Journal: bioRxiv

Article Title: Dynamic Kidney Organoid Microphysiological Analysis Platform

doi: 10.1101/2024.10.27.620552

Figure Lengend Snippet: a , The framework of our MAP’s therapeutic interventions in ARPKD pathogenesis focuses on NF-κB and oxidative stress and the use of minimal-adverse-effect drugs. Parthenolide and methylene blue were employed to regulate NF-κB and reactive oxidative stress during the early-stage cystogenesis of ARPKD. b , Differential expressions in ARPKD organoids include genes related to endogenous ROS scavengers and NF-κB regulation, illustrating the pathogenetic paradigm. c , The ARPKD organoids exhibited spontaneous development of cystic compartments with a 55±4.5% prevalence, as indicated by the arrow. These compartments were predominantly lined with E-Cadherin-positive epithelial cells, with interspersed LTL-positive cells. Additionally, secondary cyst buds were frequently observed, as denoted by the white arrow in the right confocal image. d , The therapeutic effectiveness of anti-ROS and NF-κB inhibitory agents was evaluated under conditions inducing cAMP-enhanced cystogenesis with forskolin. The heatmap on the left illustrates the frequency of cyst development within 20 ARPKD organoids. Parthenolide was administered with a one-day-long application. Methylene blue was administered for eight days at various concentrations prior to two-day-long Forskolin applications. e , The time-lapse trace presents a significant expansion of cystic compartments by forskolin application, while the therapeutic pretreatment effectively prevented pathological progression (cases depicted for 0 and 100 nM methylene blue with 500nM parthenolide prior to 30 µM forskolin). The corresponding confocal characterization with DAPI (right upper and lower panels) highlighted the exacerbation of pathological changes by cAMP signaling, whereas the therapeutic pre-treatment resulted in a normal development, comparable to their healthy organoid counterparts. The green arrows, which indicate glomerular positions, demonstrate the abnormal placement of glomeruli in cyst-containing ARPKD organoids. f , RNA-Seq comparisons among isogenic healthy, untreated ARPKD, and treated ARPK organoids are visualized in the baseline MA plot of healthy organoid vs. untreated ARPKD. The gray dots represent genes identified through the analysis of differential gene expression, while the black dots denote genes with a significance level of adjusted p<0.05. In comparison between untreated and treated ARPKD organoids, therapeutically improved genes among black-dotted genes were marked with the colored symbols as outlined in the legend. Additional details can be found in ‘Results,’ and ‘Methods.’ g , The gene ontology of the differentially expressed genes (DEG) with significance reveals PKHD1-induced modified genes that are related to skeletal muscle tissue development in biological process (BP), structural constituent of muscle in molecular function (MF), proteinaceous extracellular matrix in cellular component (CC), and striated muscle contraction in reactome (Re). Gene symbols mark the most significantly differentially expressed genes in each context. The full gene set is shown in Supplementary Table 1. h , The gene ontology of the DEG between untreated and treated ARPKD organoids highlights extracellular structure organization in BP, collagen binding in MF, proteinaceous extracellular matrix in CC, and HS-GAG degradation in Re. The complete gene set is provided in Supplementary Table 2. i , The gene set associated with therapeutic improvement was analyzed in the network analysis, resulting in an exploration of PKD-associated biology with the highlighting of blue-colored genes. The criterion applied was the significance of differentially expressed genes (DEGs) between untreated and treated ARPKD, along with the opposite fold changes between control vs. untreated ARPKD and untreated vs. treated ARPKD. Full gene names are provided in Supplementary Fig. 1.

Article Snippet: The purified RNA samples were promptly stored at -80°C and subsequently sent to a commercial RNA sequencing service (Genewiz, Azenta Life Sciences).

Techniques: RNA Sequencing Assay, Expressing, Comparison, Modification, Binding Assay, Control