Journal: Cell Reports Medicine

Article Title: Patient-derived kidney organoids recapitulate ADPKD and facilitate the identification of Rho pathway inhibitors as candidate therapeutics

doi: 10.1016/j.xcrm.2026.102720

Figure Lengend Snippet: Functional maturation and segment-specific differentiation in multi-lineage adult renal organoids (MAROs) (A) Schematic of the albumin uptake assay. Organoids were incubated with albumin-Cy3 for 24 h via the basolateral compartment of the transwell insert, followed by fixation and staining for analysis. (B) Schematic illustration of the experimental strategy for assessing P-glycoprotein (P-gp)-mediated transport in kidney organoids. (C) MAROs were incubated with Albumin-Cy3 for 24 h to assess receptor-mediated endocytosis. The top panel shows the control condition (Substrate only) with significant albumin-Cy3 internalization, while the bottom panel shows reduced uptake in the presence of an inhibitor (substrate + inhibitor). Scale bars, 50 μm. (D) Co-localization of albumin-Cy3 with CUBN and LRP2 in MAROs. Organoids were incubated with Albumin-Cy3 for 24 h, fixed, and stained for co-localization with CUBN/LTL and LRP2/LTL to confirm receptor-mediated endocytosis. Scale bars, 50 μm. (E) Live cell imaging of intracellular accumulation of calcein-AM in kidney organoids treated with the P-gp inhibitor PSC833 or vehicle control (0.2% DMSO), demonstrating P-gp transporter activity. (F) Whole-mount immunofluorescence analysis of calcein-AM accumulation in kidney organoids under the same conditions as (E), stained with ZO-1 (red), calcein (green), and DAPI (blue). Scale bars, 50 μm. (G) RT-qPCR analysis of AQP2 expression, a principal cell marker, in organoids cultured in media of indicated compositions. Gene expression was measured on day 10 post-induction. Data are presented as mean ± SEM. (H) RT-qPCR analysis of ATP6V1B1 , a marker of intercalated cells, in organoids cultured in different media. Expression levels were assessed on day 10 post-induction. Data are shown as mean ± SEM. (I) Relative expression levels of collecting duct (CD)-associated genes in organoids cultured in CD differentiation medium, as determined by RT-qPCR on day 10 post-induction. Data are presented as mean ± SEM. (J) Immunofluorescence staining comparing control organoids (left) and CD-induced organoids (right), showing localization of principal cell marker AQP2 and intercalated cell markers FOXI1 and PENDRIN. Scale bars, 50 μm. (K) Whole-mount co-immunofluorescence of CD-induced organoids, showing spatial co-localization of principal cells (AQP2) and intercalated cells (ATP6V1B1). Scale bars, 50 μm. Quantification data are expressed as mean ± SEM (∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001; ∗∗∗∗ p < 0.0001; and n.s., no statistics, Student’s t test).

Article Snippet: To assess receptor-mediated endocytosis in proximal tubule–like cells, organoids were transferred onto Transwell inserts and exposed to Albumin-Cy3 (MCE Cat. HY-NP027 final concentration 10 μg/mL) added to the basolateral compartment for 24 h at 37°C.

Techniques: Functional Assay, Incubation, Staining, Control, Live Cell Imaging, Activity Assay, Immunofluorescence, Quantitative RT-PCR, Expressing, Marker, Cell Culture, Gene Expression