Graphical Abstract Highlights d Implementing FAIR data standards requires identification of experimental confounders d Five labs performed the same experiment on mammalian cells and compared results d Several factors affecting reproducibility were explored d Biological context had an unexpected impact on the robustness of cell-based assays

Nephrotoxicity is a critical adverse event that leads to discontinuation of kinase inhibitor (KI) treatment. Here we show, through meta-analyses of FDA Adverse Event Reporting System, that dasatinib is associated with high risk for glomerular toxicity that is uncoupled from hypertension, suggesting a direct link between dasatinib and podocytes. We further investigate the cellular effects of dasatinib and other comparable KIs with varying risks of nephrotoxicity. Dasatinib treated podocytes show significant changes in focal adhesions, actin cytoskeleton, and morphology that are not observed with other KIs. We use phosphoproteomics and kinome profiling to identify the molecular mechanisms of dasatinib-induced injury to the actin cytoskeleton, and atomic force microscopy to quantify impairment to cellular biomechanics. Furthermore, chronic administration of dasatinib in mice causes reversible glomerular dysfunction, loss of stress fibers, and foot process effacement. We conclude that dasatinib induces nephrotoxicity through altered podocyte actin cytoskeleton, leading to injurious cellular biomechanics.

Podocytes are kidney cells with specialized morphology that is required for glomerular filtration. Diseases, such as diabetes, or drug exposure that causes disruption of the podocyte foot process morphology results in kidney pathophysiology. Proteomic analysis of glomeruli isolated from rats with puromycin-induced kidney disease and control rats indicated that protein kinase A (PKA), which is activated by adenosine 3′,5′-monophosphate (cAMP), is a key regulator of podocyte morphology and function. In podocytes, cAMP signaling activates cAMP response element–binding protein (CREB) to enhance expression of the gene encoding a differentiation marker, synaptopodin, a protein that associates with actin and promotes its bundling. We constructed and experimentally verified a β-adrenergic receptor–driven network with multiple feedback and feedforward motifs that controls CREB activity. To determine how the motifs interacted to regulate gene expression, we mapped multicompartment dynamical models, including information about protein subcellular localization, onto the network topology using Petri net formalisms. These computational analyses indicated that the juxtaposition of multiple feedback and feedforward motifs enabled the prolonged CREB activation necessary for synaptopodin expression and actin bundling. Drug-induced modulation of these motifs in diseased rats led to recovery of normal morphology and physiological function in vivo. Thus, analysis of regulatory motifs using network dynamics can provide insights into pathophysiology that enable predictions for drug intervention strategies to treat kidney disease.

The shape of a cell within tissues can represent the history of chemical and physical signals that it encounters, but can information from cell shape regulate cellular phenotype independently? Using optimal control theory to constrain reaction-diffusion schemes that are dependent on different surface-to-volume relationships, we find that information from cell shape can be resolved from mechanical signals. We used microfabricated 3-D biomimetic chips to validate predictions that shape-sensing occurs in a tension-independent manner through integrin β3 signaling pathway in human kidney podocytes and smooth muscle cells. Differential proteomics and functional ablation assays indicate that integrin β3 is critical in transduction of shape signals through ezrin–radixin–moesin (ERM) family. We used experimentally determined diffusion coefficients and experimentally validated simulations to show that shape sensing is an emergent cellular property enabled by multiple molecular characteristics of integrin β3. We conclude that 3-D cell shape information, transduced through tension-independent mechanisms, can regulate phenotype.

Creating a cDNA library for deep mRNA sequencing (mRNAseq) is generally done by random priming, creating multiple sequencing fragments along each transcript. A 3′-end-focused library approach cannot detect differential splicing, but has potentially higher throughput at a lower cost, along with the ability to improve quantification by using transcript molecule counting with unique molecular identifiers (UMI) that correct PCR bias. Here, we compare an implementation of such a 3′-digital gene expression (3′-DGE) approach with “conventional” random primed mRNAseq. Given our particular datasets on cultured human cardiomyocyte cell lines, we find that, while conventional mRNAseq detects ~15% more genes and needs ~500,000 fewer reads per sample for equivalent statistical power, the resulting differentially expressed genes, biological conclusions, and gene signatures are highly concordant between two techniques. We also find good quantitative agreement at the level of individual genes between two techniques for both read counts and fold changes between given conditions. We conclude that, for high-throughput applications, the potential cost savings associated with 3′-DGE approach are likely a reasonable tradeoff for modest reduction in sensitivity and inability to observe alternative splicing, and should enable many larger scale studies focusing on not only differential expression analysis, but also quantitative transcriptome profiling.