Biology subjects

Janssen, M. J.

Publications and source records attributed to Janssen, M. J..

5 recordsLinked to original sources

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

Gene surgery as a potential treatment option for Nephropathic Cystinosis in vitro

Nephropathic cystinosis is a rare monogenetic kidney disease caused by mutations in the lysosomal transporter cystinosin (encoded by CTNS) that, to date, has no cure. The hallmark of this disease is lysosomal accumulation of cystine and decline in proximal tubular function leading to kidney failure early in life. In this project, we developed a novel gene repair strategy using CRISPR/Cas9 Homology-Independent Targeted Integration (HITI) to restore CTNS. A novel, non-viral peptide-mediated approach was used to deliver the Cas9-guideRNA ribonucleoprotein (RNP) complex and repair templates to conditionally immortalized proximal tubule epithelial cell (ciPTEC) lines. The repair constructs contained either mCherry (1.7 kb), the CTNS Superexon (1.7 Kb) or both (3.2 Kb). The results demonstrated that the smaller mCherry construct achieved a higher repair efficiency (63%) compared to the CTNS-mCherry construct (16%). Clonal expansion of repaired cells showed restoration of lysosomal cystine levels in 70-75% of the clones, which was accompanied by improved mitochondrial bioenergetics. In conclusion, CRISPR/Cas9 HITI can be used to precisely insert repair templates into the genome, resulting in a functional cystinosin restoration, and a reversal of the cystinotic disease phenotype.

bioengineering↗

bioRxiv2020

The lysosomal V-ATPase B1 subunit in proximal tubule is linked to nephropathic cystinosis

Variants in ATP6V1B1, the gene encoding the B1 subunit of the vacuolar H+-ATPase lead to distal renal tubular acidosis and hearing loss of variable degree. Apart from metabolic acidosis, a 23-month-old girl with pathogenic ATP6V1B1 variants presented with increased granulocyte cystine levels, and symptoms of renal Fanconi syndrome, indicating an unknown link to proximal tubulopathy and cystinosis, a disorder of the proximal tubule caused by variants in the CTNS gene. Here, we demonstrated that ATP6V1B1 is expressed in proximal tubules of human kidney tissue, though to a lesser extent than in distal tubules. Further, we used CRISPR/Cas9 technology to selectively knockout ATP6V1B1 or CTNS in human renal proximal tubule cells and performed a full metabolomic and proteomic analysis to compare their phenotype to isogenic control cells. In line with the clinical data, the loss of ATP6V1B1 disrupted the chemiosmotic coupling between the lysosomal V ATPase B1 subunit and cystinosin and resulted in intralysosomal accumulation of cystine and autophagy activation in renal proximal tubule cells. In conclusion, we identified ATP6V1B1 as a central player in renal proximal tubule cells, regulating lysosomal cystine transport and autophagy, and its absence can lead to proximal tubule dysfunction.

pharmacology and toxicology↗

bioRxiv2020

Cysteamine-bicalutamide combination treatment restores alpha-ketoglutarate and corrects proximal tubule phenotype in cystinosis

Nephropathic cystinosis is a severe monogenetic kidney disorder caused by mutations in CTNS, encoding the lysosomal transporter cystinosin, resulting in lysosomal cystine accumulation. The sole treatment, cysteamine, slows down the disease progression, but does not correct the established proximal tubulopathy. Here, we developed a new therapeutic strategy by applying an omics-based strategy to expand our knowledge on the complexity of the disease and prioritize drug targets in cystinosis. We identified alpha-ketoglutarate as a key metabolite linking cystinosin loss, lysosomal autophagy defect and proximal tubular impairment in cystinosis. This insight offered a bicalutamide-cysteamine combination treatment as a novel dual target pharmacological approach for the phenotypical correction of cystinotic proximal tubule cells, patient-derived kidney tubuloids and cystinotic zebrafish.

pharmacology and toxicology↗