Patient-Derived ADPKD Organoids Identify Rho-Pathway Inhibitors That Reduce Cyst Growth

Adult Kidney Tissue Supplied Both Expansion and Disease Models

A Cell Reports Medicine study led by Qilu Hospital and Shandong University developed expandable adult kidney organoids and used patient-derived cystic models to test candidate treatments for autosomal dominant polycystic kidney disease, or ADPKD. The experiments connect cyst morphology with ciliary and polarity signaling, then ask whether pharmacological inhibition can change cyst growth in PKD1- and PKD2-mutant backgrounds.

The normal-tissue platform, termed multi-lineage adult renal organoids, or MAROs, was established from 69 of 76 kidney tissue samples. Control tissue came from non-tumor cortex distant from renal tumors; ADPKD organoids came from cyst walls obtained during clinically indicated procedures. Adding retinoic acid, GDNF and FGF20 to the culture formulation supported expansion for more than five months and 22 passages. Patient-derived PKD cultures had a shorter useful range and gradually lost cyst-forming capacity and viability with passage.

Single-cell analysis of 32,350 cells from three independent normal-tissue organoids identified proximal, distal and collecting-duct-associated epithelial populations. Albumin uptake, size-selective dextran internalization and increased calcein accumulation after P-glycoprotein inhibition supported transport-related functions. Collecting-duct induction increased principal- and intercalated-cell markers. Podocyte, endothelial and immune-cell signatures were absent, defining the system as a tubular epithelial model.

Cilia and Polarity Signaling Connected the Phenotype to a Drug Target

Patient-derived ADPKD organoids developed expanding lumens, elongated primary cilia and increased expression of cyst-associated chloride channels and planar cell polarity components. Single-cell profiles from PKD1 and PKD2 models showed a shift toward collecting-duct-related populations. Shared Rho GTPase signaling changes coexisted with genotype-specific patterns, including stronger metabolic and CFTR-associated signatures in PKD1 models and VEGF/Hippo-associated signatures in PKD2 models.

The researchers disrupted IFT88, a protein required for normal cilia formation. Ciliary loss reduced Rho and polarity-pathway components in PKD1 and PKD2 organoids, while corresponding effects in control organoids were limited. VANGL2 localization also changed toward the control pattern. These perturbations support a connection between cilia, the polycystin-deficient background and Rho-dependent polarity regulation; they do not establish every step of a single downstream mechanism.

Mechanical conditions mattered too. Seven days of microfluidic perfusion at 100 μL/min accelerated cyst expansion relative to static cultures and increased Rho/polarity and cilia-associated transcripts. The platform therefore exposed a dependence on culture flow that a static screening assay alone would miss.

The Screen Separated Shared Hits from Genotype-Dependent Responses

The primary screen tested 160 compounds at 10 μM in two organoid lines, one carrying a PKD1 mutation and one a PKD2 mutation. Three thousand cells were seeded per well, and 10 μM forskolin was used for seven days to promote cyst formation before drug treatment. Imaging identified 23 candidates that reduced cyst size in at least one line. Secondary testing used 0.1, 1 and 10 μM across three additional patient-derived lines, with seven-day treatment and at least five organoids analyzed per condition.

Tolvaptan and GSK2193874 were more effective in the PKD1 backgrounds, while AICAR and verteporfin performed better in PKD2 backgrounds. In contrast, the CDC42 inhibitor ML141, RAC inhibitor EHT1864 and RHOA-pathway inhibitor CCG-1423 showed activity across both genotypes. The study used a broader 0.01–36.23 μM concentration range for selected dose-response measurements. These were cyst-growth comparisons within the assay, rather than patient-level comparisons of treatment benefit.

ML141 at 30 μM caused rapid cyst shrinkage during a ten-hour exposure. After withdrawal, the response was reversible, while control organoids remained morphologically stable. Forty-eight-hour treatment with the tested Rho inhibitors did not significantly alter the fraction labeled by the DNA-synthesis marker EdU. Together, these observations favor an effect on cyst architecture under the tested conditions over simple elimination of proliferating cells.

Short-Term Cyst Responses Have a Defined Biological Context

RNA and protein measurements after ML141 treatment supported attenuation of CDC42-associated cytoskeletal and polarity programs. The reversible phenotype and preserved short-term proliferation are useful for choosing subsequent mechanistic experiments. They also distinguish the observation from irreversible cyst destruction.

The model retains endogenous patient mutations and adult tubular features, but extended passage changes its regenerative behavior. Definitive tracing of the cyst-forming lineage was not performed, and vascular, immune and systemic metabolic interactions are absent. Those features matter when moving from a short cyst-growth assay to sustained treatment: the present screen identifies compounds and genotype-dependent responses for further testing, while chronic tissue responses and whole-body exposure remain separate experimental questions.