Biology subjects

Rabelink, T. J.

Publications and source records attributed to Rabelink, T. J..

7 recordsLinked to original sources

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↗

bioRxiv2026

Partial decellularization of kidneys during prolonged acellular perfusion

Long-term ex situ machine perfusion of donor organs is an emerging clinical strategy that creates a window for advanced therapeutic interventions. Replacing donor endothelial cells during machine perfusion with recipient endothelium could conceal the allogeneic epithelium from the recipients (humoral) immune response. We postulated that brief exposure to low concentrations of decellularizing agents could selectively remove vascular endothelium. Porcine kidneys were partially decellularized with five 2-minute infusions of either 0.01%, 0.1% or 1.0% SDS during acellular perfusion. As proof-of-concept, fluorescently-labelled porcine endothelial colony forming cells (ECFCs) were infused into the renal vein and artery of a partially decellularized kidney. Tissue analysis identified 0.1% SDS effectively removed endothelial cells from glomerular and peritubular capillaries. Infused ECFCs could be found back within the glomeruli. However, partial decellularization significantly impaired renal flow and increased vascular resistance. While partial decellularization successfully removed donor endothelium, the loss of vascular patency limits its clinical potential. Future research should prioritize modifying rather than removing donor endothelial cells.

bioengineering↗

bioRxiv2025

Spatial metabolomics reveals persistent localized niche-specific metabolic failure in kidneys following ischemia-reperfusion injury

After acute kidney injury (AKI), the persistence of failed repair proximal tubule (FR-PT) cells is postulated to hamper kidney regeneration and increase the risk of chronic kidney disease. This fibrotic shift likely depends on microenvironmental interactions, which remain largely unstudied. To investigate this, we mapped the spatial metabolic architecture of post-ischemic kidneys using an untargeted semi-quantitative spatial metabolomics (qMSI) approach, integrated with high-resolution spatial transcriptomics. Unsupervised neighborhood clustering of qMSI data revealed distinct microenvironments. Lipidome profiles identified diffusely spread areas with persistent injury markers surrounding FR-PT cells. These niches exhibited decreased linoleic acid and elevated succinic acid levels, even in epithelial cells that appeared otherwise healthy. Corresponding transcriptomic profiles confirmed downregulation of oxidative phosphorylation and fatty acid {beta}-oxidation in these regions. Together, these findings point towards niche-specific metabolic failure and persistent mitochondrial dysfunction in areas considered healthy, underscoring the need to prioritize metabolic resuscitation to prevent long-term consequences of AKI.

biochemistry↗

bioRxiv2025

Normothermic human kidney preservation drives iron accumulation and ferroptosis

Ex vivo normothermic machine perfusion has been proposed to protect deceased donor organs, promoting metabolic recovery and allowing quality assessment. However, its benefits for preserving deceased donor kidneys remain ambiguous. We postulate that the use of red blood cells (RBCs) as oxygen carriers and associated secondary hemolysis may in fact cause renal injury, offsetting potential advantages. During 48-hour normothermic perfusion of seven human deceased donor kidneys, we observed progressive hemolysis, leading to iron accumulation in perfusate, tissue, and urine. Untargeted lipidomic analysis revealed profound increases in oxidized phospholipid species in perfused kidneys, pointing towards iron-dependent cell death known as ferroptosis. Next, in twelve additional human kidney perfusions, we demonstrate that either dialysis-based free hemoglobin removal or cell-free perfusion attenuates hemolysis-driven iron accumulation, phospholipid peroxidation, and acute kidney injury. Our findings highlight the pathological role of hemolysis and iron on the kidney, urging restraint in the clinical application of RBC-based kidney perfusion.

physiology↗

bioRxiv2024

Spatial quantitative metabolomics enables identification of remote and sustained ipsilateral cortical metabolic reprogramming after stroke

Mass spectrometry imaging (MSI) has become a cornerstone of spatial biology research. However, various factors that are intrinsic to the technology limit quantitative capacity of MSI-based spatial metabolomics and thus reliable interpretation. Here, we developed a quantitative MSI workflow (Q-MSI), based on isotopically 13C-labeled yeast extract as internal standards, to overcome these pitfalls. Using brain and kidney tissue, we demonstrate that this approach allows for (absolute) quantification of hundreds of metabolites and lipids. Applying our workflow to a stroke model allowed us to not only map metabolic remodeling of the infarct and peri-infarct area over time, but also discover hitherto unnoted remote metabolic remodeling in the histologically unaffected ipsilateral sensorimotor cortex. At day 7 post-stroke, increased levels of neuroprotective lysine and reduced excitatory glutamate levels were found compared to the contralateral cortex. By day 28 post-stroke, lysine and glutamate levels had been normalized, while decreased precursor pools of UDP-GlcNAc and linoleate persisted that have previously been associated with vulnerability. Importantly, traditional normalization strategies not employing internal standards were unable to visualize these differences. Using 13C-labeled yeast extracts as a normalization strategy establishes a new paradigm in quantitative MSI-based spatial metabolomics that greatly enhances reliability and interpretive strength.

biochemistry↗

bioRxiv2023

Advancing multi-day ex vivo kidney perfusion using spatially resolved metabolomics

The ability to preserve metabolically active kidneys ex vivo for multiple days may permit reconditioning, repair and regeneration of deceased donor kidneys. However, the kidneys high metabolic demand limits its functional preservation. Current approaches focus on normothermic machine perfusion (NMP) at 37{degrees}C or hypothermic machine perfusion (HMP) at 4-8{degrees}C. At normothermia, kidneys are metabolically active but ex vivo preservation is limited to hours. During hypothermia kidneys can be preserved up to 24 hours but are metabolically inactive and suffer cold-induced injury. Therefore, we revisited sub normothermic perfusion (at 25{degrees}C) as an alternative approach to preserve human kidneys in a metabolically active state for extended periods of time. In a custom-made platform that includes a cell-free perfusate enriched with TCA cycle fuels, urine recirculation, and continuous hemofiltration we perfused discarded human kidneys up to 8 days. Using spatially resolved single cell resolution isotope tracing we demonstrate active metabolism in all the different renal cell types over this period. However, beyond 4 days cell composition of nephron segments assessed with spatial lipidomics changed substantially and injury markers such as NGAL and LDH increased in the perfusate. Up to 4 days, perfused human discarded donor kidneys maintained metabolic fluxes, functional parameters and allow for reperfusion using a porcine auto transplantation model. These data underpin that extended multi-day metabolic preservation of human kidneys is achievable using a sub normothermic perfusion platform.

bioengineering↗

bioRxiv2020

Thiol-ene cross-linked alginate hydrogel encapsulation modulates the extracellular matrix of kidney organoids by reducing abnormal type 1a1 collagen deposition

Differentiated kidney organoids from induced pluripotent stem cells hold promise as a treatment for patients with kidney diseases. Before these organoids can be translated to the clinic, shortcomings regarding their cellular, extracellular compositions and developmental plateau needs to be overcome. We performed a proteomic analysis on kidney organoids cultured for a prolonged culture time and we found a specific change in the extracellular matrix composition with increased expression of types 1a1, 2 and 6a1 collagen. Such an excessive accumulation of specific collagen types is a hallmark of renal fibrosis that causes a life-threatening pathological condition by compromising key functions of the human kidney. Here we hypothesized the need for a three-dimensional environment to grow the kidney organoids, which could better mimic the in vivo surroundings of the developing kidney than standard culture on a transwell filter. Encapsulating organoids for four days in a soft, thiol-ene cross-linked alginate hydrogel resulted in decreased type 1a1 collagen expression. Furthermore, the encapsulation did not result in any changes of organoid structural morphology. Using a biomaterial to modulate collagen expression allows for a prolonged kidney organoid culture in vitro and a reduction of abnormal type 1a1 collagen expression bringing kidney organoids closer to clinical application. HIGHLIGHTSO_LIProlonging kidney organoid culture results in a developmental plateau instead of improved in vitro maturation. C_LIO_LIProteomic analyses point to an increased expression of specific collagen subtypes associated with renal fibrosis. C_LIO_LIEncapsulating kidney organoids using a soft thiol-ene cross-linked alginate hydrogel reduces collagen type 1a1 and SMA deposition. C_LI

bioengineering↗