ABSTRACT
The importance of Discoidin Domain Receptor 1 (DDR1) in renal fibrosis has been shown via gene knockout and use of antisense oligonucleotides; however, these techniques act via a reduction of DDR1 protein, while we prove the therapeutic potential of inhibiting DDR1 phosphorylation with a small molecule. To date, efforts to generate a selective small-molecule to specifically modulate the activity of DDR1 in an in vivo model have been unsuccessful. We performed parallel DNA encoded library screens against DDR1 and DDR2, and discovered a chemical series that is highly selective for DDR1 over DDR2. Structure-guided optimization efforts yielded the potent DDR1 inhibitor 2.45, which possesses excellent kinome selectivity (including 64-fold selectivity over DDR2 in a biochemical assay), a clean in vitro safety profile, and favorable pharmacokinetic and physicochemical properties. As desired, compound 2.45 modulates DDR1 phosphorylation in vitro as well as prevents collagen-induced activation of renal epithelial cells expressing DDR1. Compound 2.45 preserves renal function and reduces tissue damage in Col4a3-/- mice (the preclinical mouse model of Alport syndrome) when employing a therapeutic dosing regime, indicating the real therapeutic value of selectively inhibiting DDR1 phosphorylation in vivo. Our results may have wider significance as Col4a3-/- mice also represent a model for chronic kidney disease, a disease which affects 10% of the global population.
Subject(s)
DNA/genetics , Discoidin Domain Receptor 1/antagonists & inhibitors , Kidney/physiopathology , Nephritis, Hereditary/genetics , Animals , Autoantigens/genetics , Autoantigens/metabolism , Collagen Type IV/genetics , Collagen Type IV/metabolism , Discoidin Domain Receptor 1/metabolism , Disease Models, Animal , Epithelial Cells/metabolism , Kidney Function Tests , Mice , Mice, Knockout , Nephritis, Hereditary/physiopathology , Phosphorylation , Src Homology 2 Domain-Containing, Transforming Protein 1/metabolismABSTRACT
In pharmacological research the development of promising lead compounds requires a detailed understanding of the dynamics of disease progression. However, for many diseases, such as kidney fibrosis, gaining such understanding requires complex real-time, multi-dimensional analysis of diseased and healthy tissue. To allow for such studies with increased throughput we established a dextran hydrogel-based in vitro 3D co-culture as a disease model for kidney fibrosis aimed at the discovery of compounds modulating the epithelial/mesenchymal crosstalk. This platform mimics a simplified pathological renal microenvironment at the interface between tubular epithelial cells and surrounding quiescent fibroblasts. We combined this 3D technology with epithelial reporter cell lines expressing fluorescent biomarkers in order to visualize pathophysiological cell state changes resulting from toxin-mediated chemical injury. Epithelial cell damage onset was robustly detected by image-based monitoring, and injured epithelial spheroids induced myofibroblast differentiation of co-cultured quiescent human fibroblasts. The presented 3D co-culture system therefore provides a unique model system for screening of novel therapeutic molecules capable to interfere and modulate the dialogue between epithelial and mesenchymal cells.
Subject(s)
Cell Communication/physiology , Coculture Techniques , Kidney Diseases/metabolism , Kidney/metabolism , Cell Differentiation/physiology , Cell Line , Coculture Techniques/methods , Epithelial Cells/metabolism , Epithelial Cells/pathology , Fibrosis/metabolism , Fibrosis/pathology , Gene Expression , Humans , Kidney/pathology , Kidney Diseases/pathology , Models, Biological , Myofibroblasts/metabolism , Myofibroblasts/pathology , Tissue ScaffoldsABSTRACT
Today, novel therapeutics are identified in an environment which is intrinsically different from the clinical context in which they are ultimately evaluated. Using molecular phenotyping and an in vitro model of diabetic cardiomyopathy, we show that by quantifying pathway reporter gene expression, molecular phenotyping can cluster compounds based on pathway profiles and dissect associations between pathway activities and disease phenotypes simultaneously. Molecular phenotyping was applicable to compounds with a range of binding specificities and triaged false positives derived from high-content screening assays. The technique identified a class of calcium-signaling modulators that can reverse disease-regulated pathways and phenotypes, which was validated by structurally distinct compounds of relevant classes. Our results advocate for application of molecular phenotyping in early drug discovery, promoting biological relevance as a key selection criterion early in the drug development cascade.
Subject(s)
Computational Biology/methods , Drug Discovery/methods , Phenotype , Data Mining , Drug Evaluation, Preclinical , HumansABSTRACT
The aim of the present study was to analyze in vivo the role of zinc finger protein SNAI1 (SNAI1) on renal fibrosis. Unilateral ureteral obstruction injury was induced in Snai1 knockout mice. Snai1 gene deletion was, however, only partial and could therefore not be correlated to reduced fibrosis. Expression of SNAI1 protein and epithelial-mesenchymal transformation markers was then assessed in human chronic allograft nephropathy biopsy specimens. Significant up-regulation of SNAI1 protein was detected within cytoplasm of proximal tubules localized, for some of them, near foci of fibrosis and tubular atrophy. No concomitant epithelial-mesenchymal transformation could, however, be demonstrated analyzing the expression of the fibroblast markers vimentin, α-smooth muscle actin, and S100A4. SNAI1 cytoplasmic up-regulation was particularly evident in biopsy specimens obtained from calcineurin inhibitor-treated patients, which might be because of, as suggested by in vitro experiments, a decrease of the proteasome chimotrypsin activity. Deeper analysis on chronic allograft nephropathy biopsy specimens suggested that SNAI1 cytoplasmic up-regulation was preceded by a transient increase of phosphorylated heat shock protein 27, p38 mitogen-activated protein kinase, and glycogen synthase kinase 3ß. Concomitant down-regulation of the polyubuquitinylated conjugates was detected in SNAI1+ tubules. Altogether, these results might suggest that calcineurin inhibitor-induced tubular SNAI1 protein cytoplasmic accumulation, possibly because of impaired SNAI1 proteasomal degradation and nuclear translocation, might be a sign of a diseased profibrotic epithelial phenotype.
Subject(s)
Cytoplasm/metabolism , Epithelial Cells/metabolism , Kidney Transplantation , Kidney Tubules/metabolism , Kidney Tubules/pathology , Snail Family Transcription Factors/metabolism , Zinc Fingers , Allografts/drug effects , Animals , Biopsy , Calcineurin Inhibitors/pharmacology , Chronic Disease , Dogs , Epithelial-Mesenchymal Transition/drug effects , Female , Fibrosis , Glycogen Synthase Kinase 3 beta/metabolism , HSP27 Heat-Shock Proteins/metabolism , Kidney Transplantation/adverse effects , Kidney Tubules/drug effects , Madin Darby Canine Kidney Cells , Male , Mice, Knockout , Phenotype , Phosphorylation/drug effects , Proteasome Endopeptidase Complex/metabolism , Up-Regulation/drug effects , Ureteral Obstruction/pathology , p38 Mitogen-Activated Protein Kinases/metabolismABSTRACT
α-Smooth Muscle Actin (α-SMA), a widely characterized cytoskeletal protein, represents the hallmark of myofibroblast differentiation. Transforming growth factorß1 (TGFß1) stimulates α-SMA expression and incorporation into stress fibers, thus providing an increased myofibroblast contractile force that participates in tissue remodeling. We have addressed the molecular mechanism by which α-SMA is stably incorporated into stress fibers in human myofibroblasts following exposure to TGFß1. The unique N-terminal sequence AcEEED, which is critical for α-SMA incorporation into stress fibers, was used to screen for AcEEED binding proteins. Tropomyosins were identified as candidate binding proteins. We find that after TGFß1 treatment elevated levels of the Tpm1.6/7 isoforms, and to a lesser extent Tpm2.1, precede the increase in α-SMA. RNA interference experiments demonstrate that α-SMA fails to stably incorporate into stress fibers of TGFß1 treated fibroblasts depleted of Tpm1.6/7, but not other tropomyosins. This does not appear to be due to exclusive interactions between α-SMA and just the Tpm1.6/7 isoforms. We propose that an additional AcEEED binding factor may be required to generate α-SMA filaments containing just Tpm1.6/7 which result in stable incorporation of the resulting filaments into stress fibers.
Subject(s)
Fibroblasts/metabolism , Muscle, Smooth/metabolism , Myofibroblasts/metabolism , Protein Isoforms/metabolism , Tropomyosin/metabolism , Humans , Proteomics , Stress FibersABSTRACT
Kidney fibrosis, a scarring of the tubulo-interstitial space, is due to activation of interstitial myofibroblasts recruited locally or systemically with consecutive extracellular matrix deposition. Newly published clinical studies correlating acute kidney injury (AKI) to chronic kidney disease (CKD) challenge this pathological concept putting tubular epithelial cells into the spotlight. In this work we investigated the role of epithelial cells in fibrosis using a simple controlled in vitro system. An epithelial/mesenchymal 3D cell culture model composed of human proximal renal tubular cells and fibroblasts was challenged with toxic doses of Cisplatin, thus injuring epithelial cells. RT-PCR for classical fibrotic markers was performed on fibroblasts to assess their modulation toward an activated myofibroblast phenotype in presence or absence of that stimulus. Epithelial cell lesion triggered a phenotypical modulation of fibroblasts toward activated myofibroblasts as assessed by main fibrotic marker analysis. Uninjured 3D cell culture as well as fibroblasts alone treated with toxic stimulus in the absence of epithelial cells were used as control. Our results, with the caveats due to the limited, but highly controllable and reproducible in vitro approach, suggest that epithelial cells can control and regulate fibroblast phenotype. Therefore they emerge as relevant target cells for the development of new preventive anti-fibrotic therapeutic approaches.
Subject(s)
Epithelial Cells/pathology , Kidney Tubules/pathology , Cell Line , Cellular Microenvironment/drug effects , Cisplatin/pharmacology , Coculture Techniques , Epithelial Cells/drug effects , Fibrosis , HumansABSTRACT
Endocrine cells, such as H295R have been widely used to study secretion of steroid and other hormones. Exocytosis-dependent hormone release is accompanied by an increase in plasma membrane surface area and a decrease in vesicle content. Recovery of vesicles and decrease in plasma membrane area is achieved by endocytotic processes. These changes in the extent of the surface area lead to morphological changes which can be determined by label-free real-time impedance measurements. Exo- and endocytosis have been described to be triggered by activation of L-type Ca(2+) channels. The present study demonstrates that activation of L-type calcium channels induces prolonged oscillating changes in cellular impedance. The data support the hypothesis that a tight regulation of the intracellular Ca(2+) concentration is a prerequisite for the observed cellular impedance oscillations. Furthermore evidence is presented for a mechanism in which the oscillations depend on a Ca(2+)-triggered calmodulin-dependent cascade involving myosin light chain kinase, nonmuscle myosin II and ultimately actin polymerization, a known determinant for cell shape changes and exocytosis in secretory cells. The described assay provides a method to determine continuously prolonged changes in cellular morphology such as exo/endocytosis cycles. This article is part of a Special Issue entitled: 11th European Symposium on Calcium.
Subject(s)
Adrenal Cortex/cytology , Adrenal Cortex/metabolism , Calcium/metabolism , Cell Shape , 3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester/pharmacology , Adrenal Cortex/drug effects , Angiotensin II/pharmacology , Calcium Channels, L-Type , Calmodulin/metabolism , Cell Line , Cell Shape/drug effects , Electric Impedance , Heterocyclic Compounds, 4 or More Rings/pharmacology , Humans , Imaging, Three-Dimensional , Intracellular Space/drug effects , Intracellular Space/metabolism , Ouabain/pharmacology , RNA, Small Interfering/metabolism , Thapsigargin/pharmacology , Time FactorsABSTRACT
OBJECTIVE: The increased mortality observed with the cholesteryl ester transfer protein inhibitor torcetrapib is partly due to increased aldosterone production and blood pressure. The mechanisms underlying these effects were investigated. METHODS: Cytochrome P450 subunit 11B2 (aldosterone synthase), extracellular signal-regulated kinase (p44/42) and voltage-gated Cachannel alpha subunit mRNA profiling, aldosterone production, cytosolic calcium and RNA interference were assessed in adrenocarcinoma human cells (H295R). Telemetry was conducted in spontaneously hypertensive rats. RESULTS: Torcetrapib and angiotensin II (Ang II) but not dalcetrapib (a structurally different cholesteryl ester transfer protein inhibitor) elevated both cytochrome P450 subunit 11B2 mRNA and aldosterone production in H295R cells at 6 h. At days 1-5, torcetrapib produced a sustained increase of cytochrome P450 subunit 11B2 mRNA, unlike Ang II. Although torcetrapib and Ang II potentiated the effect of 25-OH cholesterol and raised pregnenolone levels, torcetrapib increased neither cytosolic Ca at 5 min nor extracellular signal-regulated kinase1/2 phosphorylation, suggesting initially divergent pathways. Unlike Ang II, torcetrapib steroidogenesis was not affected by Ang II type 1 receptor antagonism or voltage-gated T-type Ca channel antagonism, but was blocked by several L-type Cachannel antagonists. In unbiased genome-wide screening, Ang II and torcetrapib modulated an overlapping but distinct set of genes in H295R cells. Torcetrapib, but not Ang II, upregulated mRNA levels of the L-type Ca channel alpha 1C subunit. In spontaneously hypertensive rat, torcetrapib had a potent hypertensive effect mediated by the L-type Ca channel. CONCLUSION: The unique steroidogenic and hypertensive side effects of torcetrapib may be linked and involve voltage-gated L-type Ca channels. Structurally unrelated cholesteryl ester transfer protein inhibitors such as dalcetrapib do not share this effect.