Biology subjects

Hoenderop, J.

Publications and source records attributed to Hoenderop, J..

4 recordsLinked to original sources

bioRxiv2025

A Novel Human Distal Tubuloid-On-A-Chip Model For Investigating Sodium And Water Transport Mechanisms

The distal segments of the nephron play a central role in regulating water and electrolyte balance, making them critical targets for therapeutic interventions. Damage to these segments is associated with significant health consequences. Studying their (patho)physiology in vivo remains challenging due to the kidneys complex architecture. Recent advances led to the development of more representative in vitro models, including human tubuloids that replicate the phenotype of distal tubule segments. Additionally, novel high throughput microfluidic systems, which support 3D cell culture under flow conditions, provide a platform for closely mimicking in vivo environments. This study presents an enhanced in vitro model of human distal tubule segments by integrating tubuloid culture with the OrganoPlate(R) platform. Tubuloid cells were grown as three-dimensional tubules against a collagen-1 matrix and under alternating flow condition, then differentiated into a distal phenotype. qPCR analysis demonstrated enhanced expression of distal segment markers in 3D flow cultures compared to traditional 2D models. Immunohistochemistry confirmed the formation of a leak-tight, highly polarized epithelium with apical and basolateral localization of key electrolyte transporters. Functional integrity was verified by restricted dextran diffusion and increased transepithelial resistance. Radiolabeled sodium assays revealed active and selective sodium transport mediated by apical epithelial sodium channels (ENaC) and basolateral Na/K ATPase. Sodium transport was followed by water movement, evidenced by dome formation beneath the epithelial monolayer. The models utility was further demonstrated in toxicity studies using trimethoprim, an antibiotic that inhibits ENaC function, resulting in reduced sodium transport and dome formation. This system enables the study of primary human tubule cells with a distal phenotype under controlled flow conditions, allowing direct assessment of water and salt transport. The model provides a valuable tool for investigating distal nephron (patho)physiology and facilitates high-throughput drug development and toxicity testing.

bioengineering↗

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↗

bioRxiv2022

Insulin receptor substrate 4 deficiency mediates the insulin effect on the epithelial magnesium channel TRPM6 and causes hypomagnesemia

The kidney is the key regulator of magnesium (Mg2+) homeostasis in the human body. In the distal convoluted tubule (DCT), the apical epithelial magnesium (Mg2+) channel TRPM6, determines how much Mg2+ is excreted in the urine. To better understand the regulation of human renal Mg2+ absorption we identified novel, potential interaction partners of TRPM6 by pursuing a liquid chromatography - tandem mass spectrometry (LC-MS/MS) proteomics approach. We found insulin receptor substrate 4 (IRS4) enriched with TRPM6 tagged to glutathione S-transferase (TRPM6-GST) but not GST control. Physical interaction between IRS4 and TRPM6 was confirmed by co-immunoprecipitation. Applying microdissection of mouse tubules, we detected Irs4 mRNA expression mostly in the DCT and to a lower degree in the proximal tubule and thick ascending limb of Henle. Given the overall low abundance of Irs4 mRNA along the tubule we investigated the phenotype of Irs4 knockout mice (Irs4-/-). These Irs4-/- mice displayed significantly higher urinary and fecal Mg2+ losses and lower blood Mg2+ levels than wild-type (WT) mice. Claudin-16, claudin-19, and Hnf1b mRNA and Claudin-16 and Trpm6 protein expression was significantly higher in kidneys of 3 month old Irs4-/- mice consistent with a compensatory mechanism to conserve Mg2+. Applying whole-cell patch-clamp recording we confirmed the stimulatory role of insulin on TRPM6 channel activity and showed that IRS4 targets the two TRPM6 phosphorylation sites T1391 and S1583 to enhance TRPM6 current density. To test the effect of Mg2+ deficiency on metabolism, we performed glucose and insulin tolerance studies, which were mildly abnormal in Irs4-/- mice. SIGNIFICANCE STATEMENTMagnesium (Mg2+) is the second most abundant intracellular cation but the regulation of Mg2+ homeostasis is not well understood. The kidney is the key organ for regulating Mg2+ homeostasis. Insulin is a known stimulator of the apical epithelial Mg2+channel TRPM6. We present a novel modifier of Mg2+ absorption with insulin receptor substrate 4 (IRS4) which illuminates further, how insulin activates the TRPM6 channel and modifies Mg2+ homeostasis. Applying protein biochemistry, tubular microdissection, whole mouse physiology, and patch-clamp recording, we demonstrate that IRS4 mediates the stimulatory effect of insulin by enhancing phosphorylation of two specific TRPM6 residues. Irs4-/- mice develop increased urinary and stool Mg2+ losses, lower serum Mg2+ concentration, and display mild impairment in glucose and insulin tolerance.

physiology↗

bioRxiv2021

Functional basis for calmodulation of the TRPV5 calcium channel

Within the transient receptor potential (TRP) superfamily of ion channels, TRPV5 is a highly Ca2+-selective channel important for active reabsorption of Ca2+ in the kidney. Its channel activity is controlled by a negative feedback mechanism involving calmodulin (CaM) binding. Combining advanced microscopy techniques and biochemical assays, this study characterized the dynamic bilobal CaM regulation and binding stoichiometry. We demonstrate for the first time that functional (full-length) TRPV5 interacts with CaM in the absence of Ca2+, and this interaction is intensified at increasing Ca2+ concentrations sensed by the CaM C-lobe that achieves channel pore blocking. Channel inactivation occurs without CaM N-lobe calcification. Moreover, we reveal a 1:2 stoichiometry of TRPV5:CaM binding by implementing single molecule photobleaching counting, a technique with great potential for studying TRP channel regulation. In conclusion, our study proposes a new model for CaM- dependent regulation - calmodulation - of the Ca2+-selective TRPV5 that involves apoCaM interaction and lobe-specific actions.

cell biology↗