The Uremic Toxin Indoxyl Sulfate Accelerates Senescence in Kidney Proximal Tubule Cells

Kidney fibrosis is the common final pathway of nearly all chronic and progressive nephropathies. One cause may be the accumulation of senescent cells that secrete factors (senescence associated secretory phenotype, SASP) promoting fibrosis and inflammation. It has been suggested that uremic toxins, such as indoxyl sulfate (IS), play a role in this. Here, we investigated whether IS accelerates senescence in conditionally immortalized proximal tubule epithelial cells overexpressing the organic anion transporter 1 (ciPTEC-OAT1), thereby promoting kidney fibrosis. Cell viability results suggested that the tolerance of ciPTEC-OAT1 against IS increased in a time-dependent manner at the same dose of IS. This was accompanied by SA-β-gal staining, confirming the accumulation of senescent cells, as well as an upregulation of p21 and downregulation of laminB1 at different time points, accompanied by an upregulation in the SASP factors IL-1β, IL-6 and IL-8. RNA-sequencing and transcriptome analysis revealed that IS accelerates senescence, and that cell cycle appears to be the most relevant factor during the process. IS accelerates senescence via TNF-α and NF-ĸB signalling early on, and the epithelial-mesenchymal transition process at later time points. In conclusion, our results suggest that IS accelerates cellular senescence in proximal tubule epithelial cells

Organs-on-chip technology: a tool to tackle genetic kidney diseases

Chronic kidney disease (CKD) is a major healthcare burden that takes a toll on the quality of life of many patients. Emerging evidence indicates that a substantial proportion of these patients carry a genetic defect that contributes to their disease. Any effort to reduce the percentage of patients with a diagnosis of nephropathy heading towards kidney replacement therapies should therefore be encouraged. Besides early genetic screenings and registries, in vitro systems that mimic the complexity and pathophysiological aspects of the disease could advance the screening for targeted and personalized therapies. In this regard, the use of patient-derived cell lines, as well as the generation of disease-specific cell lines via gene editing and stem cell technologies, have significantly improved our understanding of the molecular mechanisms underlying inherited kidney diseases. Furthermore, organs-on-chip technology holds great potential as it can emulate tissue and organ functions that are not found in other, more simple, in vitro models. The personalized nature of the chips, together with physiologically relevant read-outs, provide new opportunities for patient-specific assessment, as well as personalized strategies for treatment. In this review, we summarize the major kidney-on-chip (KOC) configurations and present the most recent studies on the in vitro representation of genetic kidney diseases using KOC-driven strategies

Quantification of cystine in human renal proximal tubule cells using liquid chromatography-tandem mass spectrometry

Nephropathic cystinosis is characterized by abnormal intralysosomal accumulation of cystine throughout the body causing irreversible damage to various organs, particularly the kidneys. Cysteamine, the currently available treatment, can reduce lysosomal cystine and postpone disease progression. However, cysteamine poses serious side effects and does not address all symptoms of cystinosis. To screen for new treatment options, a rapid and reliable high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method was developed to quantify cystine in conditionally immortalized human proximal tubular epithelial cells (ciPTEC). The ciPTEC were treated with N-ethylmaleimide, lysed and deproteinized with 15% (w/v) sulfosalicylic acid. Subsequently, cystine was measured using deuterium-labeled cystine-D4, as an internal standard. The assay developed demonstrated linearity to at least 20 μmol/L with a good precision. Accuracies were between 97.3-102.9% for both cell extracts and whole cell samples. Cystine was sufficiently stable under all relevant analytical conditions. The assay was successfully applied to determine cystine levels in both healthy and cystinotic ciPTEC. Control cells showed clearly distinguishable cystine levels compared to cystinotic cells treated with or without cysteamine. The method developed provides a fast and reliable quantification of cystine, and is applicable to screen for potential drugs that could reverse cystinotic symptoms in human kidney cells

Physiological map to study kidney toxicity in the ONTOX project

editorial reviewedBackground and Objectives: Continuous improvements of computational approaches also increase the predictive performances of toxicological in silico models [1]. However, being mainly based on animal test data, these computational models lack a good correlation with human toxicity, and, being often based uniquely on chemical structures, they are unable to explain toxicological processes. To overcome these limitations, we have developed a new semi-automated strategy to build a Physiological Map (PM), a framework to study human toxicological mechanisms. Materials and Methods: Our method is useful to build a PM or to validate an existing PM. To retrieve information, a manual literature review was accompanied by computational interrogation of ontologies (e.g. Gene Ontology), thus creating a network of genes, proteins, molecules and phenotypes [2]. The network was converted manually into a PM using the CellDesigner software and visualized on the web using the MINERVA platform. The entire procedure was supported and revised by field experts. Results: We present here the human kidney PM, developed in the framework of ONTOX, a European project aimed at improving risk assessment avoiding the use of animal tests [3]. With the purpose to better understand tubular necrosis and nephrolithiasis, the PM represents the normal physiology in proximal tubule, the loop of Henle, distal tubule, and collecting duct cells, displaying the vitamin D metabolism and the urine production processes: filtration, reabsorption and secretion. Discussion and Conclusions: Our method assists the user to build a PM even starting from limited data. The PM is initially a static representation of physiological processes, also useful to study and develop new adverse outcome pathways. Subsequently, we could add kinetic parameters, transforming the PM into a dynamic model able to represent cellular perturbations. This approach presents an opportunity to investigate human toxicities, improving the toxicological predictions from a qualitative and quantitative perspective. References: [1] Manganelli S, Gamba A, Colombo E, Benfenati E (2022) 'Using VEGAHUB Within a Weight-of-Evidence Strategy'. In: Benfenati E. (eds) In Silico Methods for Predicting Drug Toxicity. Methods in Molecular Biology, vol 2425. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1960-5_18 [2] Gamba A, Salmona M, Bazzoni G (2020) 'Quantitative analysis of proteins which are members of the same protein complex but cause locus heterogeneity in disease', Sci Rep 10, 10423. https://doi.org/10.1038/s41598-020-66836-7 [3] Vinken M., et al. (2021) 'Safer chemicals using less animals: kick-off of the European ONTOX project', Toxicology 458, 152846. https://doi.org/10.1016/j.tox.2021.15284

CTNSmRNA as a potential treatment for nephropathic cystinosis

Messenger RNA (mRNA) therapies are emerging in different disease areas, but have not yet reached the kidney field. Our aim was to study the feasibility to correct the genetic defect in nephropathic cystinosis using synthetic mRNA. Cystinosis is a prototype disorder of proximal tubular dysfunction caused by mutations in the CTNS gene, encoding the lysosomal cystine-H+ symporter cystinosin, and leading to cystine accumulation in all cells of the body. The kidneys are the first and most severely affected organs, presenting glomerular and proximal tubular dysfunction. Cysteamine is the current therapeutic standard that reduces cellular cystine levels, but has many side effects and does not restore kidney function. Here, we show that synthetic mRNA is safe and effective to reintroduce functional cystinosin using lipofection in CTNS-/- kidney cells and following direct injection in ctns-/- zebrafish larvae. CTNS mRNA therapy results in prompt lysosomal expression of the functional protein and decreases cellular cystine accumulation for up to 14 days. In the ctns-/- zebrafish, CTNS mRNA therapy improves proximal tubular reabsorption, reduces proteinuria, and restores brush border expression of the multi-ligand receptor megalin. We propose that mRNA-based therapy, if sufficient kidney targeting can be achieved, may be a new approach to treat cystinosis

Co-axial Printing of Convoluted Proximal Tubule for Kidney Disease Modeling

Despite the increasing incidence of kidney-related diseases, we are still far from understanding the underlying mechanisms of these diseases and their progression. This lack of understanding is partly because of a poor replication of the diseasesin vitro,limited to planar culture. Advancing towards three-dimensional models, hereby we propose coaxial printing to obtain microfibers containing a helical hollow microchannel. These recapitulate the architecture of the proximal tubule (PT), an important nephron segment often affected in kidney disorders. A stable gelatin/alginate-based ink was formulated to allow printability while maintaining structural properties. Fine-tuning of the composition, printing temperature and extrusion rate allowed for optimal ink viscosity that led to coiling of the microfiber's inner channel. The printed microfibers exhibited prolonged structural stability (42 days) and cytocompatibility in culture. Healthy conditionally immortalized PT epithelial cells and a knockout cell model for cystinosis (CTNS-/-) were seeded to mimic two genotypes of PT. Upon culturing for 14 days, engineered PT showed homogenous cytoskeleton organization as indicated by staining for filamentous actin, barrier-formation and polarization with apical markerα-tubulin and basolateral marker Na+/K+-ATPase. Cell viability was slightly decreased upon prolonged culturing for 14 days, which was more pronounced inCTNS-/-microfibers. Finally,CTNS-/-cells showed reduced apical transport activity in the microfibers compared to healthy PT epithelial cells when looking at breast cancer resistance protein and multidrug resistance-associated protein 4. Engineered PT incorporated in a custom-designed microfluidic chip allowed to assess leak-tightness of the epithelium, which appeared less tight inCTNS-/-PT compared to healthy PT, in agreement with itsin vivophenotype. While we are still on the verge of patient-oriented medicine, this system holds great promise for further research in establishing advancedin vitrodisease models

Physiological maps and chemical-induced disease ontologies: tools to support NAMs development for next-generation risk assessment

editorial reviewedPhysiological maps (PM) can be defined as a graphical representation of cellular and molecular processes associated to specific organ functions (Vinken et al. 2021). Within the ONTOX project, we designed a total of 6 PMs describing physiological processes in the liver, the kidney and the brain. These PMs are then used as a tool to assess relevant mechanistic coverage and linkage between a specific organ function and a toxicological endpoint. Based on the Disease Maps project (Mazein et al. 2018) pipeline, we developed the first version of 6 PMs describing the following physiological processes: bile secretion & lipid metabolism (liver), vitamin D metabolism & urine composition (kidney), neural tube closure (update of the work of Heusinkveld et al. 2021) & brain development (brain). Our workflow included: (i) data collection from expert curated literature (ii) identification of the relevant biological mechanisms, (iii) screening of online databases (e.g. Wikipathways, Reactome, and KEGG) for previously described pathways, (iv) manual curation and integration of the data into a PM using CellDesigner, and (v) visualization on the MINERVA platform (Hoksza et al. 2019). These qualitative PMs represent an important tool for exploring curated literature, analyzing networks and benchmarking the development of new adverse outcome pathways (AOPs). These PMs provide the basis for developing quantitative disease ontologies, integrating different layers of pathological and toxicological information, chemical information (drug-induced pathways) and kinetic data. The resulting chemical-induced disease ontologies will provide a multi-layered platform for integration and visualization of such information. The ontologies will contribute to improving understanding of organ/disease related pathways in response to chemicals, visualize omics datasets, develop quantitative methods for computational disease modeling and for predicting toxicity, set up an in vitro & in silico test battery to detect a specific type of toxicity, and develop new animal-free approaches for next generation risk assessment

Bioengineered Cystinotic Kidney Tubules Recapitulate a Nephropathic Phenotype

Nephropathic cystinosis is a rare and severe disease caused by disruptions in the CTNS gene. Cystinosis is characterized by lysosomal cystine accumulation, vesicle trafficking impairment, oxidative stress, and apoptosis. Additionally, cystinotic patients exhibit weakening and leakage of the proximal tubular segment of the nephrons, leading to renal Fanconi syndrome and kidney failure early in life. Current in vitro cystinotic models cannot recapitulate all clinical features of the disease which limits their translational value. Therefore, the development of novel, complex in vitro models that better mimic the disease and exhibit characteristics not compatible with 2-dimensional cell culture is of crucial importance for novel therapies development. In this study, we developed a 3-dimensional bioengineered model of nephropathic cystinosis by culturing conditionally immortalized proximal tubule epithelial cells (ciPTECs) on hollow fiber membranes (HFM). Cystinotic kidney tubules showed lysosomal cystine accumulation, increased autophagy and vesicle trafficking deterioration, the impairment of several metabolic pathways, and the disruption of the epithelial monolayer tightness as compared to control kidney tubules. In particular, the loss of monolayer organization and leakage could be mimicked with the use of the cystinotic kidney tubules, which has not been possible before, using the standard 2-dimensional cell culture. Overall, bioengineered cystinotic kidney tubules recapitulate better the nephropathic phenotype at a molecular, structural, and functional proximal tubule level compared to 2-dimensional cell cultures

Bioengineered Cystinotic Kidney Tubules Recapitulate a Nephropathic Phenotype

Nephropathic cystinosis is a rare and severe disease caused by disruptions in the CTNS gene. Cystinosis is characterized by lysosomal cystine accumulation, vesicle trafficking impairment, oxidative stress, and apoptosis. Additionally, cystinotic patients exhibit weakening and leakage of the proximal tubular segment of the nephrons, leading to renal Fanconi syndrome and kidney failure early in life. Current in vitro cystinotic models cannot recapitulate all clinical features of the disease which limits their translational value. Therefore, the development of novel, complex in vitro models that better mimic the disease and exhibit characteristics not compatible with 2-dimensional cell culture is of crucial importance for novel therapies development. In this study, we developed a 3-dimensional bioengineered model of nephropathic cystinosis by culturing conditionally immortalized proximal tubule epithelial cells (ciPTECs) on hollow fiber membranes (HFM). Cystinotic kidney tubules showed lysosomal cystine accumulation, increased autophagy and vesicle trafficking deterioration, the impairment of several metabolic pathways, and the disruption of the epithelial monolayer tightness as compared to control kidney tubules. In particular, the loss of monolayer organization and leakage could be mimicked with the use of the cystinotic kidney tubules, which has not been possible before, using the standard 2-dimensional cell culture. Overall, bioengineered cystinotic kidney tubules recapitulate better the nephropathic phenotype at a molecular, structural, and functional proximal tubule level compared to 2-dimensional cell cultures