Biology subjects

van Genderen, A. M.

Publications and source records attributed to van Genderen, A. M..

5 recordsLinked to original sources

bioRxiv2026

Gentamicin impairs nephron development in iPSC-derived kidney organoids and disrupts proximal tubular lysosomal function

Gentamicin is an aminoglycoside antibiotic used to treat Gram-negative bacterial infections. However, its clinical use is limited by nephrotoxicity, affecting approximately 10 - 20% of treated patients. Nevertheless, gentamicin remains widely used due to its high potency, low cost, and low incidence of bacterial resistance and hypersensitivity. Importantly, gentamicin crosses the placental barrier and is administered to neonates, including preterm infants in whom nephrogenesis is still ongoing, raising concerns about its effects on kidney development. Current knowledge of this risk relies largely on rodent studies, which fail to fully recapitulate human kidney physiology. Here, we developed human-relevant in vitro models to investigate the impact of gentamicin on kidney development and to unravel the dynamic cellular responses underlying gentamicin-induced impairment. Kidney organoids differentiated from induced pluripotent stem cells (iPSC) and magnetically sorted proximal tubule cells were used to model immature kidney and immature proximal cells, respectively. Organoids were continuously exposed to gentamicin (0.25 - 1.5 mM) throughout development (d7+18), while megalin-expressing proximal tubule cells were exposed for 24 hours to explore the cellular mechanisms underlying gentamicin toxicity. Intracellular concentrations of the gentamicin congeners C1, C1a, and C2/C2a/C2b were quantified by LC-MS/MS. Gentamicin exposure impaired organoid growth, and nephron formation and maturation. Additionally, gentamicin induced lipid accumulation and reduced lysosomal area in proximal tubule cells, indicating disrupted cellular metabolism and homeostasis. In conclusion, iPSC-derived kidney organoids provide a suitable platform to further investigate gentamicins impact on kidney development and studying co-treatments to mitigate its adverse effects.

pharmacology and toxicology↗

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↗

bioRxiv2023

A novel KCNJ16 kidney organoid model recapitulates the disease phenotype and shows restoration of lipid accumulation upon treatment with statins

Backgroundthe KCNJ16 gene has been associated with a novel kidney tubulopathy phenotype, viz. disturbed acid-base homeostasis, hypokalemia and altered renal salt transport. KCNJ16 encodes for Kir5.1, which together with Kir4.1 constitutes a potassium channel located at kidney tubular cell basolateral membranes. Preclinical studies provided mechanistical links between Kir5.1 and a disease phenotype, however, the disease pathology remains poorly understood. Here, we aimed at generating and characterizing a novel advanced in vitro human kidney model that recapitulates the disease phenotype to investigate further the pathophysiological mechanisms underlying the disease and potential therapeutic interventions. Methodswe used CRISPR/Cas9 to generate KCNJ16 mutant (KCNJ16+/- and KCNJ16-/-) cell lines from healthy human induced pluripotent stem cells (iPSC) KCNJ16 control (KCNJ16WT). The iPSCs were differentiated following an optimized protocol into kidney organoids in an air-liquid interface. ResultsKCNJ16-depleted kidney organoids showed transcriptomic and potential functional impairment of key voltage-dependent electrolyte and water-balance transporters. We observed cysts formation, lipid droplet accumulation and fibrosis upon Kir5.1 function loss. Furthermore, a large scale, glutamine tracer flux metabolomics analysis demonstrated that KCNJ16-/- organoids display TCA cycle and lipid metabolism impairments. Drug screening revealed that treatment with statins, particularly the combination of simvastatin and C75, prevented lipid droplet accumulation and collagen-I deposition in KCNJ16-/- kidney organoids. Conclusionsmature kidney organoids represent a relevant in vitro model for investigating the function of Kir5.1. We discovered novel molecular targets for this genetic tubulopathy and identified statins as a potential therapeutic strategy for KCNJ16 defects in the kidney. Significance StatementIn this study, the use of CRISPR/Cas9 technology resulted in the establishment of a KCNJ16-depleted kidney organoid model, instrumental in elucidating the pathophysiology of the recently reported KCNJ16-associated kidney tubulopathy. Our study substantiates the role of Kir5.1 (KCNJ16) in kidney disease, confirming already described phenotypes, as well as aiding to gain insight in the causal role of Kir5.1 loss in the disease phenotype. Our approach increases the knowledge on KCNJ16-related kidney phenotype, and it states the importance of combining CRISPR/Cas9 technology and advanced in vitro models for complex disease modeling and therapy testing. Furthermore, we encourage the application of our approach to the in vitro modeling of rare and/or underrepresented genetic kidney diseases, for which the availability of patient material is limited.

cell biology↗

bioRxiv2023

Microstructured silk-fiber scaffolds with enhanced stretchability

Despite extensive research, current methods for creating three-dimensional (3D) silk fibroin (SF) scaffolds lack control over molecular rearrangement, particularly in the formation of {beta}-sheet nanocrystals, as well as hierarchical fiber organization at both micro- and macroscale. In this study, we introduce a fabrication process based on electrowriting of aqueous SF-based solutions followed by post-processing using an aqueous solution of sodium dihydrogen phosphate (NaH2PO4). This approach enables hierarchical assembly of SF chains via {beta}-sheet and -helix formation. Moreover, this process allows for precise control over micro- and macro-architectures in microfiber scaffolds, enabling the creation of 3D flat and tubular macrogeometries with square-based and crosshatch microarchitectures, featuring inter-fiber distances of 400 {micro}m and approximately 97% open porosity. Remarkably, the printed structures demonstrated restored {beta}-sheet and -helix structures, which imparted an elastic response of up to 20% deformation and the ability to support cyclic loading without plastic deformation. Furthermore, the printed constructs supported in vitro adherence and growth of human conditionally immortalized proximal tubular epithelial cells and glomerular endothelial cells, with cell viability above 95%. These cells formed uniform, aligned monolayers that deposited their own extracellular matrix. These findings represent a significant development in fabricating organized SF scaffolds with unique fiber structures, mechanical and biological properties, making them highly promising for regenerative medicine applications.

bioengineering↗

bioRxiv2020

Topographic Guidance in Melt-Electrowritten Tubular Scaffolds Enhances Engineered Kidney Tubule Performance

To advance the engineering of kidney tubules for future implantation, constructs should be both self-supportive and yet small-sized and highly porous. Here, we hypothesize that the fabrication of small-sized porous tubular scaffolds with a highly organized fibrous microstructure by means of melt-electrowriting (MEW) allows the development of self-supported kidney proximal tubules with enhanced properties. A custom-built MEW device was used to fabricate tubular fibrous scaffolds with small diameter sizes (O = 0.5, 1, 3 mm) and well-defined, porous microarchitectures (rhombus, square, and random). Human umbilical vein endothelial cells (HUVEC) and human conditionally immortalized proximal tubular epithelial cells (ciPTEC) were seeded into the scaffolds and tested for monolayer formation, integrity, and organization, as well as for extracellular matrix (ECM) production and renal transport functionality. Tubular scaffolds were successfully manufactured by fine control of MEW instrument parameters. A minimum inner diameter of 0.5 mm and pore sizes of 0.2 mm were achieved. CiPTEC formed tight monolayers in all scaffold microarchitectures tested, but well-defined rhombus-shaped pores outperformed and facilitated unidirectional cell orientation, increased collagen type IV deposition, and expression of the renal transporters and differentiation markers organic cation transporter 2 (OCT2) and P-glycoprotein (P-gp). To conclude, we present smaller diameter engineered kidney tubules with microgeometry-directed cell functionality. Due to the well-organized tubular fiber scaffold microstructure, the tubes are mechanically self-supported, and the self-produced ECM constitutes the only barrier between the inner and outer compartment, facilitating rapid and active solute transport.

bioengineering↗