Analysis of CTNS gene transcripts in nephropathic cystinosis

Nephropathic cystinosis (NC) is an autosomal recessive disorder caused by mutations of the CTNS gene that encodes for a cystine transmembrane transporter. Several mutations have been described in the coding and promoter regions of the CTNS gene in affected individuals. We selected three patients with NC from two unrelated families, in whom sequence analysis of the CTNS gene detected only one or no mutations. Total RNA was isolated from peripheral blood mononuclear cells or fibroblasts and CTNS transcripts were analyzed. We observed a skipping of exon 5 (85 bp) in two siblings and an intron 9 retention of 75 bp associated with partial replication of exon 9 in the third patient. Genomic DNA analysis of intron regions surrounding exon 5 showed a point mutation in the hypothetical lariat branch site of intron 4 at position –24 (c.141–24 T > C) in the first two patients and a duplication of 266 bp including a part of exon and intron 9 in the third patient. Analysis of CTNS gene transcripts allowed identification of mutations in patients in whom CTNS mutations could not be detected by traditional DNA sequencing. These results support the hypothesis that cystinosis is a monogenic disorder

FXYD2 and Na,K-ATPase Expression in Isolated Human Proximal Tubular Cells: Disturbed Upregulation on Renal Hypomagnesemia?

Autosomal dominant renal hypomagnesemia (OMIM 154020), associated with hypocalciuria, has been linked to a 121G to A mutation in the FXYD2 gene. To gain insight into the molecular mechanisms linking this mutation to the clinical phenotype, we studied isolated proximal tubular cells from urine of a patient and a healthy subject. Cells were immortalized and used to assess the effects of hypertonicity-induced overexpression of FXYD2 on amount, activity and apparent affinities for Na+, K+ and ATP of Na,K-ATPase. Both cell lines expressed mRNA for FXYD2a and FXYD2b, and patient cells contained both the wild-type and mutated codons. FXYD2 protein expression was lower in patient cells and could be increased in both cell lines upon culturing in hyperosmotic medium but to a lesser extent in patient cells. Similarly, hyperosmotic culturing increased Na,K-ATPase protein expression and ATP hydrolyzing activity but, again, to a lesser extent in patient cells. Apparent affinities of Na,K-ATPase for Na+, K+ and ATP did not differ between patient and control cells or after hyperosmotic induction. We conclude that human proximal tubular cells respond to a hyperosmotic challenge with an increase in FXYD2 and Na,K-ATPase protein expression, though to a smaller absolute extent in patient cells

Novel conditionally immortalized human proximal tubule cell line expressing functional influx and efflux transporters

Reabsorption of filtered solutes from the glomerular filtrate and excretion of waste products and xenobiotics are the main functions of the renal proximal tubular (PT) epithelium. A human PT cell line expressing a range of functional transporters would help to augment current knowledge in renal physiology and pharmacology. We have established and characterized a conditionally immortalized PT epithelial cell line (ciPTEC) obtained by transfecting and subcloning cells exfoliated in the urine of a healthy volunteer. The PT origin of this line has been confirmed morphologically and by the expression of aminopeptidase N, zona occludens 1, aquaporin 1, dipeptidyl peptidase IV and multidrug resistance protein 4 together with alkaline phosphatase activity. ciPTEC assembles in a tight monolayer with limited diffusion of inulin-fluorescein-isothiocyanate. Concentration and time-dependent reabsorption of albumin via endocytosis has been demonstrated, together with sodium-dependent phosphate uptake. The expression and activity of apical efflux transporter p-glycoprotein and of baso-lateral influx transporter organic cation transporter 2 have been shown in ciPTEC. This established human ciPTEC expressing multiple endogenous organic ion transporters mimicking renal reabsorption and excretion represents a powerful tool for future in vitro transport studies in pharmacology and physiology

The application of organ-on-chip models for the prediction of human pharmacokinetic profiles during drug development

Organ-on-chip (OoC) technology has led to in vitro models with many new possibilities compared to conventional in vitro and in vivo models. In this review, the potential of OoC models to improve the prediction of human oral bioavailability and intrinsic clearance is discussed, with a focus on the functionality of the models and the application in current drug development practice. Multi-OoC models demonstrating the application for pharmacokinetic (PK) studies are summarized and existing challenges are identified. Physiological parameters for a minimal viable platform of a multi-OoC model to study PK are provided, together with PK specific read-outs and recommendations for relevant reference compounds to validate the model. Finally, the translation to in vivo PK profiles is discussed, which will be required to routinely apply OoC models during drug development

Cystinosis: practical tools for diagnosis and treatment

Cystinosis is the major cause of inherited Fanconi syndrome, and should be suspected in young children with failure to thrive and signs of renal proximal tubular damage. The diagnosis can be missed in infants, because not all signs of renal Fanconi syndrome are present during the first months of life. In older patients cystinosis can mimic idiopathic nephrotic syndrome due to focal and segmental glomerulosclerosis. Measuring elevated white blood cell cystine content is the corner stone for the diagnosis. The diagnosis is confirmed by molecular analysis of the cystinosin gene. Corneal cystine crystals are invariably present in all patients with cystinosis after the age of 1 year. Treatment with the cystine depleting drug cysteamine should be initiated as soon as possible and continued lifelong to prolong renal function survival and protect extra-renal organs. This educational feature provides practical tools for the diagnosis and treatment of cystinosis

Role of P-Glycoprotein Expression and Function in Cystinotic Renal Proximal Tubular Cells

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Fluorescence-Based Transport Assays Revisited in a Human Renal Proximal Tubule Cell Line

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 presence or absence of model inhibitors for BCRP (KO143), P-gp (PSC833) or MRP's (MK571). Overexpression cell lines MDCKII-BCRP and MDCKII-P-gp were used as positive controls and membrane vesicles over-expressing 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 while 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 MRP's, while PSC833 appears a potent inhibitor for BCRP and P-gp, but not the MRP's. Furthermore, ciPTEC accumulate 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

The use of CDME in cystinosis research.

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Recellularized native kidney scaffolds as a novel tool in nephrotoxicity screening

Drug-induced kidney injury (DIKI) in medicinal compound development accounts for over 20% of clinical trials failure and involves damage to different nephron segments, mostly the proximal tubule. Yet, currently applied cell models fail to reliably predict nephrotoxicity nor are easy to establish. Here, we developed a novel three-dimensional (3D) nephrotoxicity platform based on decellularized rat kidneys scaffolds (DS) recellularized with conditionally immortalized human renal proximal tubule epithelial cells overexpressing the organic anion transporter 1 (ciPTEC-OAT1). A 5-days sodium dodecyl sulfate (SDS)-based decellularization protocol was used to generate DS , of which 100 um slices were cut and used for cell seeding. After 8 days of culturing, recellularized scaffolds (RS) demonstrated 3D tubules formation along with tubular epithelial characteristics, including drug transporter funcion. Exposure of RS to cisplatin (CDDP), tenofovir (TFV) or cyclosporin A (CsA) as prototypical nephrotoxic drugs, revealed concentration-dependent reduction in cell viability, as assessed by PrestoBlue and Live/Dead staining assays. CDDP, TFV and CsA TC50-values were 108 ± 1 and 12 ± 1, 212 ± 1 and 97 ± 1, 129 ± 2 and 294 ± 2 μM in 2D and 3D cultures, respectively. This was most likely due to specific uptake of CDDP by the organic cation transporter 2 (OCT2), TFV through organic anion transporter 1 (OAT1) and CsA competing for P-glycoprotein-mediated efflux. As compared to 2D cultures, RS showed an increased sensitivity to cisplatin and tenofovir toxicity after 24h exposure (9 and 2.2 fold, respectively). In conclusion, we developed a physiologically-relevant 3D nephrotoxicity screening platform that could potentially be suitable as a novel alternative in drug development

Epithelial-to-mesenchymal transition in fibrosis:Collagen type I expression is highly upregulated after EMT, but does not contribute to collagen deposition

<p>The hallmark of fibrosis is an accumulation of fibrillar collagens, especially of collagen type I. There is considerable debate whether in vivo type II epithelial-to-mesenchymal transition (EMT) is involved in organ fibrosis. Lineage tracing experiments by various groups show opposing data concerning the relative contribution of epithelial cells to the pool of myofibroblasts. We hypothesized that EMT-derived cells might directly contribute to collagen deposition. To study this. EMT was induced in human epithelial lung and renal cell lines in vitro by means of TGF-beta 1 stimulation, and we compared the collagen type I (COL1A1) expression levels of transdifferentiated cells with that of myofibroblasts obtained by TGF-beta 1 stimulation of human dermal and lung fibroblasts. COL1A1 expression levels of transdifferentiated epithelial cells appeared to be at least one to two orders of magnitude lower than that of myofibroblasts. This was confirmed at immunohistochemical level: in contrast to myofibroblasts, collagen type I deposition by EMT-derived cells was not or hardly detectable. We postulate that, even when type II EMT occurs in vivo, the direct contribution of EMT-derived cells to collagen accumulation is rather limited. (C) 2013 Published by Elsevier Inc.</p>