49 research outputs found
Development of a high-throughput small molecule screening assay for phenotypical characterization of lysosomal storage disorder-affected cells, with infantile cystinosis as a proof of principle
No full textTogether with the Pivot Park Screening Centre we performed a drug screen on CTNS-/- proximal tubule cells. For this we developed an assay to evaluate LC3-II positive puncta, and which may be applied for any disease in which autophagy plays an important role. The screen was optimized by the hotel for a 384 well format, making it useful for high throughput screening. The screen was performed with 1280 compounds from the Prestwick library.</p
CRISPRing the way to Advanced in Vitro Modelling for Genetic Kidney Disease
No full textUnderstanding the genetic basis of hereditary kidney diseases is crucial for diagnosis, management, and treatment of patients. Furthermore, this can provide valuable insights into the fundamental biological processes involved in kidney development and its functions. Therefore, this thesis aims to generate, validate, and characterize advanced in vitro models for genetic kidney disorders to provide novel insights in the disease pathophysiology and to eventually offer novel therapies for these diseases. The approach includes developing advanced, microphysiological culture systems and kidney organoid models, along with the application of gene editing technologies (CRISPR/Cas system) for gene mutation as well as restoration. Following this introduction, in chapter 2, a novel 3D microfluidic system is presented to model nephropathic cystinosis in vitro. We employed a HFM system as 3D scaffold, and we used a CRISPR-engineered (CTNS-/-) and a patient-derived (CTNSPatient) cystinotic conditionally immortalized PTEC line (ciPTECs) to generate cystinotic tubules. We aimed to offer a novel advanced model to study cystinosis at a proximal tubule level that fully recapitulates the disease phenotype, viz. renal Fanconi syndrome. In chapter 3, CRISPR technology is used to generate kidney organoids depleted for the KCNJ16 gene, a novel candidate gene recently described to be related to a tubular disease phenotype in the kidney. We focused on the characterization of described and novel phenotypes upon KCNJ16 depletion, as well as on the potential phenotype restoration using a pharmacological approach. Similarly, chapter 4 presents NPHP1-depleted kidney organoids using CRISPR technology with the aim of phenotypically characterizing and further investigating the missing link between NPHP1 loss and the characteristics and symptoms of nephronophthisis-1. In chapter 5, a novel gene editing method to restore defects in the CTNS gene in the two cystinotic ciPTEC lines mentioned earlier is described. Our novel approach includes the use of Homology-Independent Targeted Integration (HITI), a novel enhanced CRISPR version for highly efficient and precise DNA insertions, coupled with a novel non-viral peptide-mediated delivery method (LAH5), as well as several repair constructs designed to be able to repair any mutation within the first 10 exons of the CTNS gene. We aimed to introduce the CRISPR repair system into both cystinotic ciPTEC lines genome efficiently and precisely, which would lead to the downstream lysosomal cystine restoration. Additionally, we aimed to explore whether the phenotypical restoration upon gene repair extends beyond lysosomal cystine accumulation by evaluating the mitochondrial bioenergetics before and after gene repair of selected clones. In chapter 6, the results presented in this thesis are summarized and discussed in the context of the latest innovations in the field of advanced in vitro modelling for kidney diseases, providing a comprehensive overview of the findings of this thesis as well as current developments in the field. Lastly, the future directions to further develop CRISPR and kidney organoids as a platform for disease modelling and their value in future medicine are presented from a reflective perspective
Insights into protein-bound uremic toxins in proximal tubule cell senescence and kidney fibrosis
No full textKidney fibrosis leads to kidney failure by an excessive accumulation of extracellular matrix (ECM), which is the common endpoint for a variety of progressive chronic kidney diseases (CKD). In healthy kidneys, protein-bound uremic toxins (PBUTs) are cleared from the systemic circulation by proximal tubule cells through the concerted action of plasma membrane transporters that facilitate their urinary excretion, but the endogenous metabolites are hardly removed with kidney dysfunction. Accumulating evidence suggests that senescence of kidney tubule cells influences kidney fibrosis. Senescence is a special state of cells characterized by permanent cell cycle arrest and limitation of proliferation, which promotes fibrosis by releasing senescence-associated secretory phenotype (SASP) factors. This thesis demonstrated the effects related to senescence of PBUTs in vitro by using conditionally immortalized proximal tubule epithelial cell line overexpressing the organic anion transporter 1 (ciPTEC-OAT1), which gives a new angle to the role that PBUTs play in CKD. These observations thus pave the way for investigating novel strategies for CKD treatment, such as kidney-targeted delivery of senolytic drugs. Chapter 1 gives a general introduction of kidney fibrosis, senescence and PBUTs. A hypothesis is proposed that PBUTs may promote kidney fibrosis by accelerating senescence, possibly via mitochondrial dysfunction, cell cycle arrest, and the production of SASP factors. The findings described in Chapter 2 suggest that ciPTEC-OAT1 develops a senescence phenotype in a time dependent manner at 37℃, including a cell cycle arrest, resistance to apoptosis, SASP factors production and responsiveness to senolytics treatment. Therefore, ciPTEC-OAT1 represents a valid model for studying kidney senescence by simply adjusting culture conditions. Chapter 3 and Chapter 4 indicate that PBUTs could induce SASP factors release, and trigger oxidative stress possibly causing mitochondrial dysfunction, thus driving kidney senescence. The results in Chapter 3 demonstrate that PBUTs induce inflammasome-mediated IL-1β production in proximal tubule cells via oxidative stress and NF-κB signalling. Chapter 4 indicates that IS may contribute to kidney disease by accelerating senescence, through the regulation of senescence markers and by modulating inflammatory and profibrotic processes, as evidenced by changes in the TNF-α/NF-ĸB pathway and the EMT process. Together, the findings revealed that PBUTs, and especially IS, are drivers of kidney senescence, and that they do so by inducing oxidative stress, promoting inflammatory response, and via increased resistance to cell death. Chapter 5 provides insight into possible approaches for kidney-targeted senolytic therapy (Nav-Lx-LZM conjugates) for more efficient clearance of senescent cells and the reduction of systemic side effects. The results obtained suggest that ciPTEC-OAT1 appears suitable to take up the rhodamine-LZM conjugates, indicating that this is a valuable in vitro model to evaluate the efficacy of senolytic-LZM conjugates. Finally, Chapter 6 gives an overview on how senescence contributes to CKD and the involvement of PBUTs, how kidney senescence can be modelled and studied, the ways of improving current senolytic therapies, along with future perspectives
Bioengineered Cystinotic Kidney Tubules Recapitulate a Nephropathic Phenotype
No full textNephropathic 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
The Uremic Toxin Indoxyl Sulfate Accelerates Senescence in Kidney Proximal Tubule Cells
No full textKidney 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
The Uremic Toxin Indoxyl Sulfate Accelerates Senescence in Kidney Proximal Tubule Cells
No full textKidney 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
No full textChronic 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
Bioengineered Cystinotic Kidney Tubules Recapitulate a Nephropathic Phenotype
No full textNephropathic 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
Loss of Heterozygosity Is Present in SEC63 Germline Carriers with Polycystic Liver Disease
No full textContains fulltext :
108556.pdf (Publisher’s version ) (Open Access)Polycystic liver disease (PCLD) is an autosomal dominant disorder characterised by multiple fluid filled cysts in the liver. This rare disease is caused by heterozygous germline mutations in PRKCSH and SEC63. We previously found that, in patients with a PRKCSH mutation, over 76% of the cysts acquired a somatic 'second-hit' mutation in the wild type PRKCSH allele. We hypothesise that somatic second-hit mutations are a general mechanism of cyst formation in PCLD which also plays a role in PCLD patients carrying a SEC63 germline mutation. We collected cyst epithelial cells from 52 liver cysts from three different SEC63 patients using laser microdissection. DNA samples were sequenced to identify loss of heterozygosity (LOH) mutations and other somatic mutations in cyst epithelial DNA. We discovered somatic SEC63 mutations in patient 3 (1/14 cysts), but not in patient 1 and 2 (38 cysts). Upon review we found that the germline mutation of patient 1 and 2 (SEC63 c.1703_1705delAAG) was present in the same frequency in DNA samples from healthy controls, suggesting that this variant is not causative of PCLD. In conclusion, as somatic second-hit mutations also play a role in cyst formation in patients with a SEC63 germline mutation, this appears to be a general mechanism of cyst formation in PCLD
