Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a multipotent cytokine involved in the production and function of hematopoietic cells, and GM-CSF plays in particular a major role in responses to infection and physiological and pathological inflammatory processes. GM-CSF is produced in many cell types, and increases in the intracellular Ca2+ concentration are, like in many other systems, of major importance in the intracellular signaling that determines GM-CSF expression after receptor stimulation of the cells. Previous studies have shown that the Ca2+/calmodulin-dependent phosphatase calcineurin (CN) mediates stimulation of GM-CSF transcription in response to Ca2+. This thesis shows that Ca2+ signaling also regulates GM-CSF transcription negatively through Ca2+/calmodulin-dependent kinase II (CaMK II) phosphorylation of serines in the autoinhibitory domain for DNA binding of the transcription factor Ets1. Mutation of the CaMK II target serines increased transactivation of the GM-CSF promoter/enhancer and decreased the sensitivity to inhibition by increased Ca2+ or constitutively active CaMK II. The Ca2+-dependent phosphorylation of Ets1 was also shown to reduce the binding of Ets1 to the GM-CSF promoter in vivo. RUNX1, also known as acute myeloid leukemia 1 (AML1), is one of three mammalian RUNX transcription factors and has many essential functions in hematopoiesis. RUNX1 has also many important roles in the immune system, and RUNX1 is the most frequent target for chromosomal translocation of genes in acute human leukemias. This thesis shows that RUNX1 directly interacts with both subunits of CN and that the strongest interaction is localised to the regulatory CN subunit and the DNA binding domain of the RUNX protein. Constitutively active CN was shown to activate the promoter/enhancer of GM-CSF synergistically with RUNX1, RUNX2 or RUNX3, and the Ets1 binding site of the promoter was shown to be essential for the synergy between RUNX1 and CN in Jurkat T cells. The analysis suggests that Ets1 phosphorylated by the protein kinase glycogen synthase kinase-3β is the target of RUNX1-recruited CN phosphatase at the GM-CSF promoter. Transforming growth factor-β (TGF-β) is another multipotent cytokine that often has a role opposite to that of GM-CSF in inflammatory responses since it is a potent suppressor of immune cells and therefore is anti-inflammatory. This thesis shows that TGF-β can decrease transcription from a GM-CSF promoter/enhancer. Certain constitutively active TGF-β receptors and the TGF-β activated transcription factor Smad3 could also repress GM-CSF transcription, whereas several other Smad proteins did not have this inhibitory effect. The inhibition required intact DNA binding ability of Smad3, and the 125 bp upstream of the transcription initiation site, which was sufficient for the inhibition, contains several weak Smad binding sites near the TATA box next to an Ets1 site of the promoter. Smad3 was able to bind to the promoter DNA together with Ets1 and could also be in complex with Ets1 in the absence of DNA. Surface plasmon resonance analysis revealed that Ets1 interacted with the DNA binding domain of Smad3, and the binding constant of this interaction was about 1 µM. The results identify a negative regulation of the GM-CSF promoter by TGF-β signaling through direct Smad3 binding and indicate that the mechanism is by Smad3 interaction with Ets1 and perhaps other proteins around the TATA box of the promoter. This thesis also identifies a novel transactivation domain in the N-terminal of RUNX1 including the N-terminal α-helix in the DNA binding domain. The domain was also required for RUNX2 and RUNX3 transactivation. Despite this, the N-terminal domain of RUNX1 was not essential for RUNX1 function in megakaryocytopoiesis in vitro from mouse embryonic stem cells.

Transforming growth factor-β (TGF-β) is the proto-type member of a super family of secreted proteins comprised of several structurally related, but functionally divergent proteins like the BMP, activin, inhibin, mullerian inhibitory substance etc. TGF-β was originally identified as a secreted factor, which in the presence of EGF was capable of transforming normal rat kidney fibroblasts. Studies over the years have shown that this protein is multifunctional that influences several processes including development, immune function, epithelial cell growth and motility, wound healing etc. TGF-β plays important role in the normal physiology as well as in pathological conditions in mammals. There are three mammalian isoforms that are involved in several developmental processes as has been shown by the knockout mice models. An important role for TGF-β has been implicated in several disease processes like fibrotic disorders (of liver, lung, kidney), inflammatory disorders (rheumatoid arthritis), autoimmune disorders (systemic lupus erythematosus) and cancer. TGF-β has a dual role in carcinogenesis. Initially it acts as a tumor suppressor and causes growth arrest of epithelial cells and cells in the early stages of cancer. But in an established tumor, TGF-β exerts an effect which is favorable for the survival, progression and metastasis of the tumor by promoting epithelial-mesenchymal transition (EMT), angiogenesis and escape from immune surveillance. Studies using mouse models have shown that an intact TGF-β signaling is essential for the metastasis of breast cancer. These observations indicate that the normal epithelial cells show differential response to TGF-β as compared to the tumor they give rise to. Supporting this, it has been shown that prostate tumor cells show invasive behavior in response to TGF-β and not non-tumorigenic cells. Most actions of TGF-β are brought about by regulation of gene expression and differential gene expression mediated by TGF-β has been reported in tumor cells and normal cells. For example, in response to TGF-β, tumorcells show increase in the production of proteases like uPA, MMPs etc and down regulation of the inhibitors of proteases TIMP isoforms, whereas this is not observed in the normal cells. However, there is no clear understanding of the mechanism (s) responsible for differential responses of various cell types to TGF-β. Since a role for TGF-β has been established in several pathological conditions particularly cancer and fibortic disorders, this pathway are a very attractive target for therapeutic intervention. Hence, if the TGF-β pathway has to be targeted for therapy of any disease, it becomes essential to identify the targets of TGF-β in different cell-types and their mechanism of regulation, particularly in un-transformed and transformed cells. Over the past few years, there have been several independent transcriptome analyses of cells in response to TGF-β treatment in various cell types such as HaCaT, fibroblasts, corneal epithelial cells etc. From a comparison of these studies, it is noted that TGF-β regulates genes in a cell type specific manner. Considering the dual role of TGF-β on normal and transformed cells, identification of genes and/or biochemical pathways regulated by TGF-β in these cells may allow identification of therapeutic targets for diseases involving TGF-β signaling pathway. With this background, the following objectives were set for the current investigation: 1. Identification of targets of TGF-β in normal and tumor cells and also the genes differentially regulated by TGF-β 2. Understand the mechanism of regulation of a few selected genes 3. Characterize novel targets of TGF-β with respect to their regulation by TGF-β and also their function Towards the aim of identification of targets of TGF-β in different cell-lines, expression profiling of genes in response to TGF-β was performed in a lung adenocarcinoma cell line (A549) and a matched immortalized lung epithelial cell line (HPL1D). Our data showed similar regulation of 267 genes in HPL1D and A549 cells by TGF-β. This suggests that the genes commonly regulated in both HPL1D and A549 are not tumor specific. Some of these genes were also reported to be regulated by TGF-β in other studies using micro array in various cell types. While 1757 genes are exclusively regulated by TGF-β in A549, only 733 genes are exclusively regulated in HPL1D cells. The reasons for this differential response are not known. However, some of the genes exclusively regulated in A549 such as Integrin αV, thrombospondin 1 have been shown to aid tumor survival, maintenance and metastasis. In contrast, in HPL1D, TGF-β regulates tumor suppressor genes like WT1, ECM proteins like collagen which are responsible for arrest of cell growth and apoptosis. This differential gene regulation in normal and tumor cells may explain the dual role of TGF-β in carcinogenesis. The differences in the effects of TGF-β on these two cell-lines could be due to the phenotypic properties of these cells, HPL1D being a non-transformed cell-line and A549 being a transformed cell-line. It is also possible that the differences are due to cell-type specific effects. In order to address this question, expression profiling in response to TGF-β was carried out using another cell-line namely HaCaT, which is an immortalized skin keratinocyte cell-line. When the expression profiles of the three celllines namely HPL1D, HaCaT and A549 in response to TGF-β treatment were compared, it was found that the genes regulated by TGF-β can be divided into seven categories based on the cell-line in which they are regulated. In this comparison, it was seen that there were several genes which were regulated by TGF-β in A549 and HaCaT despite the fact that these two cell-lines have little in common. The reason for these two celllines to show similarities in their gene expression profile in response to TGF-β is unclear. When the genes regulated by TGF-β in the three cell-lines were categorized based on their annotated functions using the DAVID tool, it was found that signaling pathways like MAP kinas, focal adhesion, Wnt signaling are regulated by TGF-β in all the celllines. On the other hand, Integrin αV was found to be regulated in A549 and HaCaT cells and very marginal regulation was seen in HPL1D cells. This could be one of the reasons for the similarities between A549 and HaCaT. There are studies which show the role of Integrin αV in some of the TGF-β mediated actions although the mechanism by which Integrin signaling modulates gene expression is not well understood. Our data shows that indeed thrombospondin 1 which is regulated by TGF-β in A549 and HaCaT is regulated through the integrin signaling pathways as blocking this pathway partially blocks the induction of this gene by TGF-β. TGF-β actions on cells are to a large extent are carried out by the phosphorylation of SMAD 2/3 by activated TGF-β type I receptor upon TGF-β signaling. Several genes that are transcriptionally regulated by TGF-β contain a SMAD complex binding element (SBE). However, over the last few years, evidences have accumulated which suggest that some actions of TGF-β could be independent of SMADs, mediated by the other signaling pathways like the MAP kinas, PKC and others. In order to understand the mechanism of regulation of a few selected genes by TGF−β, inhibitors for the three MAP kinas pathways (p38, ERK and JNK) were used prior to treatment with TGF-β. The expression of these genes was assessed by qRT-PCR analyses. These studies showed that most of the genes regulated by TGF-β require one or more of the MAP kinas pathways. In HaCaT and A549, the number of genes dependent on the MAP kinas pathways is more compared to HPL1D. Based on our data, we propose that activated MAP kinas pathway could be one of the essential determining factors for the various differential actions of TGF-β in tumor cells. However, the reason for the behaviour of HaCaT cells, which are untransformed cells in a manner similar to the A549 cells, is still unclear. One of the reasons for the similarity could be the activation of the integrin signaling pathway as described before. The expression profiling data identified several novel targets of TGF-β. One such target is S100A2, a calcium binding protein containing an EF hand motif that has been implicated in cancer. A progressive reduction in the expression of this gene has been reported with increasing grade of the tumor. Our studies show that this gene is regulated by TGF-β in HaCaT and HPl1D, but not in A549 cells. The induction of S100A2 by TGF-β in HaCaT cells is likely to be transcriptional as it is sensitive to actinomycin treatment. We further investigated role of other signaling pathways in the regulation of S100A2 by TGF-β and found that the regulation of this gene by TGF-β depends on the ERK and also the integrin signaling pathways. In order to characterize this gene with respect to its functions, A549 cells were chosen as they have very low endogenous expression of S100A2. Hence, in order to explore if there is any role for the loss of S100A2 expression in the progression of A549 cells, we cloned the DNA of S100A2 in a mammalian expression vector, transected A549 cells with this and isolated clones stably expressing this gene. We performed assays to assess cell proliferation, cell migration and potential to form colonies in soft agar. The data suggests phenotypic differences in the colonies that formed in soft agar and no major differences in other assays. Overall, our data has identified several novel targets regulated by TGF-β other than S100A2 like IGFBP7, FGFR1, and SPUVE etc. Further, regulation of several genes was found to be in a cell type specific manner involving MAP kinase and integrin signaling pathways. This study also identified major differences in the genes regulated by TGF-β in transformed and non-transformed lung epithelial cells.

TGF-β isoforms are important signalling molecules in wound repair in the skin. Transforming growth factor β3 (TGF-β3) has been implicated in scarless healing. In both animal and human models the application of exogenous TGF-β3 causes a reduction in the inflammatory response and improves the architecture of the neodermis. Research into the influence of TGF-β on scarring has tended to focus on fibroblasts. However, keratinocytes play a major role in scarring both indirectly, as a result of their influence over the behaviour of fibroblasts and also by directly influencing wound contraction. Thus, experiments were carried out to investigate the influence of TGF-β3 on the behaviours of a keratinocyte cell line (HaCaT). Incubation with TGF-β3 increased cell spreading and appeared to reduce cell-surface contacts indicated by both SPR imaging and a detachment assay. TGF-β3 also caused a decreased cell alignment response to microcontact printed protein patterns, in part due to the deposition of laminin which is associated with the TGF-β induced cell migration. There is evidence that TGF-β isoforms differentially influence the outcome of wound healing. Similar to the results produce following addition of exogenous TGF-β3, the neutralisation of TGF-β1 and 2 has been shown to reduce scar formation in the adult wounds. During reepithelialisation keratinocytes experience a dynamic environment. Both extracellular matrix proteins and growth factors influence the progression of wound repair which includes both cell migration and proliferation. Few studies have examined collective cell behaviour in response to TGF-β isoforms and ECM coated substrates. Thus both wound closure and cell proliferation assays were conducted for different ECM proteins fibronectin, laminin and collagen type I and for TGF-β1, 2 and 3. Rates of wound closure were significantly reduced on laminin coated substrates while cell proliferation rates were increased. TGF-β2 and 3 induced significant increases in wound closure rates. This appeared to correspond with an increase in the number of cells independently migrating out from the wound margins. Only TGF-β3 caused a significant decrease in cell proliferation over a 4 day period. Laminin332 deposition is central to the reepithelialisation process and is known to be induced in response to TGF-β. Thus experiments were carried out to investigate HaCaT cell laminin332 deposition in response to TGF-β1, 2 and 3. Both an immunofluorescence staining technique and an ELISA based semi-quantification method was used. Following 4 day incubation all TGF-β isoforms significantly increased laminin332 deposition; however TGF-β2 and 3 caused the most significant increases. Integrin receptors enable cell-matrix interactions during wound repair. TGF-β is known to influence the expression of integrin subunits. Thus, experiments were carried out to compare the influence of each TGF-β isoform on the expression of subunits β3, β2, β5, β1 and β4. All TGF-β isoforms significantly increased all subunit expression. TGF-β3 caused the most significant increase in β4 and both TGF-β2 and 3 caused the most significant increase in β2. While there were differences in cell responses to each isoforms, TGF-β3 did not stand out from the other two isoforms. Interestingly, TGF-β2 shared more similarities with TGF-β3 than it did with TGF-β1, in its role in enhancing wound closure and LN332 deposition. These comparative studies have shown that differences exist in the way TGF-β isoforms influence HaCaT cell behaviour, namely migration, laminin deposition and integrin expression.

Mesenchymal stem cells (MSCs) are bone marrow derived multipotent cells with the ability to self-renew and differentiate into multiple connective cell lineages. In vivo, MSCs travel from the bone-marrow to the inflammatory sites and actively participate in remodeling and regeneration process under the influence of soluble growth factors. Due to these inherent properties, MSCs have emerged as an ideal candidate for diverse regenerative therapeutic applications. The development of MSC-based therapies requires in vitro expansion of MSCs; however, MSC expansion results in phenotypical changes that have limited its efficacy upon reintroduction in vivo. In order to increase the efficacy of MSC-based therapeutics, it is critical for us to improve the current understanding of MSC interactions with its niche specific factors and explore new methods to enhance MSC function in vivo. We used tumor conditioned media, which contains soluble factors secreted by tumor cells in culture (TCM), and inflammatory niche-specific soluble factors, such as platelet derived growth factor (PDGF) and transforming growth factor-β1 (TGF-β1), to characterize the mechanical response of MSCs. The intracellular mechanical properties of MSCs were dramatically altered in response to soluble factors and MSCs displayed cytosolic stiffening in response to TCM and TGF-β1. Although PDGF treated cells did not elicit any mechanical response, blocking PDGF signaling with a small molecule inhibitor reversed the stiffening response in TGF-β1 treated cells, indicating crosstalk between these two pathways is essential in TGF-β1 mediated cell stiffening. Furthermore, a genome-wide microarray analysis revealed TGF-β1 dependent regulation of cytoskeletal actin-binding protein (ABP) genes. Actin crosslinking and bundling protein genes, which regulate cytosolic rheology through changes in semiflexible actin polymer meshworks, were upregulated with TGF-β1 treatment. Since TGF-β1 treatment profoundly altered the MSC phenotype after relatively short exposure times, we sought to understand if pretreated cells could sustain these enhanced characteristics leading to higher efficacy in vivo. We found that MSCs pretreated with TGF-β1 displayed enhanced adhesive properties while maintaining the expression profile of surface adhesion molecules even after removal of stimulus. Additionally, pretreated MSCs exposed to lineage specific induction media, demonstrated superior differentiation potential along multiple lineages. Based on the large number of sustained changes, TGF-β1 pretreated cells were used to treat full thickness skin wounds for in vivo wound healing model to determine their therapeutic efficacy. TGF-β1 pretreated MSCs increased wound closure rate and displayed enhanced migration of MSCs towards the center of the wound compared to the control cells. In conclusion, soluble factor pretreated MSCs with altered mechanical properties displayed significantly improved cell functions leading to highly efficient tissue regeneration in vivo. Mechanical priming of MSCs with niche specific factors prior to transplantation can become a viable strategy to maximize their therapeutic potential.

Η χρόνια νεφρική ανεπάρκεια οφείλεται σε διάφορα αίτια βλάβης του σπειράματος και του διαμεσοσωληναριακού χώρου. Χαρακτηρίζεται ιστολογικά από την παρουσία σοβαρού βαθμού σπειραματικής σκλήρυνσης, ίνωσης του διάμεσου ιστού, ατροφίας των ουροφόρων σωληναρίων και υαλίνωσης των αρτηριδίων. Η αρχική βλάβη του σπειράματος που προκαλείται από διάφορα είδη ερεθισμάτων μπορεί να ακολουθήσει την οδό της αποκατάστασης ή να εξελιχθεί προς σκλήρυνση, διαδικασίες στις οποίες συμμετέχουν κυτταροκίνες και αυξητικοί παράγοντες που προέρχονται από ενδοθηλιακά, μεσαγγειακά, επιθηλιακά σωληναριακά κύτταρα, μονοκύτταρα και ινοβλάστες1 . Ο Transforming Growth Factor-β1 (TGF-β1) είναι ο κυριότερος αυξητικός παράγοντας που μέσω πολλαπλών μηχανισμών συμμετέχει στην ανάπτυξη σκληρυντικών αλλοιώσεων2. Προάγει την παραγωγή συστατικών της εξωκυττάριας θεμέλιας ουσίας και μειώνει την αποικοδόμησή τους, προκαλεί ενεργοποίηση των μυοϊνοβλαστών, δηλαδή κυττάρων με μεταναστευτικές ιδιότητες που παράγουν κολλαγόνο, συμμετέχει στη διαφοροποίηση των επιθηλιακών σωληναριακών κυττάρων προς ινοβλάστες και ευοδώνει την κυτταρική απόπτωση η οποία οδηγεί στην απογύμνωση του νεφρικού ιστού από τα φυσιολογικά του κύτταρα. Η χρήση αντισωμάτων κατά του TGF-β1 και αναστολέων του μετατρεπτικού ενζύμου της αγγειοτενσίνης σε διάφορα πειραματικά μοντέλα νεφρικής βλάβης έχει συμβάλλει στην κατανόηση μηχανισμών που συμμετέχουν στη διαδικασία εξέλιξής της. Σε πειραματικά μοντέλα έχει διαπιστωθεί ότι ο TGF–β ευθύνεται για την αυξημένη παρουσία μυοινοβλαστών, την εναπόθεση κολλαγόνου και την απώλεια του σωληναριακού επιθηλίου. Πιο πρόσφατα, ένα μέλος της υπερ-οικογένειας των ΤGF–β, η ΒΜΡ7, φάνηκε να εξουδετερώνει την ίνωση που προκαλείται μέσω του TGF–β. Οι δραστηριότητα των παραγόντων αυτών ελέγχεται από άλλες πρωτείνες οι οποίες μπορούν να αυξήσουν ή να καταστείλουν τη διέγερση των υποδοχέων των παραγόντων αυτών. Τα BMPs είναι ενδογενή μόρια που προστατεύουν το νεφρό από διάφορα είδη βλάβης όπως γενετικές βλάβες, ανοσολογικές αντιδράσεις, περιβαλλοντικοί παράγοντες, μεταβολικά αίτια και καταστάσεις οξείας ή χρόνιας νεφρικής βλάβης. / -

In the exploration into new therapeutic agents for human disease, medicinal herbs offer an enormous resource due to their wide range of biologically active components. However, because of these biologically active components, medicinal herbs can also have toxic side effects. The focus of this thesis is the effect of herbal therapies, both good and bad, on chronic kidney disease (CKD) and tubulointerstitial fibrosis. Tubulointerstitial fibrosis is considered one of the defining characteristics of CKD. In Chapter 1, the literature regarding the pathogenesis of tubulointerstitial fibrosis is reviewed, beginning with the mechanisms of its development, the main structural and functional features, and the molecular mediators. The structural features include activation of resident fibroblasts and transition of tubular epithelial cells into myofibroblasts, deposition of extracellular matrix proteins, increased apoptosis of normal cells of the renal nephron and development of tubular atrophy, increased renal oxidative stress, and hypoxia of renal tissues. Molecular mediators that are explored include angiotensin II, transforming growth factor-ß1 and numerous other cytokines and growth factors. Pharmacological manipulation of these features and their molecular mediators for regression of tubulointerstitial fibrosis is then discussed. Currently, the gold standard of therapy for people with CKD is blockade of the renin-angiotensin-aldosterone system with angiotensin converting enzyme inhibitors (ACEIs) and/or angiotensin receptor blockers (ARBs). Because of the complexity of the pathogenesis of renal fibrosis and the multiple mechanisms by which ACEIs and ARBs work, this portion of the thesis focuses on the qualities that additional agents should have to complement their actions. These additional agents could work by decreasing oxidative damage, by decreasing fibroblast numbers through apoptosis, through the interruption of inflammatory, fibrotic mediators, by increasing anti-inflammatory mediators or through other mechanisms. The literature review therefore continues with a discussion of the historical use of medicinal herbs in genitourinary pathologies and the known contributions that medicinal herbs can make to the treatment or development of tubulointerstitial fibrosis and CKD. From this review, a large number of herbs were identified as having traditional use in renal disorders or as being of interest, currently, to researchers of renal pathologies. However, much information is lacking regarding the mechanisms by which the hypothesised benefits occur, making it impossible to assess which herb(s) may offer valuable additive support or alternative treatments to the therapies currently given to people with CKD. Furthermore, there is a lack of information regarding toxicity of these herbs; many herbs have never been assessed in cell culture or in animal toxicity studies. It was apparent that preliminary in vitro work was necessary before in vivo pharmacological work could be undertaken. This thesis, therefore, aimed to test the following hypotheses: (1) That medicinal herbs used currently for treatment of renal dysfunction have high anti-oxidant properties that can be further enhanced by specific extraction processes; (2) That the in vitro testing of selected extracts from medicinal herbs, identified in (1), will reveal some anti-oxidant benefits or indications of toxicity that need careful analysis in animal studies; (3) That careful in vivo testing of specific toxic medicinal herbs identified in these leadup studies will define specific pathological processes that predict an outcome of CKD; and (4) That careful in vivo testing of selected medicinal herbs, used in conjunction with more conventional medicines for CKD, will show an additive benefit when used to ameliorate development of CKD induced using an established animal model. The subsequent laboratory work was designed to test the validity of these hypotheses and the results are then presented in Chapters that each comprise a publication. The aim of Chapter 2 was to present a systematic analysis of the oxidant properties of 55 medicinal herbs that have been used traditionally to treat kidney and urinary disorders or have been of recent interest to researchers of renal disorders. Since different extraction processes yield different constituents, each of the herbs was sequentially extracted with three solvents of decreasing polarity. An assay was performed on each of the fractions to determine the oxygen radical absorbance capacity. The aim of Chapter 3 was to test the benefit or otherwise of each of the three extracts of the chosen herbs using an in vitro cell study. Each extract was tested for potential toxic, apoptotic, mutagenic and antioxidant activity on normal mammalian renal tubular epithelial cells (NRK-52E). The effect of the extracts on renal fibroblasts (NRK-49F) was also analysed. Several specific hypotheses arose from the combination of the systematic analyses and the literature review regarding benefits and toxicities of a number of the extracts. The subsequent in vivo work was designed to test the validity of two of these hypotheses. The aim of Chapter 4 was to test the hypotheses developed from the results of the previous Chapter. The herb Dioscorea villosa had demonstrated extreme cytotoxicity to mammalian renal epithelial cells and had caused transdifferentiation of epithelial cells into fibroblasts. An in vivo rodent model was used to test chronic dosage with this herb and its toxicity and predisposition for induction of CKD verified. The aim of Chapter 5 was to determine whether a herbal preparation (Angelica sinensis and Astragalus membranaceus) that had some support from the literature and the results from Chapters 2 and 3, could complement the actions of ACEIs in a rodent model of renal fibrosis (unilateral ureteral obstruction). The combination of herbal medicines and the ACEI was significantly more effective than the ACEI alone in ameliorating several characteristics of CKD development. To conclude the thesis, Chapter 6 provides an overview discussion of the results and a critical analysis of the methods used. Further, Chapter 6 looks towards future experiments that are planned to further resolve issues of concern about effects on renal health from use of medicinal herbs. .

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2007. / Härtill 4 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst., 2005. / Härtill 5 uppsatser.

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.

La fibrose pulmonaire (FP) idiopathique est une maladie sans traitement efficace caractérisée par une prolifération de myofibroblastes et par un départ sous-pleural suggérant un rôle de la plèvre. Le transforming growth factor (TGF)-ß1 induit un processus de transformation des cellules mésothéliales pleurales (CMP) en cellules de type myofibroblaste. Les protéines de choc thermique régulent la voie du TGF-ß1. L’importance de l’axe caspase-1/IL-1ß, a été décrite dans les modèles animaux de FP.La protéine de stress AlphaB-crystallin a été étudiée dans la FP au niveau des CMP et l’importance de l’axe caspase-1/IL-1ß a été recherchée au niveau des cellules structurales pulmonaires dans la toxicité de la bléomycine (BLM).aB-crystallin est surexprimée dans la FP idiopathique au niveau des CMP. Son inhibition empêche la transformation et la migration des CMP dans la fibrose pleuro-pulmonaire. Dans un modèle de FP induite par la BLM chez la souris, la voie caspase-1 est activée dans les régions pleurales. In vitro, la caspase-1 a un rôle crucial dans la transformation des CMP. Son activation induit une réaction fibrosante chez la souris. Dans une seconde partie, nous montrons qu’une forme déglycosylée de la BLM, incapable d’activer la caspase-1, n’induit pas de FP mais a une activité anti-cancéreuse. La déglyco-BLM n’entraine pas la pyroptose, mort caspase-1 dépendante, chez les cellules épithéliales alvéolaires. Nos résultats suggèrent qu’AlphaB-crystallin et la voie caspase-1/IL-1ß pourraient être des cibles thérapeutiques dans la FP idiopathique ou induite par la BLM. Nous apportons aussi une preuve de concept de l’utilisation de la déglyco-BLM comme alternative non toxique à la bléomycine. / Idiopathic Pulmonary Fibrosis (PF) is a rare and devastating disease without efficient treatment at this time. Idiopathic FP is characterized by accumulation of myofibroblasts and has a typical sub-pleural onset suggesting a role of the pleura in the disease. Transforming Growth Factor (TGF)-ß1 induces transformation of pleural mesothelial cells (PMC) into active cells exhibiting myofibroblast phenotype. Heat shock proteins can act as regulator of the TGF-ß1 signaling. A role for caspase-1/IL-1ß axis has already been described in animal models of PF.The heat shock protein AlphaB-crystallin has been studied in PF at the PMC level and the importance of caspase-1/IL-1ß axis has been investigated specifically in lung structural cells in the context of bleomycin (BLM) toxicity.AlphaB-crystallin is overexpressed by PMC during idiopathic PF. Its inhibition in mice interferes with PMC transformation and subsequent migration in pleuro-pulmonary fibrosis. In BLM-induced PF in mice, caspase-1 is activated in sub-pleural areas. In vitro, caspase-1 has a crucial role in the transformation process of PMC. Activation of caspase-1 triggers fibrotic response in mice. In a second part, we show that a deglycosylated form of BLM, which failed to promote caspase-1 activation, is unable to trigger PF but stills have an anti-tumor activity. Deglyco-BLM does not induce pyroptosis, a caspase-1 dependent cell death, in alveolar epithelial cells.Our data suggest that AlphaB-crystallin and caspase-1/IL-1ß could represent interesting therapeutic targets in idiopathic as well as BLM-induced PF. We also bring a proof of concept for the use of deglyco-BLM as a less toxic alternative to BLM in cancer therapy.