Le TGFβ (Transforming Growth Factor-β) est une cytokine ayant de nombreusesfonctions au cours de la vie embryonnaire et de la vie adulte. Au cours de la cancérogenèse,le TGFβ a un effet anti-tumoral sur les épithelia sains ou immortalisés, et acquière despropriétés facilitant la progression tumorale des épithélia transformés. Afin d’étudier cettedualité fonctionnelle du TGFβ, nous avons choisi comme modèle d’étude l’adénocarcinomedu pancréas, une tumeur de très mauvais pronostic, qui représente la cinquième cause demortalité par cancer dans les pays développés. Les cancers du pancréas, dans leur grandemajorité, présentent des mutations activatrices de l’oncogène Kras, sécrètent de grandequantités de TGFβ et présentent des mutations inactivatrices au niveau de gènes régulateursde la voie du TGFβ. L’objectif général de mes travaux de thèse était de comprendre le rôle duTGFβ au cours des différentes phases de la cancérogenèse pancréatique grâce à l'utilisationde souris génétiquement modifiées. Tout d’abord, nous avons montré que l’activation cibléede la voie du TGFβ dans le pancréas coopérait avec l’oncogène Kras afin d’induire unepancréatite, une inflammation du pancréas favorisant le développement tumoral. Nous avonségalement démontré le rôle suppresseur de tumeur de TIF1γ, une protéine dont la fonction estméconnue mais qui a été proposée pour réguler la voie du TGFβ. En conclusion, mes travauxont tout d’abord contribué à une meilleure compréhension des mécanismes à l’origine del’inflammation du pancréas. Ceci ouvre de nouvelles perspectives de traitement visant àinactiver le programme pro-inflammatoire du TGFβ et ainsi d’inhiber l’effet pro-tumoral dela pancréatite. D’autre part, mes travaux ont permis de mettre en évidence une nouvelle voiesuppresseur de tumeur dans le pancréas. La caractérisation des programmes anti-tumorauxmis en jeu par TIF1γ devrait permettre de définir de nouvelles stratégies thérapeutiques. / The TGFβ (Transforming Growth Factor-β) belongs to a wide family of cytokinesinvolved in numerous functions during embryogenesis and adult life. During tumorigenesis,TGFβ is considered as a double-edge-sword preventing tumor initiation in normal orimmortalized epithelia but, in contrast, facilitating tumor progression in transformedepithelia. We have studied this dual functionality of TGFβ in Pancreatic DuctalAdenocarcinoma (PDAC), a devastating disease representing the fifth leading-cause ofrelated-cancer death in industrialized countries. Most of pancreatic cancers present activatingKras oncogene mutations, high expression level of secreted TGFβ and inactivating mutationsof affecting major mediators of the TGFβ signaling. The main objective of my thesis was tounderstand the role of TGFβ during pancreatic tumorigenesis using genetically modifiedmouse models, then mimicking the human disease. First, we showed that targeted activationof TGFβ signaling in the pancreas could cooperate with Kras oncogene to induce pancreatitis,an inflammation of the pancreas described as a tumor-promoting environment. Second, wedemonstrated the tumor suppressor role of TIF1γ, a protein recently involved in the TGFβsignaling. In conclusion, this work has contributed to a better understanding of the molecularmechanisms responsible for pancreatitis initiation. Our results open new therapeuticsperspectives leading to the inhibition of the TGFβ-mediated program of thus inhibiting prooncogeniceffect of pancreatitis. Moreover, we defined a new tumor suppressor pathwayactivated in the pancreas. The molecular characterization of programs engaged by TIF1γcould allow defining new therapeutic strategies.

The amphibian species Xenopus laevis, along with mouse and chicken is a very important model system, used widely to dissect the molecular intricacies of various aspects of vertebrate development. Study with Xenopus has clear advantages in terms of various technical considerations including the ease of handling early stage of embryos and due to the remarkable documentation of several early molecular events during development. The concept of inductive interactions between various cell types during early development was first revealed by the studies performed in Xenopus, and among the various factors proposed for mesoderm induction, the members of transforming growth factor-β (TGF- β) superfamily have been considered to be the most probable candidates. About forty different members of the TGF-β superfamily have been cloned and characterized from various organisms. The superfamily members like activins and BMPs have been studied extensively with respect to their functional role during development. While BMPs were assigned as candidates for inducing ventral mesoderm, activins oppose the role of BMPs by inducing dorsal mesoderm. Studies that helped in delineating their roles were performed using three approaches that utilized the ligands, receptors or down stream signaling components (Smads). All the three components were studied with respect to their endogenous expression pattern and effects of ectopic expressions of the wild type or dominant negative mutants. These approaches led to the accumulation of evidences supporting the importance of these signaling molecules. All the above mentioned studies were only possible due to the cloning and characterization of cDNAs of the various proteins involved in the signaling pathway including the ligands. TGF-β2 and 5 are the two isoforms of TGF-β cloned from the amphibian system. We have earlier cloned and characterized the promoter for TGF-β5 gene, which suggested possible regulation of this factor by tissue specific transcription factors. Messenger RNA in situ hybridization analysis to study the TGF-β5-expression pattern during Xenopus development, showed spatial and temporal expression pattern. The expression was confined to specific regions that include notochord, somites, and tail bud among others, in the various stages analyzed. This suggested a possible role for TGF-β5 in organogenesis during the amphibian development. To better understand the role of TGF-β in Xenopus development, studies to examine the specific receptor expression pattern for this growth factor is very essential. With the lack of any reports on cloning of TGF-β receptors from this system, the aim of the present study was to isolate and characterize the receptors for TGF-β from Xenopus laevis. PCR cloning using degenerate primers based on the conserved kinase domains of this class of receptors, coupled to library screenings enabled the identification of two novel receptor cDNAs of the TGF-β receptor superfamily. Characterization of the isolated cDNAs suggested that one of them codes for a type II receptor for TGF-β. Further the cDNAs were found to be ubiquitously expressed during development, as judged by RT-PCR analysis. The cloned cDNAs can now be employed as tools, to study the expression pattern by means of mRNA in situ hybridization, on the various developmental stage embryos and to perform studies using antisense and dominant negative mRNA injection experiments in vivo. Such studies will greatly assist in delineating the role of TGF-β ligands and receptors during amphibian development.

Diss. (sammanfattning) Linköping : Linköpings universitet, 2008. / Härtill 4 uppsatser.

Diss. (sammanfattning) Linköping : Linköpings universitet, 2007. / Härtill 4 uppsatser.

Thesis (Ph.D. in Pharmacology) -- University of Colorado Denver, 2007. / Typescript. Non-Latin script record Includes bibliographical references (leaves 112-128). Free to UCD affiliates. Online version available via ProQuest Digital Dissertations;

La fibrose pulmonaire idiopathique (FPI) est de pronostic sombre et sans traitement efficace. Elle est caractérisée par un début sous pleural et la présence de myofibroblastes responsables de la synthèse excessive de la matrice extracellulaire. La voie de signalisation du Transforming Growth Factor (TGF)-β1, facteur clé de la genèse de la fibrose et sa progression, passe par les Smads, notamment Smad4. Le TGF-β1 induit la différenciation des fibroblastes pulmonaires et des cellules épithéliales et mésothéliales en myofibroblastes. AB-crystallin est une protéine de choc thermique surexprimée dans la fibrose du foie, du rein et la fibrose vasculaire. Elle peut être induite par le TGF-β1. Dans ce travail, nous avons étudié le rôle d’αB-crystallin dans la fibrose pleurale et pulmonaire. Nous montrons qu’αB-crystallin est surexprimée dans les poumons et la plèvre de patients atteints de FPI. In vivo, dans trois modèles de fibrose pulmonaire (bléomycine, surexpression de TGF-β1 ou d’IL-1β) les souris KO pour αB-crystallin sont protégées de la fibrose avec une inhibition de la voie du TGF-β. In vitro, dans les cellules épithéliales, mésothéliales ou les fibroblastes, αB-crystallin augmente la localisation nucléaire de Smad4. En interagissant avec TIF1γ, responsable de l’export nucléaire de Smad4, elle favorise la séquestration nucléaire de Smad4 et son activité pro-fibrosante. Au contraire, son inhibition permet la formation du complexe Smad4/TIF1γ et l’export nucléaire de Smad4 inhibant son activité. Ce travail montre l’importance d’αB-crystallin dans la fibrose pleuro-pulmonaire et son rôle sur la voie du TGF-. AB-crystallin pourrait être une cible thérapeutique de la FPI. / Idiopathic pulmonary fibrosis (IPF) has no effective current treatment. It is characterized by a sub-pleural onset and the presence of myofibroblasts, responsible for the excessive extracellular matrix synthesis. Transforming Growth Factor (TGF)-β1 is considered as the major profibrotic cytokine. Its signaling pathway occurs through the Smads proteins, including Smad4. TGF-β1 allows the differentiation of lung fibroblasts and epithelial and mesothelial cells into myofibroblasts. AB-crystallin is a small heat shock protein overexpressed in liver, renal and vascular fibrosis and can be induced by TGF-β1. In this study, we assessed the role of αB-crystallin in pleural and pulmonary fibrosis. We show that αB-crystallin is overexpressed in the lung and the pleura of IPF patients. In vivo, in three pulmonary fibrosis models (bleomycin, TGF-β1 or IL-1β overexpression) αB-crystallin KO mice are protected from fibrosis with an inhibition of the TGF-β pathway. In vitro, in epithelial and mesothelial cells or fibroblasts, αB-crystallin increases Smad4 nuclear localization. Interacting with TIF1γ, responsible for the nuclear export of Smad4, it promotes the nuclear sequestration of Smad4 and thus its profibrotic activity. Instead, αB-crystallin inhibition allows the formation of the Smad4/TIF1γ complex and promotes Smad4 nuclear export an profibrotic activity. This work shows the importance of αB-crystallin in pleuro-pulmonary fibrosis and its role on the TGF-β1 pathway. AB-crystallin appears as a putative therapeutic target for IPF.

Die Knochenhomöostase erfolgt durch das Zusammenspiel mehrerer Zelltypen. Während die Osteoblasten für den Knochenaufbau verantwortlich sind, resorbieren die Osteoklasten Knochengewebe. Beide Vorgänge werden durch die Osteozyten streng reguliert. Eine Störung im strikt regulierten Gleichgewicht zwischen Knochenabbau und Knochenaufbau kann daher zu Knochenkrankheiten wie Osteoporose führen. Auf molekularer Ebene erfolgt die Kommunikation zwischen den einzelnen Zelltypen über zwei wichtige Signalwege, den der „Bone Morphogenetic Protein“-Superfamilie (BMPs) und den der Wnt-Proteine. Die Signalübertragung wird hierbei durch sekretierte Faktoren induziert, die an Rezeptoren auf der Zelloberfläche binden. Deren Aktivierung führt zu einem intrazellulären Signal, welches letztlich die Expression von Zielgenen reguliert. Beide Signalwege werden auf mehreren Ebenen, extrazellulär, membranständig und intrazellulär reguliert. Das 2003 identifizierte Sclerostin ist ein Vertreter der extrazellulären Regulatorproteine und wurde aufgrund seiner Zugehörigkeit zur DAN-Familie zunächst fälschlicherweise als direkter Inhibitor des BMP-Signalwegs eingestuft. Mittlerweile wird allerdings davon ausgegangen, dass Sclerostin den Wnt-Signalweg negativ reguliert, indem es die Wnt Ko-Rezeptoren LRP5 und LRP6 bindet, die beide zu der Familie der „Low-density lipoprotein receptors“ gehören. Über den molekularen Inhibitionsmechanismus von Sclerostin war jedoch zum Startpunkt dieser Dissertationsarbeit wenig bekannt. Daher wurde Sclerostin im Rahmen dieser Arbeit biophysikalisch und biochemisch charakterisiert. Die Aufklärung mittels NMR-Spektroskopie ergab für Sclerostin eine Struktur, die sich in drei Regionen gliedert: den Cystinknoten, sowie einen „Loop“-Bereich und die Fingerregion. Vom zentralen Cystinknoten gehen drei Peptid-Schleifen in zwei entgegengesetzte Richtungen aus. Schleife eins und drei bilden eine definierte ß-Faltblattstruktur und ähneln zwei Fingern einer Hand. Die zweite Schleife, welche vom Cystinknoten isoliert in die entgegengesetzte Richtung verläuft („Loop“), ist wie die beiden langen N- und C-Termini flexibel und unstrukturiert. Die in Zusammenarbeit mit der Firma AbD-Serotec entstandenen Fab-Fragmente ermöglichten die Bestimmung des Bindeepitops der Sclerostin/LRP5-Interaktion im Bereich der unstrukturierten dritten Schleife von Sclerostin. Die Struktur von Sclerostin und die Identifikation des Bindeepitops auf Sclerostinseite geben nun erste Einblicke in den molekularen Mechanismus der Sclerostin/LRP5-Interaktion. Diese Kenntnis kann für die Entwicklung von Kleinmolekülinhibitoren mittels rationalem Drugdesign genutzt werden, welche, wie auch der in Kooperation entwickelte die Sclerostinaktivität neutralisierende Antikörper AbD09097, hochinteressante Ansätze für neuartige anabole Therapien von Krankheiten mit Knochenschwund darstellen. / Different cell types like osteoblasts, osteoclasts and osteocytes maintain bone homeostasis. While osteoblasts build up bone, osteoclasts resorb bone tissue and both actions are tightly regulated by the osteocytes. Imbalance between bone formation and resorption will lead to various bone diseases, e.g. osteoporosis. On a molecular level communication between these cell types occurs through two major signalling pathways, i.e. the bone morphogenetic proteins (BMPs) and the Wnt-factors. In both pathways signal transduction is induced by secreted factors, which bind to cell surface receptors. This activation leads to an intracellular signal that finally regulates expression of target genes. Both pathways are tightly regulated at various cellular levels, extracellular, at the membrane as well as intracellular. Sclerostin, which was identified in 2003, is a member of the extracellular modulator proteins. Initially it was wrongly classified as a direct inhibitor of the BMP-signalling pathway due to its classification as a member of the DAN-family. Meanwhile it became apparent that sclerostin targets the Wnt-pathway by binding to the Wnt co-receptors LRP5 and LRP6, which belong to the family of low-density lipoprotein receptors. At the beginning of this work very little was known about the molecular mechanism how sclerostin inhibits the Wnt-pathway. The structure analysis of sclerostin employing NMR-spectroscopy revealed in a modular architecture, which can be divided into three regions: the central, characteristic cystine knot, the loop-region and the two fingers. From the cystine knot three loops emanate in two opposite directions. Loop one and loop three form defined ß-sheet structures resembling two fingers of a hand. Loop two, which runs into the opposite direction, is unstructured and highly flexible like the long N- and C-termini. Antibody fab-fragments, which were generated in collaboration with AbD-Serotec, facilitated the mapping of the binding-epitop of sclerostin to LRP5/6, highlighting an extended area of the unstructured loop region of sclerostin as the LRP5/6 binding site. The high-resolution structure of sclerostin and the identification of the LRP5-binding-epitop yield first insights into the molecular mechanism of sclerostin-LRP5 interaction. This knowledge can now be used to develop small-molecule inhibitors by rational drug design, which are, like the sclerostin activity neutralising fab-fragment AbD09097, highly interesting targets for new bone-anabolic therapies of diseases characterised by bone loss.

Bone Morphogenetic Proteins (BMPs) are secreted multifunctional signaling proteins that play an important role during development, maintenance and regeneration of tissues and organs in almost all vertebrates and invertebrates. BMPs transmit their signals by binding to two types of serine-/threonine-kinase receptors. BMPs bind first to their high affinity receptor, thereby recruiting their low affinity receptor into the complex. This receptor assembly starts a Smad (Small mothers against decapentaplegic) protein signaling cascade which regulates the transcription of responsive genes. Up to date, only seven type I and five type II receptors are known for more than 30 ligands. Therefore, many BMP ligands can recruit more than one receptor subtype. Vice versa, receptors can bind to several ligands, indicating a highly promiscuous ligand-receptor interaction. This raises the following questions: (i) How are BMPs able to induce ligand-specific signals, despite forming complexes with identical receptor composition and (ii) how are they able to recognize and bind various binding partners in a highly specific manner. From the ligand’s point of view, heterodimeric BMPs are valuable tools for studying the interplay between different sets of receptors, thereby providing new insights into how the various BMP signals can be generated. This study describes the expression and purification of the heterodimers BMP-2/6 and -2/7 from E.coli cells. BIAcore interaction studies and various in vitro cell activity assays revealed that the generated heterodimers are biologically active. Furthermore, BMP-2/6 and -2/7 exhibit a higher biological activity in most of the cell assays compared to their homodimeric counterparts. In addition, the BMP type I receptor BMPR-IA is involved in heterodimeric BMP signaling. However, the usage of other type I receptor subtypes (e.g. ActR-I) building a heteromeric ligand-receptor type I complex as indicated in previous works could not be determined conclusively. Furthermore, BMP heterodimers seem to require only one type I receptor for signaling. From the receptors’ point of view, the BMP type I receptor BMPR-IA is a prime example for its promiscuous binding to different BMP ligands. The extracellular binding interface of BMPR-IA is mainly unfolded in its unbound form, requiring a large induced fit to adopt the conformation when bound to its ligand BMP-2. In order to unravel whether the binding promiscuity of BMPR-IA is linked to structural plasticity of its binding interface, the interaction of BMPR-IA bound to an antibody Fab fragment was investigated. The Fab fragment was selected because of its ability to recognize the BMP-2 binding epitope on BMPR-IA, thus neutralizing the BMP-2 mediated receptor activation. This study describes the crystal structure of the complex of the extracellular domain of BMPR-IA bound to the antibody Fab fragment AbyD1556. The crystal structure revealed that the contact surface of BMPR-IA overlaps extensively with the contact surface of BMPR-IA for BMP-2 interaction. Although the contact epitopes of BMPR-IA to both binding partners coincide, the three-dimensional structures of BMPR-IA in both complexes differ significantly. In contrast to the structural differences, alanine-scanning mutagenesis of BMPR-IA showed that the functional determinants for binding to both the antibody and BMP-2 are almost identical. Comparing the structures of BMPR-IA bound to BMP-2 or to the Fab AbyD1556 with the structure of unbound BMPR-IA revealed that binding of BMPR-IA to its interaction partners follows a selection fit mechanism, possibly indicating that the ligand promiscuity of BMPR-IA is inherently encoded by structural adaptability. / „Bone Morphogenetic Proteins” (BMPs) sind sezernierte multifunktionelle Signalproteine, die eine wichtige Rolle während der Entwicklung, Aufrechterhaltung und Regeneration von Geweben und Organen in fast allen Vertebraten und wirbellosen Tieren spielen. Die BMP-Signalgebung wird durch die Bindung an zwei Typen von Serin/Threonin Rezeptorkinasen eingeleitet. Hierbei binden BMPs zuerst an ihren hochaffinen Rezeptor, bevor der niederaffine Rezeptor in den Komplex eingefügt wird. Durch das Zusammenfügen beider Rezeptortypen wird eine von Smad (Small mothers against decapentaplegic)-Proteinen gesteuerte Signalkaskade gestartet, die letztendlich die Transkription responsiver Gene reguliert. Aktuell sind nur sieben Typ I und fünf Typ II Rezeptoren für mehr als 30 Liganden bekannt. Viele BMP-Liganden können demzufolge mehr als einen Rezeptorsubtyp rekrutieren. Umgekehrt jedoch können auch Rezeptoren an unterschiedliche Liganden binden, was auf eine im hohen Maße promiske Ligand-Rezeptor-Interaktion hinweist. Dabei stellen sich folgende Fragen: (i) Wie können BMPs ligandspezifische Signale erzeugen, obwohl sie dafür die gleichen Rezeptoren benutzen? (ii) Und wie können BMPs unterschiedliche Bindungspartner erkennen und trotzdem hochspezifisch an diese binden? Von Blickwinkel der Liganden aus betrachtet stellen heterodimere BMPs wertvolle Hilfsmittel dar, um das Zusammenspiel zwischen den verschiedenen Rezeptortypen zu studieren. Darüber hinaus können sie neue Einblicke in die Entstehung von unterschiedlichen BMP-Signalen gewähren. In dieser Doktorarbeit wird die Expression und Aufreinigung von heterodimeren BMP-2/6 und -2/7 aus E.coli Zellen beschrieben. Mittels BIAcore Interaktionsstudien und in vitro Aktivitätsassays in Säugerzellen konnte gezeigt werden, dass die hergestellten Heterodimere biologisch aktiv sind. Darüber hinaus zeigen BMP-2/6 and -2/7 in den meisten Zellassays eine höhere biologische Aktivität als ihre homodimeren Gegenstücke. Außerdem konnte nachgewiesen werden, dass der BMP Typ I Rezeptor BMPR-IA an der Signalgebung von heterodimeren BMPs involviert ist. Eine Beteiligung weiterer Typ I Rezeptoren (wie z.B. die von ActR-I), die einen heteromeren Ligand-Rezeptor Typ I Signalkomplex bilden, wie es bereits in früheren Studien gezeigt wurde, konnte jedoch experimentell nicht eindeutig belegt werden. Des Weiteren lassen die Ergebnisse darauf schließen, dass heterodimere BMPs für eine erfolgreiche Signalweiterleitung nur die Präsenz eines einzelnen Typ I Rezeptors benötigen. Von Blickwinkel der Rezeptoren aus betrachtet, ist der BMP Typ I Rezeptor BMPR-IA ein Paradebeispiel für promiskes Bindeverhalten an verschiedene BMP-Liganden. Das extra-zelluläre Kontaktepitop von BMPR-IA ist im Wesentlichen ungefaltet, wenn BMPR-IA in freier ungebundener Form vorliegt. Infolge dessen durchläuft die Binderegion in BMPR-IA weit reichende strukturelle Veränderungen, um die erforderliche Konformation auszubilden, die für die Bindung an BMP-2 essentiell ist. Um herauszufinden, ob das promiske Binde-verhalten von BMPR-IA mit einer strukturellen Plastizität seiner Binderegion einhergeht, wurde die Interaktion zwischen BMPR-IA und einem Antikörper Fab Fragment experimentell untersucht. Das Fab Fragment wurde aufgrund folgender Eigenschaft ausgewählt, nämlich an das BMP-2 Bindeepitop des Rezeptors anzudocken, um so eine BMP-2 vermittelte Rezeptoraktivierung zu verhindern. In dieser Doktorarbeit wird die Kristallstruktur des Komplexes, bestehend aus der extrazellulären Domäne von BMPR-IA und dem Antikörper Fab Fragment AbyD1556 beschrieben. Die Kristallstruktur zeigt, dass die Kontaktoberfläche von BMPR-IA zu einem sehr großen Teil mit der Kontaktoberfläche bei der Interaktion mit BMP-2 übereinstimmt. Obwohl das Kontaktepitop von BMPR-IA zu beiden Bindungspartnern weitestgehend deckungsgleich ist, unterscheiden sich die dreidimensionalen Strukturen von BMPR-IA in beiden Komplexen sehr stark voneinander. Im Gegensatz zu den strukturellen Differenzen zeigt jedoch eine Mutationsanalyse, bei der wichtige Aminosäuren mit Alanin ausgetauscht wurden, dass die funktionellen Determinanten, die die Bindung an den Antikörper und an BMP-2 bestimmen, beinahe die gleichen sind. Wenn man die Strukturen von BMPR-IA, das an BMP-2 bzw. an das Fab Fragment AbyD1556 gebunden ist, mit der Struktur von ungebundenem BMPR-IA vergleicht, so fällt auf, dass die Bindung von BMPR-IA an seine Bindungspartner einem sog. „Selektions-Anpassungsmechanismus“ folgt, was möglicherweise zeigt, dass das promiske Ligand-Bindeverhalten von BMPR-IA von Natur aus durch seine strukturelle Anpassungsfähigkeit festgelegt wird.

目的： 转化生长因子－1（TGF-β1）通过与II型受体结合而引起I型受体活化，进一步激活其下游信号分子蛋白Smad2 和Smad3，它们与Smad4（Co-Smad）结合后形成Smad复合体并发生核转移，从而发挥广泛的生物学效应。同时，整个TGF-β信号通路又受到其抑制因子Smad7的负反馈调节。研究结果显示Smad3是肾脏炎症和纤维化中重要的致病分子，相反，Smad7在多种肾脏疾病中起保护作用。然而，由于转化生长因子II型受体（TβRII），Smad2 或Smad4基因敲除的小鼠无法存活，这些分子在TGF-β1介导的肾脏炎症和纤维化中的功能尚未见报道。因此，本研究旨在剖析TβRII、Smad2 和Smad4 在肾脏疾病发生发展中的作用及机制。 / 方法：本研究利用Cre/LoxP系统分别靶向敲除小鼠肾小管上皮细胞的TβRII、Smad2 或者Smad4，通过结扎小鼠单侧输尿管建立梗阻性肾病模型，观察这些分子对肾脏炎症和纤维化的影响，并用体外实验进行验证。具体实验结果请参见本论文第III,IV, V章。 / 结果：通过分析，本论文取得以下新的发现： / (1) TβRII在TGF-β1介导的肾脏炎症和纤维化的双向调节中起到了决定性的作用：研究结果显示条件性敲除TβRII明显抑制TGF-β/Smad3介导的肾脏纤维化，同时增强NF-κB引起的肾脏炎症反应。由此可见，TRII不仅仅是TGF-β/Smad信号通路的启动因子，更决定了TGF-β1对肾脏炎症和纤维化的双向性调节。(参见第III章) / (2)尽管Smad2和Smad3结构相似并共同介导了TGF-β1的生物学效应，本研究意外发现Smad2可反向调节Smad3引起的纤维化。体内和体外实验共同证实，敲除Smad2基因增强了Smad3的磷酸化，核转位及其转录子活性，并能促进Smad3与I型胶原转录子的结合，进而加重肾脏纤维化（参见第IV章）。 / (3)我们还发现Smad4不仅作为TGF-β/Smad信号通路的共有蛋白，它在TGF-β1介导肾脏炎症和纤维化中起到了重要的双向性调节作用：条件敲除Smad4显著降低了Smad7对NF-κB介导肾脏炎症的抑制作用，同时在转录水平（而非磷酸化水平）抑制Smad3的功能，从而减轻纤维化。（参见第V章） / 结论：TβRII和Smad4 在TGF-β1介导肾脏炎症和纤维化中起到了重要的双向性作用；Smad2通过抑制Smad3信号传导和功能，在肾脏纤维化中起保护作用。 / Objectives: TGF-β1 binds its receptor II (TβRII) and then activates receptor I to initiate the downstream Smad signaling, called Smad2 and Smad3 which bind a common Smad4 to form the Smad complex and then translocate to nucleus to exert its biological activities. This process is negatively regulated by an inhibitory Smad7. While the pathogenic role of Smad3 and the protective role of Smad7 in renal fibrosis and inflammation are clearly understood, the functional role of TβRII, Smad2 and Smad4 in kidney diseases remains largely unexplored due to the lethality of these knockout mice. Therefore, the aim of present study is to dissect the functional role of these TGF-β/Smad signaling molecules in renal inflammation and fibrosis. / Methods: Kidney conditional knockout (KO) mice for TβRII, Smad2 and Smad4 were generated by crossing the FloxFlox mice with the kidney specific promoter driven Cre (KspCre) mice, in which TβRII, Smad2 or Smad4 were specifically deleted from the kidney tubular epithelial cells (TEC) respectively. Then, a well-characterized progressive renal inflammation and fibrosis mouse model of Unilateral ureteral obstructive (UUO) nephropathy was induced in these conditional KO mice and the specific roles for TβRII, Smad2, and Smad4 in renal inflammation and fibrosis were investigated in vivo and in vitro as described in the Chapter III, IV and V of this thesis. / Results: There were several novel findings through this thesis: / 1. TGF-β1 signals through its TβRII to diversely regulate renal fibrosis and inflammation. We found that disrupted TRII suppressed Smad3-dependent renal fibrosis while enhancing NF-κB-driven renal inflammation. Thus, TβRII not only acts as a binding receptor for initiating the TGF-β signaling, but also determines the diverse role of TGF-β1 in inflammation and fibrosis, which was described in the Chapter III. / 2. As shown in the Chapter IV, an unexpected finding from this thesis was that although Smad2 and Smad3 were homologically similar and bound together in response to TGF-β1 stimulation, Smad2 counter-regulated Smad3-mediated renal fibrosis. This was evidenced by the findings that conditional deletion of Smad2 enhanced Smad3 signaling including phosphorylation, nuclear translocation, the Smad3 responsive promoter activity, and the binding of Smad3 to Col1A2 promoter. Thus, disrupted Smad2 from the kidney significantly enhanced Smad3-mediated renal fibrosis in the UUO kidney and in cultured TEC. / 3. Finally, we also showed that that Smad4 acted not only as a common Smad in TGF-β signaling, but exerted its regulatory role in determining the diverse role of TGF-β1 in renal inflammation and fibrosis. Disruption of Smad4 significantly enhanced renal inflammation by impairing inhibitory effect of Smad7 on NF-κB-driven renal inflammation. In contrast, disrupted Smad4 inhibited renal fibrosis by blocking Smad3 functional activity without influencing Smad3 signaling. Because deletion of Smad4 inhibited TGF-β1-induced Smad3 responsive promoter activity and the binding of Smad3 to the Col1A2 promoter without altering the phosphorylation and nuclear translocation of Smad3 (Chapter V). / Conclusions: TβRII and Smad4 may function as key regulators of TGF-β signaling and diversely regulate the renal inflammation and fibrosis. Smad2 plays a protective role in renal fibrosis by counter-regulating Smad3 signaling. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Meng, Xiaoming. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 202-231). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese. / Abstract --- p.i / Declaration --- p.viii / Acknowledgement --- p.ix / Table of Contents --- p.xii / List of Abbreviations --- p.xxvii / List of Figures/Tables --- p.xxix / Chapter CHAPTER I --- INTRODUCTION --- p.1 / Chapter 1.1 --- TGF-β signaling pathway --- p.2 / Chapter 1.1.1 --- TGF-β superfamily --- p.2 / Chapter 1.1.2 --- TGF-β signaling transduction --- p.3 / Chapter 1.1.2.1 --- Smad-dependent TGF-β signaling --- p.4 / Chapter 1.1.2.2 --- Smad-independent TGF-β signaling --- p.10 / Chapter 1.2 --- Chronic Kideny disease (CKD) --- p.12 / Chapter 1.2.1 --- Epidemiology of CKD --- p.12 / Chapter 1.2.2 --- Pathophysiology of CKD --- p.12 / Chapter 1.3 --- TGF-β signaling in renal diseases --- p.13 / Chapter 1.3.1 --- Role of TGF-β1 in renal diseases --- p.13 / Chapter 1.3.2 --- Potential role of TβRII in renal diseases --- p.15 / Chapter 1.3.3 --- Potential role of Smad2 in renal diseases --- p.17 / Chapter 1.3.4 --- Potential role of Smad4 in renal diseases --- p.20 / Chapter 1.3.5 --- Role of Smad7 in renal diseases --- p.23 / Chapter 1.3.6 --- Role of Smad-independent TGF-β signaling in renal disease --- p.24 / Chapter CHAPTER II --- MATERIALS AND METHODS --- p.26 / Chapter 2.1 --- MATERIALS --- p.27 / Chapter 2.1.1 --- Reagents and Equipments --- p.27 / Chapter 2.1.1.1 --- General reagents and equipments for cell culture --- p.27 / Chapter 2.1.1.2 --- General reagents and equipments for real-time RT-PCR --- p.28 / Chapter 2.1.1.3 --- General reagents and equipments for Masson Trichrome Staining --- p.28 / Chapter 2.1.1.4 --- General reagents and equipments for Immunohistochemistry --- p.29 / Chapter 2.1.1.5 --- General reagents and equipments for Immunofluorescence --- p.29 / Chapter 2.1.1.6 --- General reagents and equipments for Western Blot --- p.29 / Chapter 2.1.1.7 --- General reagents and equipments for Promoter assay --- p.31 / Chapter 2.1.1.8 --- General reagents and equipments for ChIP assay --- p.32 / Chapter 2.1.2 --- Buffers --- p.32 / Chapter 2.1.2.1 --- Buffers for Immunohistochemistry --- p.32 / Chapter 2.1.2.2 --- Buffers for Western blot --- p.35 / Chapter 2.1.3 --- Sequences of Primers and siRNAs --- p.40 / Chapter 2.1.4 --- Antibodies --- p.42 / Chapter 2.2 --- METHODS --- p.44 / Chapter 2.2.1 --- Animal model of Unilateral Ureteral Obstruction (UUO) --- p.44 / Chapter 2.2.2 --- Cell culture --- p.44 / Chapter 2.2.2.1 --- NRK52E cell line --- p.44 / Chapter 2.2.2.2 --- Smad2 WT/KO mouse embryonic fibroblasts (MEFs) --- p.45 / Chapter 2.2.2.3 --- Primary culture of kidney fibroblasts --- p.45 / Chapter 2.2.2.4 --- Primary culture of peritoneal macrophages --- p.46 / Chapter 2.2.3 --- PAS staining --- p.47 / Chapter 2.2.3.1 --- Tissue Handling and Fixation --- p.47 / Chapter 2.2.3.2 --- Tissue embedding and sectioning --- p.47 / Chapter 2.2.3.3 --- Preparation of Paraffin Tissue Sections for PAS staining --- p.48 / Chapter 2.2.3.4 --- PAS staining --- p.48 / Chapter 2.2.4 --- Real-time RT-PCR --- p.48 / Chapter 2.2.4.1 --- Total RNA isolation --- p.48 / Chapter 2.2.4.2 --- Reverse Transcription --- p.49 / Chapter 2.2.4.3 --- Real-time PCR --- p.50 / Chapter 2.2.4.4 --- Analysis of Real-time PCR --- p.50 / Chapter 2.2.5 --- Masson Trichrome Staining --- p.51 / Chapter 2.2.6 --- Immunohistochemistry --- p.52 / Chapter 2.2.6.1 --- Preparation of Paraffin Tissue Sections for IHC --- p.52 / Chapter 2.2.6.2 --- Antigen-Antibody Reaction --- p.52 / Chapter 2.2.6.3 --- Signal Detection --- p.53 / Chapter 2.2.6.4 --- Semi-quantification of Immunohistochemistry --- p.53 / Chapter 2.2.7 --- Immunofluorescence --- p.54 / Chapter 2.2.8 --- Western blot analysis --- p.54 / Chapter 2.2.8.1 --- Protein preparation --- p.55 / Chapter 2.2.8.2 --- SDS-PAGE --- p.56 / Chapter 2.2.8.3 --- Transmembrane of protein --- p.56 / Chapter 2.2.8.4 --- Incubation of first and second antibody --- p.57 / Chapter 2.2.8.5 --- Signal capture and analysis --- p.57 / Chapter 2.2.8.6 --- Stripping --- p.57 / Chapter 2.2.9 --- Promoter assay --- p.58 / Chapter 2.2.10 --- ChIP assay --- p.61 / Chapter 2.2.11 --- Statistical analysis --- p.62 / Chapter CHAPTER III --- THE DIVERSE ROLE OF TGF-BETA RECEPTOR II IN RENAL INFLAMMATION AND FIBROSIS --- p.63 / Chapter 3.1 --- INTRODUCTION --- p.64 / Chapter 3.2 --- AIMS --- p.64 / Chapter 3.3 --- MATERIALS AND METHODS --- p.66 / Chapter 3.3.1 --- Generation and characterization of TβRII conditional Knockout mice --- p.66 / Chapter 3.3.2 --- Generation and characterization of TβRII disrupted tubular epithelial cell line (NRK52E) and kidney interstitial fibroblasts --- p.67 / Chapter 3.3.3 --- Animal model of Unilateral Ureteral Obstruction --- p.67 / Chapter 3.3.4 --- Cell culture --- p.67 / Chapter 3.3.5 --- Real-time RT-PCR --- p.68 / Chapter 3.3.6 --- Masson Trichrome Staining --- p.68 / Chapter 3.3.7 --- Immunohistochemistry --- p.68 / Chapter 3.3.8 --- PAS staining --- p.69 / Chapter 3.3.9 --- Immunofluorescence --- p.69 / Chapter 3.3.10 --- Western blot analysis --- p.70 / Chapter 3.3.11 --- Promoter assay --- p.70 / Chapter 3.3.12 --- Statistical analysis --- p.70 / Chapter 3.4 --- RESULTS --- p.71 / Chapter 3.4.1 --- Characterization of TβRII conditional Knockout mice and TβRII disrupted cells --- p.71 / Chapter 3.4.2 --- Disruption of TβRII suppresses renal interstitial damage in the UUO kidney --- p.72 / Chapter 3.4.3 --- Disruption of TβRII suppresses renal fibrosis in UUO kidney and TGF-β1-induced fibrotic response in vitro --- p.76 / Chapter 3.4.3.1 --- Conditional knockout of TβRII from the kidney decreases the collagen I level in UUO kidney --- p.76 / Chapter 3.4.3.2 --- Disruption of TβRII inhibits TGF-β1 induced collagen I level in vitro --- p.79 / Chapter 3.4.3.3 --- Conditional knockout of TβRII from the kidney decreases the α-SMA positive cells infiltration in vivo --- p.81 / Chapter 3.4.3.4 --- Disruption of TβRII inhibits TGF-β1-induced α-SMA expression in vitro --- p.83 / Chapter 3.4.3.5 --- Conditional knockout of TβRII from the kidney decreases the FN level in UUO nephropathy --- p.85 / Chapter 3.4.3.6 --- Disruption of TβRII decreases TGF-β1-induced FN expression in vitro --- p.87 / Chapter 3.4.4 --- Disruption of TβRII impairs the TGF-β/Smad signaling in vivo in the UUO kidney and in vitro in TGF-β1 treated tubular epithelial cells and kidney fibroblasts --- p.89 / Chapter 3.4.4.1 --- Conditional knockout of TβRII decreases the UUO induced TGF-β1 expression in vivo and the TGF-β1 auto-induction in vitro --- p.89 / Chapter 3.4.4.2 --- Disrupted TβRII decreases CTGF level in the UUO nephropathy in vivo and the TGF-β1 induced CTGF mRNA level in vitro --- p.91 / Chapter 3.4.4.3 --- Conditional knockout of TβRII impairs the Smad3 signaling in the injured kidney --- p.93 / Chapter 3.4.4.4 --- Disrupted TβRII inhibits TGF-β1-induced Smad3 phosphorylation, P-Smad3 nuclear translocation and Smad3 responsive promoter activity in vitro --- p.95 / Chapter 3.4.4.5 --- Conditional knockout of TβRII doesn’t alter the activation of ERK and P38 signaling in the UUO kidney --- p.97 / Chapter 3.4.4.6 --- Disrupted TβRII inhibits TGF-β1-induced ERK and P38 phosphorylation in vitro --- p.99 / Chapter 3.4.5 --- Disruption of TβRII enhances inflammatory cytokines expression in the UUO kidney and impairs the anti-inflammatory effect of TGF-β1 in response to IL-1β triggered inflammatory response in the TEC cells --- p.101 / Chapter 3.4.5.1 --- Conditional knockout of TβRII increases the TNF-α expression in the UUO nephropathy --- p.101 / Chapter 3.4.5.2 --- Conditional knockout of TβRII increases the IL-1β expression in the UUO nephropathy --- p.103 / Chapter 3.4.5.3 --- Conditional knockout of TβRII doesn’t enhance the MCP-1 expression and macrophages infiltration in the UUO nephropathy --- p.104 / Chapter 3.4.5.4 --- Disruption of TβRII in TECs decreases the anti-inflammatory effect of TGF-β1 in response to IL-1β --- p.106 / Chapter 3.4.6 --- Disruption of TβRII enhances NFκB activation in vivo and in vitro --- p.108 / Chapter 3.5 --- DISCUSSION --- p.110 / Chapter 3.6 --- CONCLUSION --- p.114 / Chapter CHAPTER IV --- Smad2 protects against TGF-β/Smad3 mediated renal fibrosis --- p.115 / Chapter 4.1 --- INTRODUCTION --- p.116 / Chapter 4.2 --- AIMS --- p.117 / Chapter 4.3 --- MATERIALS AND METHODS --- p.117 / Chapter 4.3.1 --- Generation and characterization of Smad2 conditional Knockout mice --- p.117 / Chapter 4.3.2 --- Generation and characterization of Smad2 KO MEFs and Smad2 knockdown/overexpression tubular epithelial cell line (NRK52E) --- p.118 / Chapter 4.3.3 --- Animal model of Unilateral Ureteral Obstruction --- p.118 / Chapter 4.3.4 --- Cell culture --- p.118 / Chapter 4.3.5 --- Real-time RT-PCR --- p.119 / Chapter 4.3.6 --- Western blot analysis --- p.119 / Chapter 4.3.7 --- Immunohistochemistry --- p.119 / Chapter 4.3.8 --- Masson Trichrome Staining --- p.119 / Chapter 4.3.9 --- Immunofluorescence --- p.120 / Chapter 4.3.10 --- Promoter assay --- p.120 / Chapter 4.3.11 --- ChIP assay --- p.120 / Chapter 4.3.12 --- Statistical analysis --- p.120 / Chapter 4.4 --- RESULTS --- p.121 / Chapter 4.4.1 --- Characterization of Smad2 disrupted mice and cells --- p.121 / Chapter 4.4.1.1 --- Characterization of Smad2 conditional Knockout mice --- p.121 / Chapter 4.4.1.2 --- Characterization of Smad2 knockout MEFs, Smad2 knockdown/overexpression TECs --- p.123 / Chapter 4.4.2 --- Disruption of Smad2 further enhances renal fibrosis in vivo and in vitro --- p.124 / Chapter 4.4.2.1 --- Conditional knockout of Smad2 increases total collagen deposition and Col.I level in the UUO kidney --- p.124 / Chapter 4.4.2.2 --- Disruption of Smad2 in MEFs and TECs increases Col.I production in a time- and dosage-dependent manner in response to TGF-β1 --- p.126 / Chapter 4.4.2.3 --- Conditional knockout of Smad2 increases Col.III level in the UUO kidney --- p.128 / Chapter 4.4.2.4 --- Disruption of Smad2 in MEFs and TECs increases Col.III production in a time- and dosage-dependent manner in response to TGF-β1 --- p.130 / Chapter 4.4.3 --- Disruption of Smad2 further enhances renal fibrosis by suppressing the collagen degradation system in vivo and in vitro --- p.132 / Chapter 4.4.3.1 --- Conditional knockout of Smad2 inhibits the MMP2 mRNA while enhances TIMP-1 production in UUO kidney --- p.132 / Chapter 4.4.3.2 --- Disruption of Smad2 in MEFs and TECs decreases the MMP2 level while enhances TIMP-1 production in response to TGF-β1 --- p.133 / Chapter 4.4.4 --- Disruption of Smad2 further increases renal fibrosis by increasing TGF-β1 auto-induction and CTGF level in vivo and in vitro --- p.135 / Chapter 4.4.4.1 --- Disruption of Smad2 increases TGF-β1 auto-induction in vivo and in vitro --- p.135 / Chapter 4.4.4.2 --- Disruption of Smad2 increases CTGF synthesis in vivo and in vitro --- p.137 / Chapter 4.4.5 --- Disruption of Smad2 further increases renal fibrosis by enhancing Smad3 signaling in vivo and in vitro --- p.139 / Chapter 4.4.5.1 --- Conditional knockout of Smad2 further enhances Smad3 phosphorylation and nuclear translocation --- p.139 / Chapter 4.4.5.2 --- Disruption of Smad2 in MEFs and TECs further enhances Smad3 phosphorylation, nuclear translocation, Smad3 responsive promoter activity and the binding to the Col1A2 promoter --- p.141 / Chapter 4.4.6 --- Overexpression of Smad2 suppresses Smad3 signaling therefore ameliorates the TGF-β1-induced fibrotic response in TECs --- p.144 / Chapter 4.4.6.1 --- Overexpression of Smad2 ameliorates the TGF-β1- induced fibrotic response in TECs --- p.144 / Chapter 4.4.6.2 --- Overexpression of Smad2 suppresses Smad3 phosphorylation --- p.146 / Chapter 4.5 --- DISCUSSION --- p.147 / Chapter 4.6 --- CONCLUSION --- p.150 / Chapter CHAPTER V --- THE DISTINCT ROLE OF SMAD4 IN RENAL INFLAMMATION AND FIBROSIS --- p.151 / Chapter 5.1 --- INTRODUCTION --- p.152 / Chapter 5.2 --- AIMS --- p.152 / Chapter 5.3 --- MATERIALS AND METHODS --- p.153 / Chapter 5.3.1 --- Generation and characterization of Smad4 conditional Knockout mice --- p.153 / Chapter 5.3.2 --- Generation and characterization of Smad4 disrupted kidney interstitial fibroblasts and peritoneal macrophages --- p.153 / Chapter 5.3.3 --- Animal model of Unilateral Ureteral Obstruction (UUO) --- p.154 / Chapter 5.3.4 --- Cell culture --- p.154 / Chapter 5.3.5 --- Real-time RT-PCR --- p.155 / Chapter 5.3.6 --- Western blot analysis --- p.155 / Chapter 5.3.7 --- Immunohistochemistry --- p.155 / Chapter 5.3.8 --- Masson Trichrome Staining --- p.155 / Chapter 5.3.9 --- Promoter assay --- p.156 / Chapter 5.3.10 --- ChIP assay --- p.156 / Chapter 5.3.11 --- Statistical analysis --- p.156 / Chapter 5.4 --- RESULTS --- p.157 / Chapter 5.4.1 --- Characterization of Smad4 conditional Knockout mice and Smad4 disrupted cells --- p.157 / Chapter 5.4.2 --- Disruption of Smad4 suppresses renal fibrosis in the UUO nephropathy in vivo and TGF-β1-induced fibrotic response in vitro --- p.160 / Chapter 5.4.2.1 --- Conditional knockout of Smad4 from the kidney decreases the total collagen deposition in the UUO nephropathy --- p.160 / Chapter 5.4.2.2 --- Conditional knockout of Smad4 from the kidney decreases the Col.I production in the UUO nephropathy --- p.161 / Chapter 5.4.2.3 --- Disruption of Smad4 inhibits TGF-β1-induced Col.I production in vitro --- p.163 / Chapter 5.4.3 --- Disruption of Smad4 impairs the Smad3 function in vivo and in vitro --- p.164 / Chapter 5.4.3.1 --- Conditional knockout of Smad4 doesn’t decrease Smad3 phosphorylation and P-Smad3 nuclear translocation in vivo and in vitro --- p.164 / Chapter 5.4.3.2 --- Disruption of Smad4 inhibits TGF-β1 induced Smad3 promoter activity and the Smad3 binding to Col1A2 promoter --- p.166 / Chapter 5.4.3.3 --- Disruption of Smad4 has minimal effect on the activation of ERK signaling in vivo and in vitro --- p.167 / Chapter 5.4.4 --- Disruption of Smad4 enhances renal inflammation and impairs the anti-inflammatory effect of TGF-β1 in response to IL-1β triggered inflammatory response in vitro --- p.169 / Chapter 5.4.4.1 --- Conditional knockout of Smad4 increases the inflammatory cells infiltration --- p.169 / Chapter 5.4.4.2 --- Conditional knockout of Smad4 increases the TNFα expression in the UUO nephropathy --- p.171 / Chapter 5.4.4.3 --- Conditional knockout of Smad4 increases the IL-1β expression in the UUO nephropathy --- p.172 / Chapter 5.4.4.4 --- Conditional knockout of Smad4 increases the MCP-1 expression in the UUO nephropathy --- p.173 / Chapter 5.4.4.5 --- Conditional knockout of Smad4 increases the ICAM-1 level in the UUO nephropathy --- p.174 / Chapter 5.4.4.6 --- Time and dosage dependent experiments in response to IL-1β in macrophages --- p.175 / Chapter 5.4.4.7 --- Disruption of Smad4 in macrophages decreases the anti-inflammatory effect of TGF-β1 in response to IL-1β --- p.176 / Chapter 5.4.5 --- Disruption of Smad4 impairs the inhibitory effect of Smad7 on NFκB activation in vivo and in vitro --- p.178 / Chapter 5.4.5.1 --- Conditional knockout of Smad4 largely inhibits Smad7 level in UUO kidney --- p.178 / Chapter 5.4.5.2 --- Conditional knockout of Smad4 suppresses IκBα and further increases NF-κB p65 activation in UUO kidney --- p.180 / Chapter 5.4.5.3 --- Disruption of Smad4 inhibits Smad7 synthesis in macrophages --- p.182 / Chapter 5.4.5.4 --- Conditional knockout of Smad4 impair the inhibition effect of TGF-β1 on the activation of NFκB p65 in macrophages --- p.184 / Chapter 5.5 --- DISCUSSION --- p.186 / Chapter 5.6 --- CONCLUSION --- p.189 / Chapter CHAPTER VI --- SUMMARY AND DISCUSSION OF THE MAJOR FINDINGS --- p.190 / Chapter 6.1 --- SUMMARY AND DISCUSSION --- p.192 / Chapter 6.1.1 --- The diverse role of TβRII in renal inflammation and fibrosis both in vivo and in vitro --- p.192 / Chapter 6.1.2 --- Smad2 protects renal fibrosis by counter-regulating Smad3 signaling --- p.192 / Chapter 6.1.3 --- Disruption of Smad4 increased renal inflammation while suppressed the renal fibrosis in vivo and in vitro --- p.194 / Chapter 6.1.4 --- Comparative analysis of functions and related mechanisms between TβRII and Smad4 in renal disease --- p.195 / Chapter 6.1.5 --- Inadequacies of current work and future plan --- p.197 / Chapter 6.1.6 --- Perspectives (1) : The balance within the TGF-b/Smad signaling may determine the fate of renal diseases --- p.197 / Chapter 6.1.7 --- Perspectives(2)：The balance within the TGF-β/Smad signaling may determine the fate of renal diseases --- p.198 / Chapter 6.2 --- CONCLUSION --- p.201 / REFERENCES --- p.202 / PUBLICATION LIST --- p.232 / HONORS AND AWARDS --- p.237

Introdução: A podocitopenia e a podocitúria são marcadores de injúria glomerular em podocitopatias (POD) e glomerulonefrites proliferativas (GNsP), e mesmo em fases iniciais destas doenças mecanismos pró-fibróticos indutores de glomeruloesclerose e fibrose renal progressiva estão ativados. Objetivo: Avaliar pacientes portadores de glomerulopatias biopsiados em diferentes tempos de evolução clínica, correlacionando lesões morfológicas dos compartimentos glomerular e túbulo-intersticial com a expressão dos RNAm de proteínas associadas ao podócito e de fatores pró-fibróticos no tecido renal. Materiais e Métodos: Foram incluídos no estudo oitenta e quatro pacientes adultos portadores de glomerulopatias de diferentes etiologias submetidos à biópsia renal por indicação clínica. As lesões histológicas foram individualizadas e a porcentagem de fibrose intersticial e atrofia tubular foi quantificada na coloração de Tricrômio de Masson. Foram mensurados no tecido renal o log 10 do RNAm pela reação em cadeia da polimerase em tempo real das proteínas associadas ao podócito alfa actinina-4, podocina e podocalixina e dos fatores pró-fibróticos fator de crescimento transformador ₁ (TGF₁), fator de crescimento do tecido conectivo (CTGF) e fator de crescimento derivado do endotélio A (VEGF-A). A secção livre de neoplasia de rins removidos por câncer renal foram usados como controles da expressão dos RNAm. Resultados: No grupo POD, os diagnósticos histopatológicos foram: Glomeruloesclerose segmentar e focal (n=20), GN membranosa (n=12), Nefropatia diabética (n=9) e Lesões mínimas (n=7); no grupo GNsP foram Nefropatia por IgA (n=15), GN membranoproliferativa (n=5), Nefrite lúpica (n=5) e GN proliferativa mesangial (n=4), e outros diagnósticos (n=7). O RNAm do tecido renal nos pacientes com POD e GNsP foi significativamente menor comparado ao dos controles para todos os genes estudados. A presença de crescentes, independente do estágio evolutivo, foi associada à maior expressão do RNAm de alfa actinina-4 (p=0,04), podocina (p=0,01) e podocalixina (p=0,038). O RNAm dos genes pró-fibróticos também estava inibido comparado a sua expressão no rim normal. Nas GNsP, o VEGF-A (p<0,001) e o CTGF (p<0,001) foram os genes com menor nível de expressão comparado aos controles. Em relação às biópsias com lesões crescênticas, tanto o RNAm do TGFβ1 (p=0,001) como do CTGF (p=0,041) tiveram maior expressão comparado ao RNAm das biópsias sem crescentes. Nas biópsias com fibrose intersticial superior a 30%, a expressão do RNAm de TGFβ1, (p=0,038) e do VEGF-A (p=0,040) foi maior do que nas biópsias com fibrose leve. O maior tempo entre o início da doença clínica e a realização da biópsia renal não teve influência detectável na expressão tecidual do RNAm dos biomarcadores estudados. Conclusões: Pacientes com podocitopatias ou glomerulonefrites proliferativas apresentaram inibição da expressão do RNAm de proteínas associadas ao podócito e de fatores indutores de fibrose renal, achados compatíveis com injúria podocitária e podocitopenia. Nas biópsias renais com maior grau de fibrose intersticial e atrofia tubular, assim também como naquelas com lesões crescênticas, a expressão do RNAm de fatores fibrogênicos como TGF-β1 e CTGF foi significativamente aumentada, o que pode sugerir supra-regulação de moléculas associadas a mecanismos de fibrose renal e patologia glomerular. / Introduction: Both podocitopenia and podocyturia are markers of glomerular injury in podocytopathies (POD) and proliferative glomerulonephritis (PGNs), and even in the early stages of these diseases pro-fibrotic mechanisms leading to glomerulosclerosis and progressive renal fibrosis are running. Objective: This study evaluated patients with glomerulopathies who were biopsied at different times of clinical evolution, correlating morphological lesions of the glomerular and tubulointerstitial compartments with renal messenger RNA (mRNA) expression of podocyteassociated proteins and pro-fibrotic factors. Materials and Methods: The study included eighty-four adult patients with glomerulopathies of different etiologies undergoing kidney biopsy as clinically indicated. The histological lesions were individualized and the percentage of interstitial fibrosis and tubular atrophy was quantified on Trichrome Masson staining. Tissue log 10 mRNA of the podocyte proteins alpha-actinin-4, podocin and podocalyxin and of the pro-fibrotic factors transforming growth factor β₁ (TGFβ₁), connective tissue growth factor (CTGF) and vascular endothelium growth factor A (VEGF-A) was measured by real time polymerase chain reaction. The sections free of neoplasia of kidneys removed for renal cancer were used as controls for the mRNA tissue expression. Results: Results: In the POD group, the histopathological diagnoses were: focal segmental glomerulosclerosis (n=20), membranous (n=12), diabetic nephropathy (n=9) and minimal changes (n=7); in PGNs group were IgA nephropathy (n=15), membranoproliferative (n=5), lupus nephritis (n=5) and mesangial proliferative (n=4), and other diagnoses (n=7). Messenger RNA expression of POD and PGNs groups was significantly lower compared to controls for all the studied genes. The presence of crescents, regardless of their evolutive stage, was associated with higher mRNA expression of alpha-actinin-4 (p=0.04), podocin (p=0.01) and podocalyxin (p=0.038). The mRNA of pro-fibrotic genes was also inhibited compared to their expression in normal kidneys. In PGNs, VEGF-A (p<0.001) and CTGF (p<0.001) were the genes with lowest mRNA levels compared to controls. Regarding the biopsies with crescentic lesions, both the mRNA of TGFβ1 (p=0.001) and CTGF (p=0.041) were highly expressed as compared to those of biopsies without crescents. In biopsies with moderate to severe interstitial fibrosis (more than 30%), the mRNA expression of TGFβ1 (p=0.038) and VEGF-A (p=0.040) was highly expressed compared to biopsies with mild fibrosis. A longer interval between the clinical disease and the performance of kidney biopsy did not have a detectable influence on tissue mRNA expression of the studied biomarkers. Conclusions: Patients with POD or PGNs presented inhibition of the mRNA expression of podocyte-associated proteins and pro-fibrotic factors, findings that are consistent with podocyte injury and podocitopenia. In renal biopsies with a higher degree of interstitial fibrosis and tubular atrophy, as well as those with crescentic lesions, the mRNA expression of fibrogenic factors such as TGF-β1 and CTGF was significantly increased, which may suggest upregulation of molecules associated with mechanisms of renal fibrosis and glomerular pathology.