The occurrence of a functional intracellular renin-angiotensin system (RAS) has emerged as a new paradigm. Recently, we and others demonstrated intracellular synthesis of ANG II in cardiac myocytes and vascular smooth muscle cells that was dramatically stimulated in high glucose conditions. Cardiac fibroblasts significantly contribute to diabetes-induced diastolic dysfunction. The objective of the present study was to determine the existence of the intracellular RAS in cardiac fibroblasts and its role in extracellular matrix deposition. Neonatal rat ventricular fibroblasts were serum starved and exposed to isoproterenol or high glucose in the absence or presence of candesartan, which was used to prevent receptor-mediated uptake of ANG II. Under these conditions, an increase in ANG II levels in the cell lysate represented intracellular synthesis. Both isoproterenol and high glucose significantly increased intracellular ANG II levels. Confocal microscopy revealed perinuclear and nuclear distribution of intracellular ANG II. Consistent with intracellular synthesis, Western analysis showed increased intracellular levels of renin following stimulation with isoproterenol and high glucose. ANG II synthesis was catalyzed by renin and angiotensin-converting enzyme (ACE), but not chymase, as determined using specific inhibitors. High glucose resulted in increased transforming growth factor-β and collagen-1 synthesis by cardiac fibroblasts that was partially inhibited by candesartan but completely prevented by renin and ACE inhibitors. In conclusion, cardiac fibroblasts contain a functional intracellular RAS that participates in extracellular matrix formation in high glucose conditions, an observation that may be helpful in developing an appropriate therapeutic strategy in diabetic conditions.

Patients with cystic fibrosis who are infected with the pathogen Pseudomonas aeruginosa have shown favorable responses to the drug azithromycin (AZM). This drug works in an anti-inflammatory capacity, improving clinical outcomes and improving quality of life in this population. The drug has also been shown to affect macrophage polarization by shifting these cells away from an inflammatory phenotype toward an alternatively activated anti-inflammatory phenotype. The full impact of this phenotypic change is not well understood in the context of the response to P. aeruginosa infection, or the overall immune response in cystic fibrosis. To understand how the AZM-polarized macrophage affects other types of cells, we utilized a co-culture in vitro system, with macrophages and fibroblasts incubating together. In this system, we determined that AZM causes upregulation of the pro-fibrotic mediator transforming growth factor-β as well as the extracellular matrix (ECM) protein fibronectin. The mediator of ECM turnover, matrix metalloproteinase (MMP)-9 was upregulated in this system as well. In an in vivo model of P. aeruginosa infection, MMP- 9 and fibronectin were increased in the bronchoalveolar lavage 7 days post-infection in mice that were treated with AZM. This was accompanied by a decrease in damage to the lung tissue, determine by histological examination. To determine if these changes would continue in human subjects with cystic fibrosis, a clinical study was done in this population. Subjects with AZM treatment had decreased TGF-β levels, but no differences in MMP-9 or fibronectin. Interestingly, correlations between certain fibrotic mediators and inflammatory cytokines, specifically interleukin -1β, were different in subjects with AZM treatment compared to subjects without AZM therapy. Together, these data indicate that AZM alters the fibrotic response from the macrophages, as well as the interaction of the inflammatory response and fibrosis development.

Idiopathic pulmonary fibrosis is a chronic and fatal disease that causes the stiffening of lung tissue and gradual lung function decline. Currently, there are no effectives therapies for this disease. Fibrotic lungs are characterized by accumulation of smooth muscle α actin- (SMA) expressing myofibroblasts and excessive deposition of a collagen rich extracellular matrix. The differentiation of lung fibroblasts into myofibroblasts is stimulated by numerous growth factors, including transforming growth factor β (TGFβ), and potentiated by a stiff mechanical environment. Our laboratory has identified a secreted matrix protein, aortic carboxypeptidase-like protein (ACLP), which is upregulated in idiopathic pulmonary fibrosis. Additionally, ACLP knockout mice are protected from experimentally induced fibrosis. This led to the hypothesis that ACLP promotes the fibroblast to myofibroblast transition, and the goal of this research was to characterize the mechanism of ACLP action. ACLP expression preceded SMA and collagen type I expression in rapidly differentiating primary mouse lung myofibroblasts. In gain of function studies, recombinant ACLP induced SMA and collagen I expression in both primary differentiating myofibroblasts as well as IMR90 human lung fibroblasts. ACLP knockdown by siRNA slowed myofibroblast differentiation and partially reverted fully differentiated myofibroblasts into fibroblasts. Because of the similarities among ACLP targets and TGFβ targets, it was hypothesized that ACLP stimulates TGFβ signaling. In lung fibroblasts, ACLP induced Smad3 phosphorylation and nuclear translocation, a feature of TGFβ signaling. The effects of ACLP on myofibroblast differentiation were dependent on TGFβ receptor (TβR) kinase activity and ACLP interacted directly with T&betaR II to promote myofibroblast differentiation. A recombinant TβR II Fc chimera was used to inhibit ACLP-induced SMA expression, but this reagent had no effect on ACLP-induced collagen type I expression, which suggests a differential regulation of SMA and collagen by ACLP. Additionally, ACLP modulated changes in differentiation between cells grown on softer versus stiffer matrices. Using recombinant fragments of the ACLP protein, the N-terminal thrombospondin repeat domain was found to be necessary and sufficient to promote myofibroblast differentiation. Taken together, these studies identified a novel mechanism of ACLP action in fibroblasts and may lead to new therapeutic strategies to treat fibrotic disease.

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.

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.

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. / -

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.

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.

No description available.

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 α3, α2, α5, 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 α2. 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. / EPSRC and DTA grant