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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.

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.

Nephropathic cystinosis is a rare disease caused by mutations of the CTNS gene that encodes for cystinosin, a lysosomal cystine/H+ symporter. The disease is characterized by early-onset chronic kidney failure and progressive development of extra-renal complications related to cystine accumulation in all tissues. At the cellular level, several alterations have been demonstrated, including enhanced apoptosis, altered autophagy, defective intracellular trafficking, and cell oxidation, among others. Current therapy with cysteamine only partially reverts some of these changes, highlighting the need to develop additional treatments. Among compounds that were identified in a previous drug-repositioning study, disulfiram (DSF) was selected for in vivo studies. The cystine depleting and anti-apoptotic properties of DSF were confirmed by secondary in vitro assays and after treating Ctns-/- mice with 200 mg/kg/day of DSF for 3 months. However, at this dosage, growth impairment was observed. Long-term treatment with a lower dose (100 mg/kg/day) did not inhibit growth, but failed to reduce cystine accumulation, caused premature death, and did not prevent the development of renal lesions. In addition, DSF also caused adverse effects in cystinotic zebrafish larvae. DSF toxicity was significantly more pronounced in Ctns-/- mice and zebrafish compared to wild-type animals, suggesting higher cell toxicity of DSF in cystinotic cells. ispartof: CELLS vol:10 issue:12 ispartof: location:Switzerland status: published

Contains fulltext : 208411.pdf (Publisher’s version ) (Open Access) End-stage kidney disease represents irreversible kidney failure. Dialysis and transplantation, two main treatment options currently available, present various drawbacks and complications. Innovative cell-based therapies, such as a bioartificial kidney, have not reached the clinic yet, mostly due to safety and/or functional issues. Here, we assessed the safety of conditionally immortalized proximal tubule epithelial cells (ciPTECs) for bioartificial kidney application, by using in vitro assays and athymic nude rats. We demonstrate that these cells do not possess key properties of oncogenically transformed cells, including anchorage-independent growth, lack of contact inhibition and apoptosis-resistance. In late-passage cells we did observe complex chromosomal abnormalities favoring near-tetraploidy, indicating chromosomal instability. However, time-lapse imaging of ciPTEC-OAT1, confined to a 3D extracellular matrix (ECM)-based environment, revealed that the cells were largely non-invasive. Furthermore, we determined the viral integration sites of SV40 Large T antigen (SV40T), human telomerase (hTERT) and OAT1 (SLC22A6), the transgenes used for immortalization and cell function enhancement. All integrations sites were found to be located in the intronic regions of endogenous genes. Among these genes, early endosome antigen 1 (EEA1) involved in endocytosis, and BCL2 Like 1 (BCL2L1) known for its role in regulating apoptosis, were identified. Nevertheless, both gene products appeared to be functionally intact. Finally, after subcutaneous injection in athymic nude rats we show that ciPTEC-OAT1 lack tumorigenic and oncogenic effects in vivo, confirming the in vitro findings. Taken together, this study lays an important foundation towards bioartificial kidney (BAK) development by confirming the safety of the cell line intended for incorporation.

In this review we provide an overview of the expanding molecular toolbox that is available for gene based therapies and how these therapies can be used for a large variety of kidney diseases. Gene based therapies range from restoring gene function in genetic kidney diseases to steering complex molecular pathways in chronic kidney disorders, and can provide a treatment or cure for diseases that otherwise may not be targeted. This approach involves the delivery of recombinant DNA sequences harboring therapeutic genes to improve cell function and thereby promote kidney regeneration. Depending on the therapy, the recombinant DNA will express a gene that directly plays a role in the function of the cell (gene addition), that regulates the expression of an endogenous gene (gene regulation), or that even changes the DNA sequence of endogenous genes (gene editing). Some interventions involve permanent changes in the genome whereas others are only temporary and leave no trace. Efficient and safe delivery are important steps for all gene based therapies and also depend on the mode of action of the therapeutic gene. Here we provide examples on how the different methods can be used to treat various diseases, which technologies are now emerging (such as gene repair through CRISPR/Cas9) and what the opportunities, perspectives, potential and the limitations of these therapies are for the treatment of kidney diseases.

Contains fulltext : 172570.pdf (Publisher’s version ) (Closed access) Apical transport is key in renal function, and the activity of efflux transporters and receptor-mediated endocytosis is pivotal in this process. The conditionally immortalized proximal tubule epithelial cell line (ciPTEC) endogenously expresses these systems. Here, we used ciPTEC to investigate the activity of three major efflux transporters, viz., breast cancer resistance protein (BCRP), multidrug resistance protein 4 (MRP4), and P-glycoprotein (P-gp), as well as protein uptake through receptor-mediated endocytosis, using a fluorescence-based setup for transport assays. To this end, cells were exposed to Hoechst33342, chloromethylfluorescein-diacetate (CMFDA), and calcein-AM in the presence or absence of model inhibitors for BCRP (KO143), P-gp (PSC833), or MRPs (MK571). Overexpression cell lines MDCKII-BCRP and MDCKII-P-gp were used as positive controls, and membrane vesicles overexpressing one transporter were used to determine substrate and inhibitor specificities. Receptor-mediated endocytosis was investigated by determining the intracellular accumulation of fluorescently labeled receptor-associated protein (RAP-GST). In ciPTEC, BCRP and P-gp showed similar expressions and activities, whereas MRP4 was more abundantly expressed. Hoechst33342, GS-MF, and calcein are retained in the presence of KO143, MK571, and PSC833, showing clearly redundancy between the transporters. Noteworthy is the fact that both KO143 and MK571 can block BCRP, P-gp, and MRPs, whereas PSC833 appears to be a potent inhibitor for BCRP and P-gp but not the MRPs. Furthermore, ciPTEC accumulates RAP-GST in intracellular vesicles in a dose- and time-dependent manner, which was reduced in megalin-deficient cells. In conclusion, fluorescent-probe-based assays are fast and reproducible in determining apical transport mechanisms, in vitro. We demonstrate that typical substrates and inhibitors are not specific for the designated transporters, reflecting the complex interactions that can take place in vivo. The set of tools we describe are also compatible with innovative kidney culture models and allows studying transport mechanisms that are central to drug absorption, disposition, and detoxification.

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 of the 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 the internal standard. The assay developed demonstrated linearity to at least 20 μmol/L with a good precision. Accuracies were between 97.3 and 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 with 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.

Contains fulltext : 182172.pdf (Publisher’s version ) (Open Access) As current kidney replacement therapies are not efficient enough for end-stage renal disease (ESRD) treatment, a bioartificial kidney (BAK) device, based on conditionally immortalized human proximal tubule epithelial cells (ciPTEC), could represent an attractive solution. The active transport activity of such a system was recently demonstrated. In addition, endocrine functions of the cells, such as vitamin D activation, are relevant. The organic anion transporter 1 (OAT-1) overexpressing ciPTEC line presented 1alpha-hydroxylase (CYP27B1), 24-hydroxylase (CYP24A1) and vitamin D receptor (VDR), responsible for vitamin D activation, degradation and function, respectively. The ability to produce and secrete 1alpha,25-dihydroxy-vitamin D(3), was shown after incubation with the precursor, 25-hydroxy-vitamin D(3). The beneficial effect of vitamin D on cell function and behavior in uremic conditions was studied in the presence of an anionic uremic toxins mixture. Vitamin D could restore cell viability, and inflammatory and oxidative status, as shown by cell metabolic activity, interleukin-6 (IL-6) levels and reactive oxygen species (ROS) production, respectively. Finally, vitamin D restored transepithelial barrier function, as evidenced by decreased inulin-FITC leakage in biofunctionalized hollow fiber membranes (HFM) carrying ciPTEC-OAT1. In conclusion, the protective effects of vitamin D in uremic conditions and proven ciPTEC-OAT1 endocrine function encourage the use of these cells for BAK application.