ECM Characterization Reveals a Massive Activation of Acute Phase Response during FSGS.

The glomerular basement membrane (GBM) and extra-cellular matrix (ECM) are essential to maintain a functional interaction between the glomerular podocytes and the fenestrated endothelial cells in the formation of the slit diaphragm for the filtration of blood. Dysregulation of ECM homeostasis can cause Focal segmental glomerulosclerosis (FSGS). Despite this central role, alterations in ECM composition during FSGS have not been analyzed in detail yet. Here, we characterized the ECM proteome changes in miR-193a-overexpressing mice, which suffer from FSGS due to suppression of Wilms' tumor 1 (WT1). By mass spectrometry we identified a massive activation of the acute phase response, especially the complement and fibrinogen pathways. Several protease inhibitors (ITIH1, SERPINA1, SERPINA3) were also strongly increased. Complementary analysis of RNA expression data from both miR-193a mice and human FSGS patients identified additional candidate genes also mainly involved in the acute phase response. In total, we identified more than 60 dysregulated, ECM-associated genes with potential relevance for FSGS progression. Our comprehensive analysis of a murine FSGS model and translational comparison with human data offers novel targets for FSGS therapy.

MicroRNA-193a Regulates the Transdifferentiation of Human Parietal Epithelial Cells toward a Podocyte Phenotype.

Parietal epithelial cells have been identified as potential progenitor cells in glomerular regeneration, but the molecular mechanisms underlying this process are not fully defined. Here, we established an immortalized polyclonal human parietal epithelial cell (hPEC) line from naive human Bowman's capsule cells isolated by mechanical microdissection. These hPECs expressed high levels of PEC-specific proteins and microRNA-193a (miR-193a), a suppressor of podocyte differentiation through downregulation of Wilms' tumor 1 in mice. We then investigated the function of miR-193a in the establishment of podocyte and PEC identity and determined whether inhibition of miR-193a influences the behavior of PECs in glomerular disease. After stable knockdown of miR-193a, hPECs adopted a podocyte-like morphology and marker expression, with decreased expression levels of PEC markers. In mice, inhibition of miR-193a by complementary locked nucleic acids resulted in an upregulation of the podocyte proteins synaptopodin and Wilms' tumor 1. Conversely, overexpression of miR-193a in vivo resulted in the upregulation of PEC markers and the loss of podocyte markers in isolated glomeruli. Inhibition of miR-193a in a mouse model of nephrotoxic nephritis resulted in reduced crescent formation and decreased proteinuria. Together, these results show the establishment of a human PEC line and suggest that miR-193a functions as a master switch, such that glomerular epithelial cells with high levels of miR-193a adopt a PEC phenotype and cells with low levels of miR-193a adopt a podocyte phenotype. miR-193a-mediated maintenance of PECs in an undifferentiated reactive state might be a prerequisite for PEC proliferation and migration in crescent formation.

Computational drug repositioning of clopidogrel as a novel therapeutic option for focal segmental glomerulosclerosis.

Focal segmental glomerulosclerosis (FSGS) is a glomerular lesion often associated with nephrotic syndrome. It is also associated with a high risk of progression to end-stage kidney disease. Current treatment of FSGS is limited to systemic corticosteroids or calcineurin inhibition, along with inhibitors of the renin-angiotensin-aldosterone system. FSGS is heterogeneous in etiology, and novel therapies targeting specific, dysregulated molecular pathways represent a major unmet medical need. We have generated a network-based molecular model of FSGS pathophysiology using previously established systems biology workflows to allow computational evaluation of compounds for their predicted interference with molecular processes contributing to FSGS. We identified the anti-platelet drug clopidogrel as a therapeutic option to counterbalance dysregulated FSGS pathways. This prediction of our computational screen was validated by testing clopidogrel in the adriamycin FSGS mouse model. Clopidogrel improved key FSGS outcome parameters and significantly reduced urinary albumin to creatinine ratio (P < 0.01) and weight loss (P < 0.01), and ameliorated histopathological damage (P < 0.05). Clopidogrel is used to treat several cardiovascular diseases linked to chronic kidney disease. Clopidogrel's favorable safety profile and its efficacy in the adriamycin mouse FSGS model thus recommend it as an attractive drug repositioning candidate for clinical trial in FSGS.

miR-29a suppresses tristetraprolin, which is a regulator of epithelial polarity and metastasis.

Several microRNAs (miRNAs) have recently been described as crucial regulators of epithelial-to-mesenchymal transition (EMT) and metastasis. By comparing the expression profiles of miRNAs, we found upregulation of miR-29a in mesenchymal, metastatic RasXT cells relative to epithelial EpRas cells. Overexpression of miR-29a suppressed the expression of tristetraprolin (TTP), a protein involved in the degradation of messenger RNAs with AU-rich 3'-untranslated regions, and led to EMT and metastasis in cooperation with oncogenic Ras signalling. We also observed enhanced miR-29a and reduced TTP levels in breast cancer patient samples, indicating relevance for human disease. Previously, miR-29 family members were shown to have tumour-suppressive effects in haematopoietic, cholangiocytic and lung tumours. Therefore, miRNAs can act as either oncogenes or tumour suppressors, depending on the context.

Podocyte RNA sequencing reveals Wnt- and ECM-associated genes as central in FSGS.

Loss of podocyte differentiation can cause nephrotic-range proteinuria and Focal and Segmental Glomerulosclerosis (FSGS). As specific therapy is still lacking, FSGS frequently progresses to end-stage renal disease. The exact molecular mechanisms of FSGS and gene expression changes in podocytes are complex and widely unknown as marker changes have mostly been assessed on the glomerular level. To gain a better insight, we isolated podocytes of miR-193a overexpressing mice, which suffer from FSGS due to suppression of the podocyte master regulator Wt1. We characterised the podocytic gene expression changes by RNAseq and identified many novel candidate genes not linked to FSGS so far. This included strong upregulation of the receptor tyrosine kinase EphA6 and a massive dysregulation of circadian genes including the loss of the transcriptional activator Arntl. By comparison with podocyte-specific changes in other FSGS models we found a shared dysregulation of genes associated with the Wnt signaling cascade, while classical podocyte-specific genes appeared widely unaltered. An overlap with gene expression screens from human FSGS patients revealed a strong enrichment in genes associated with extra-cellular matrix (ECM) and metabolism. Our data suggest that FSGS progression might frequently depend on pathways that are often overlooked when considering podocyte homeostasis.

A systems pharmacology workflow with experimental validation to assess the potential of anakinra for treatment of focal and segmental glomerulosclerosis.

Focal and Segmental Glomerulosclerosis (FSGS) is a severe glomerulopathy that frequently leads to end stage renal disease. Only a subset of patients responds to current therapies, making it important to identify alternative therapeutic options. The interleukin (IL)-1 receptor antagonist anakinra is beneficial in several diseases with renal involvement. Here, we evaluated the potential of anakinra for FSGS treatment. Molecular process models obtained from scientific literature data were used to build FSGS pathology and anakinra mechanism of action models by exploiting information on protein interactions. These molecular models were compared by statistical interference analysis and expert based molecular signature matching. Experimental validation was performed in Adriamycin- and lipopolysaccharide (LPS)-induced nephropathy mouse models. Interference analysis (containing 225 protein coding genes and 8 molecular process segments) of the FSGS molecular pathophysiology model with the drug mechanism of action of anakinra identified a statistically significant overlap with 43 shared molecular features that were enriched in pathways relevant in FSGS, such as plasminogen activating cascade, inflammation and apoptosis. Expert adjudication of molecular signature matching, focusing on molecular process segments did not suggest a high therapeutic potential of anakinra in FSGS. In line with this, experimental validation did not result in altered proteinuria or significant changes in expression of the FSGS-relevant genes COL1A1 and NPHS1. In summary, an integrated bioinformatic and experimental workflow showed that FSGS relevant molecular processes can be significantly affected by anakinra beyond the direct drug target IL-1 receptor type 1 (IL1R1) context but might not counteract central pathophysiology processes in FSGS. Anakinra is therefore not suggested for extended preclinical trials.

Beta-catenin/LEF-1 signalling in breast cancer--central players activated by a plethora of inputs.

Although the role of Wnt signalling in breast cancer is far from being fully understood, in the last years its importance has been reported frequently. Besides stimulation by canonical Wnt signalling, the downstream effectors beta-catenin and the transcriptional modulators of the T-cell factor/lymphoid enhancer-binding factor (TCF/LEF) family can also be activated by other inputs including the TGF-beta pathway. Wnt and TGF-beta signalling are both major signal transduction pathways, which provide important cues during development and tumor progression. However, particularly TGF-beta has a complicated influence on oncogenesis, which ranges from suppressive to promoting activity. Signalling pathways activated in parallel with TGF-beta might determine the oncogenic influence, and therefore place signals cooperating with TGF-beta into the limelight. During early development Wnt and TGF-beta signalling collaborate extensively. Here we provide an overview of the known interactions of Wnt with TGF-beta signalling in development and metastasis, particularly in breast cancer. We want to focus on the Wnt-activated transcription factor complex beta-catenin/LEF-1, its upstream activators, its downstream targets and consequences on the cellular level in response to beta-catenin/LEF-1 activation.

Focal segmental glomerulosclerosis is induced by microRNA-193a and its downregulation of WT1.

Focal segmental glomerulosclerosis (FSGS) is a frequent and severe glomerular disease characterized by destabilization of podocyte foot processes. We report that transgenic expression of the microRNA miR-193a in mice rapidly induces FSGS with extensive podocyte foot process effacement. Mechanistically, miR-193a inhibits the expression of the Wilms' tumor protein (WT1), a transcription factor and master regulator of podocyte differentiation and homeostasis. Decreased expression levels of WT1 lead to downregulation of its target genes PODXL (podocalyxin) and NPHS1 (nephrin), as well as several other genes crucial for the architecture of podocytes, initiating a catastrophic collapse of the entire podocyte-stabilizing system. We found upregulation of miR-193a in isolated glomeruli from individuals with FSGS compared to normal kidneys or individuals with other glomerular diseases. Thus, upregulation of miR-193a provides a new pathogenic mechanism for FSGS and is a potential therapeutic target.

Annexin A1 attenuates EMT and metastatic potential in breast cancer.

Metastasis is the major cause of carcinoma-induced death, but mechanisms involved are poorly understood. Metastasis crucially involves epithelial-to-mesenchymal transition (EMT), causing loss of epithelial polarity. Here we identify Annexin A1 (AnxA1), a protein with important functions in intracellular vesicle trafficking, as an efficient suppressor of EMT and metastasis in breast cancer. AnxA1 levels were strongly reduced in EMT of mammary epithelial cells, in metastatic murine and human cell lines and in metastatic mouse and human carcinomas. RNAi-mediated AnxA1 knockdown cooperated with oncogenic Ras to induce TGFß-independent EMT and metastasis in non-metastatic cells. Strikingly, forced AnxA1 expression in metastatic mouse and human mammary carcinoma cells reversed EMT and abolished metastasis. AnxA1 knockdown stimulated multiple signalling pathways but only Tyk2/Stat3 and Erk1/2 signalling were essential for EMT.

The impact of target site accessibility on the design of effective siRNAs.

Small-interfering RNAs (siRNAs) assemble into RISC, the RNA-induced silencing complex, which cleaves complementary mRNAs. Despite their fluctuating efficacy, siRNAs are widely used to assess gene function. Although this limitation could be ascribed, in part, to variations in the assembly and activation of RISC, downstream events in the RNA interference (RNAi) pathway, such as target site accessibility, have so far not been investigated extensively. In this study we present a comprehensive analysis of target RNA structure effects on RNAi by computing the accessibility of the target site for interaction with the siRNA. Based on our observations, we developed a novel siRNA design tool, RNAxs, by combining known siRNA functionality criteria with target site accessibility. We calibrated our method on two data sets comprising 573 siRNAs for 38 genes, and tested it on an independent set of 360 siRNAs targeting four additional genes. Overall, RNAxs proves to be a robust siRNA selection tool that substantially improves the prediction of highly efficient siRNAs.