A proof-of-concept assay for quantitative and optical assessment of drug-induced toxicity in renal organoids

Abstract

Kidneys are complex organs, and reproducing their function and physiology in a laboratory setting remains difficult. During drug development, potential compounds may exhibit unexpected nephrotoxic effects, which imposes a significant financial burden on pharmaceutical companies. As a result, there is an ongoing need for more accurate model systems. The use of renal organoids to simulate responses to nephrotoxic insults has the potential to bridge the gap between preclinical drug efficacy studies in cell cultures and animal models, and the stages of clinical trials in humans. Here we established an accessible fluorescent whole-mount approach for nuclear and membrane staining to first provide an overview of the organoid histology. Furthermore, we investigated the potential of renal organoids to model responses to drug toxicity. For this purpose, organoids were treated with the chemotherapeutic agent doxorubicin for 48 h. When cell viability was assessed biochemically, the organoids demonstrated a significant, dose-dependent decline in response to the treatment. Confocal microscopy revealed visible tubular disintegration and a loss of cellular boundaries at high drug concentrations. This observation was further reinforced by a dose-dependent decrease of the nuclear area in the analyzed images. In contrast to other approaches, in this study, we provide a straightforward experimental framework for drug toxicity assessment in renal organoids that may be used in early research stages to assist screen for potential adverse effects of compounds.

Figure 1

(A) H&E stain of a renal organoid. 17-day-old renal organoid; cell nuclei in dark purple, extracellular matrix, and cytoplasm in pink. The organoid resembles tubular epithelial structures similar to in vivo nephrons. Epithelial cells are demarcated by a basement membrane (light pink color) and arranged to form a lumen on their apical side. (B) Bar graphs showing fold change for expression of kidney marker genes and hiPSC marker genes (SOX2, OCT4) in organoids relative to undifferentiated IMR90 hiPSC determined with qPCR. Data are mean of 3 biological replicates (2 replicates for SOX2), with 3 technical replicates for each analyzed gene. Displayed are mean and s.e.m. (C) Orthogonal projection of two-photon excitation microscopy analysis of 13-day-old whole-mounted organoid. Displayed are frontal XY, sagittal YZ, and transversal XZ directions. For z-stacks, the sample was imaged from the dish bottom up to 140 µm in z-direction with a slice thickness of 1 µm. The renal organoid showed tubular structures recognizable by circularly arranged nuclei and membranous structures, stained by Hoechst 33342 and WGA TMR, respectively. A lumen within the tubules is evident. (D) Organoid with LTL ⁺proximal tubules. (E) Confocal images of detection of NPHS1 and NPHS2 mRNA with RNAscope in an FFPE d 14 renal organoid. Focus-like distribution of hybridization signals for NPHS1 and NPHS2. 20×magnification. (F) High-resolution detail of fluorescence signal distribution for NPHS1 and NPHS2. Strong nuclear hybridization signal for NPHS1 and fine dot-like and evenly distributed signals for NPHS2. 63 × magnification with Airyscan 2. Scale bars: 100 µm (A,C,D,E), 10 μm (F). WGA Wheat germ agglutinin, TMR Tetramethylrhodamine, LTL Lotus tetragonolobus lectin, FFPE Formalin-fixed paraffin-embedded.

Figure 2

(A) Schematic illustration of the course DOX toxicity assay. HiPSCs were seeded in Stage-I medium at day 0. EBs formed, differentiated, and showed renal tubule structures as kidney organoids. By day 11, organoids were singled into a 96-well plate and treated with DOX (0.08–5 µg/mL) and medium controls for 48 h. On day 13, organoids underwent either a histological (a) or a biochemical (b) analysis. (a) DOX-treated organoids and medium control whole-mounted organoids were fluorescently stained as described and morphology was assessed with confocal imaging. (b) Cell viability of DOX-treated organoids was assessed with a luciferase-ATP assay. (B) Confocal imaging of DOX-treated renal organoids after 48 h. Whole-mount staining of organoids with Hoechst 33342 (blue, cell nuclei) and WGA 633 (red, cell membrane) enabled visualization of DOX-induced toxicity in organoids. Structural analysis showed visible tubular damage and loss of membrane integrity at higher drug concentrations (1.25–5 µg/mL) compared to untreated controls, which is less observable for lower concentrations. Scale bar: 100 µm. (C) Organoid cell viability of DOX-treated kidney organoids and medium control in % (relative to untreated medium control). Renal organoids were treated with DOX concentrations ranging from 5 to 0.08 µg/mL for 48 h. Cell viability as an inverted measure of DOX toxicity was determined with an ATP-luciferase assay. Organoids were lysed, and ATP concentrations were measured luminometrically. High ATP levels equal high metabolic activity of cells, hence high viability. Treated organoids showed significantly lower cell viability compared to the medium control. The viability decreased with increasing DOX concentrations, with the lowest viability (5%) corresponding to the highest DOX concentration (5 µg/mL). Data is plotted as boxplot with whiskers and data points depicted as dots using the ggplot2 v3.4.1 package in R v3.6.3. p-values were calculated by one-way ANOVA (p < 2*10⁻¹⁶) and post-hoc Tukey multiple comparison of means, *** represents statistical significance versus medium control with p < 1*10⁻⁷, n = 4 independent experiments. (D) Measurement of nuclear area of DOX-treated organoids. p-values were calculated by one-way ANOVA (p < 2*10⁻¹⁶) and post-hoc Tukey multiple comparison of means; *** represents statistical significance versus medium control with p < 1*10⁻⁷, n = 4 independent experiments. DOX Doxorubicin, HiPSCs Human induced pluripotent stem cells, EBs Embryoid bodies, ATP Adenosine triphosphate, Ctrl Medium control, WGA Wheat germ agglutinin.