation. Catalase-Tg mice that specifically overexpress catalase in proximal tubules will be tested. Such Tg mice with catalase overexpression represent a model for exploring our hypothesis on the role of ROS in gestational diabetes