Biermans, S. | Search bioRxiv

bioRxiv2026

Scalable proxiloids enable human-relevant assessment of kidney proximal tubule toxicity

Drug-induced injury to the human proximal tubule (PT) is a leading cause of acute kidney injury and drug attrition, yet remains difficult to predict preclinically. PT toxicity arises from the coupling of transporter-mediated xenobiotic accumulation and high oxidative metabolic demand. Current models lack key aspects of PT physiology or are difficult to scale for toxicity testing. New Approach Methodologies (NAMs) address this challenge through human-relevant in vitro systems. Here we introduce proxiloids, a scalable suspension-based human induced pluripotent stem cell differentiation strategy. Within 14 days, proxiloids form lumenized, polarized tubular organoids enriched for PT identity, with functional transport and oxidative metabolic competence. Proxiloids are compatible with genetically encoded reporters and standard multiwell assays, enabling detection of defined stress responses. They recapitulate aminoglycoside nephrotoxicity with greater sensitivity than matched two-dimensional cultures and detect adefovir-induced mitochondrial toxicity not predicted in rodents. Together, proxiloids provide a scalable, human-relevant NAM for PT nephrotoxicity assessment.

cell biology↗

bioRxiv2025

Loss of nephronophthisis-associated nephrocystin-1 impairs DNA damage repair in kidney organoids

Nephronophthisis (NPH) is a heterogeneous, autosomal recessive ciliopathy and an important cause of end-stage renal disease (ESRD) in children and young adults. Since its classification as ciliopathy in 2003, NPH disease causal attribution had been focused primarily on ciliary dysfunction. The finding that ciliopathy players are involved in the DNA damage response (DDR) signaling resulted in a paradigm shift in thinking on NPH disease aetiology. Mutations in NPHP1 are the leading cause of NPH, but the underlying mechanisms that lead to the disease phenotype remain poorly understood. Here, nephrocystin-1 depleted kidney organoids were generated and characterized to address this knowledge gap. We used CRISPR/Cas9 to generate NPHP1 control (NPHP1WT) and two mutant (NPHP1ko1 and NPHP1ko2) cell lines from healthy human induced pluripotent stem cells (iPSC), which then were differentiated into kidney organoids in an air-liquid interface following an optimized protocol. Upon loss of nephrocystin-1, kidney organoids showed impaired nephron structures and loss of glomerular mesangial and distal tubular cells. Furthermore, NPHP1 depleted organoids exhibited a persistent inability to repair DNA lesions and showed increased senescence and fibrosis characteristics. Dynamic subcellular localization of nephrocystin-1 in NPHP1WT, particularly its translocation to nuclei 15 min post-UVC light exposure, suggested its direct involvement in the DDR. In conclusion, a novel NPHP1-depleted kidney organoid model was established, providing a platform to comprehensively study DNA damage, senescence and fibrosis simultaneously upon nephrocystin-1 loss. This advanced model aids in the understanding of the pathophysiology of NPH and paves the way towards identifying novel druggable targets.

molecular biology↗