Drug-induced nephrotoxicity still hampers drug development, because current translation from in vitro or animal studies to human lacks high predictivity. Often, renal adverse effects are recognized only during clinical stages of drug development. The current study aimed to establish a robust and a more complete human cell model suitable for screening of drug-related interactions and nephrotoxicity. In addition to endogenously expressed renal organic cation transporters and efflux transporters, conditionally immortalized proximal tubule epithelial cells (ciPTEC) were completed by transduction of cells with the organic anion transporter (OAT) 1 or OAT3. Fluorescence-activated cell sorting upon exposure to the OAT substrate fluorescein successfully enriched transduced cells. A panel of organic anions was screened for drug-interactions in ciPTEC-OAT1 and ciPTEC-OAT3. The cytotoxic response to the drug-interactions with antivirals was further examined by cell viability assays. Upon subcloning, concentration-dependent fluorescein uptake was found with a higher affinity for ciPTEC-OAT1 (Km = 0.8 ± 0.1 μM) than ciPTEC-OAT3 (Km = 3.7 ± 0.5 μM). Co-exposure to known OAT1 and/or OAT3 substrates (viz. para-aminohippurate, estrone sulfate, probenecid, furosemide, diclofenac, and cimetidine) in cultures spanning 29 passage numbers revealed relevant inhibitory potencies, confirming the robustness of our model for drug-drug interactions studies. Functional OAT1 was directly responsible for cytotoxicity of adefovir, cidofovir, and tenofovir, while a drug interaction with zidovudine was not associated with decreased cell viability. Our data demonstrate that human-derived ciPTEC-OAT1 and ciPTEC-OAT3 are promising platforms for highly predictive drug screening during early phases of drug development.Electronic supplementary materialThe online version of this article (doi:10.1208/s12248-016-9871-8) contains supplementary material, which is available to authorized users.

The conversion of the alpha-helical, protease sensitive and noninfectious form of the prion protein (PrP(C)) into an insoluble, protease resistant, predominantly beta-sheeted and infectious form (PrP(Sc)) is the fundamental event in prion formation. In the present work, two soluble and stable intermediate structural states are newly identified for recombinant Syrian hamster PrP(90-231) (recPrP), a dimeric alpha-helical state and a tetra- or oligomeric, beta-sheet rich state. In 0.2% SDS at room temperature, recPrP is soluble and exhibits alpha-helical and random coil secondary structure as determined by circular dichroism. Reduction of the SDS concentration to 0.06% leads first to a small increase in alpha-helical content, whereas further dilution to 0.02% results in the aquisition of beta-sheet structure. The reversible transition curve is sigmoidal within a narrow range of SDS concentrations (0.04 to 0.02%). Size exclusion chromatography and chemical crosslinking revealed that the alpha-helical form is dimeric, while the beta-sheet rich form is tetra- or oligomeric. Both the alpha-helical and beta-sheet rich intermediates are soluble and stable. Thus, they should be accessible to further structural and mechanistic studies. At 0.01% SDS, the oligomeric intermediates aggregated into large, insoluble structures as observed by fluorescence correlation spectroscopy. Our results are discussed with respect to the mechanism of PrP(Sc) formation and the propagation of prions.

Membrane transporters and receptors are responsible for balancing nutrient and metabolite levels to aid body homeostasis. Here, we report that proximal tubule cells in kidneys sense elevated endogenous, gut microbiome-derived, metabolite levels through EGF receptors and downstream signaling to induce their secretion by up-regulating the organic anion transporter-1 (OAT1). Remote metabolite sensing and signaling was observed in kidneys from healthy volunteers and rats in vivo, leading to induced OAT1 expression and increased removal of indoxyl sulfate, a prototypical microbiome-derived metabolite and uremic toxin. Using 2D and 3D human proximal tubule cell models, we show that indoxyl sulfate induces OAT1 via AhR and EGFR signaling, controlled by miR-223. Concomitantly produced reactive oxygen species (ROS) control OAT1 activity and are balanced by the glutathione pathway, as confirmed by cellular metabolomic profiling. Collectively, we demonstrate remote metabolite sensing and signaling as an effective OAT1 regulation mechanism to maintain plasma metabolite levels by controlling their secretion.

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The increasing prevalence of end‐stage renal disease and persistent shortage of donor organs call for alternative therapies for kidney patients. Dialysis remains an inferior treatment as clearance of large and protein‐bound waste products depends on active tubular secretion. Biofabricated tissues could make a valuable contribution, but kidneys are highly intricate and multifunctional organs. Depending on the therapeutic objective, suitable cell sources and scaffolds must be selected. This study provides a proof‐of‐concept for stand‐alone kidney tubule grafts with suitable mechanical properties for future implantation purposes. Porous tubular nanofiber scaffolds are fabricated by electrospinning 12%, 16%, and 20% poly‐ε‐caprolactone (PCL) v/w (chloroform and dimethylformamide, 1:3) around 0.7 mm needle templates. The resulting scaffolds consist of 92%, 69%, and 54% nanofibers compared to microfibers, respectively. After biofunctionalization with L‐3,4‐dihydroxyphenylalanine and collagen IV, 10 × 106 proximal tubule cells per mL are injected and cultured until experimental readout. A human‐derived cell model can bridge all fiber‐to‐fiber distances to form a monolayer, whereas small‐sized murine cells form monolayers on dense nanofiber meshes only. Fabricated constructs remain viable for at least 3 weeks and maintain functionality as shown by inhibitor‐sensitive transport activity, which suggests clearance capacity for both negatively and positively charged solutes.

Introduction: To date, tubular tissue engineering relies on large, non-porous tubular scaffolds (Ø > 2 mm) for mechanical self-support, or smaller (Ø 150–500 μm) tubes within bulk hydrogels for studying renal transport phenomena. To advance the engineering of kidney tubules for future implantation, constructs should be both self-supportive and yet small-sized and highly porous. Here, we hypothesize that the fabrication of small-sized porous tubular scaffolds with a highly organized fibrous microstructure by means of melt-electrowriting (MEW) allows the development of self-supported kidney proximal tubules with enhanced properties.Materials and Methods: A custom-built melt-electrowriting (MEW) device was used to fabricate tubular fibrous scaffolds with small diameter sizes (Ø = 0.5, 1, 3 mm) and well-defined, porous microarchitectures (rhombus, square, and random). Human umbilical vein endothelial cells (HUVEC) and human conditionally immortalized proximal tubular epithelial cells (ciPTEC) were seeded into the tubular scaffolds and tested for monolayer formation, integrity, and organization, as well as for extracellular matrix (ECM) production and renal transport functionality.Results: Tubular fibrous scaffolds were successfully manufactured by fine control of MEW instrument parameters. A minimum inner diameter of 1 mm and pore sizes of 0.2 mm were achieved and used for subsequent cell experiments. While HUVEC were unable to bridge the pores, ciPTEC formed tight monolayers in all scaffold microarchitectures tested. Well-defined rhombus-shaped pores outperformed and facilitated unidirectional cell orientation, increased collagen type IV deposition, and expression of the renal transporters and differentiation markers organic cation transporter 2 (OCT2) and P-glycoprotein (P-gp).Discussion and Conclusion: Here, we present smaller diameter engineered kidney tubules with microgeometry-directed cell functionality. Due to the well-organized tubular fiber scaffold microstructure, the tubes are mechanically self-supported, and the self-produced ECM constitutes the only barrier between the inner and outer compartment, facilitating rapid and active solute transport.

Facing the problems of limited renal regeneration capacity and the persistent shortage of donor kidneys, dialysis remains the only treatment option for many end-stage renal disease patients. Unfortunately, dialysis is only a medium-term solution because large and protein-bound uremic solutes are not efficiently cleared from the body and lead to disease progression over time. Current strategies for improved renal replacement therapies (RRTs) range from whole organ engineering to biofabrication of renal assist devices and biological injectables for in vivo regeneration. Notably, all approaches coincide with the incorporation of cellular components and biomimetic micro-environments. Concerning the latter, hydrogels form promising materials as scaffolds and cell carrier systems due to the demonstrated biocompatibility of most natural hydrogels, tunable biochemical and mechanical properties, and various application possibilities. In this review, the potential of hydrogel-based cell therapies for kidney regeneration is discussed. First, we provide an overview of current trends in the development of RRTs and in vivo regeneration options, before examining the possible roles of hydrogels within these fields. We discuss major application-specific hydrogel design criteria and, subsequently, assess the potential of emergent biofabrication technologies, such as micromolding, microfluidics and electrodeposition for the development of new RRTs and injectable stem cell therapies.

Cisplatin is a cytostatic drug used for treatment of solid organ tumors. The main adverse effect is organic cation transporter 2 (OCT2)-mediated nephrotoxicity, observed in 30% of patients. The contribution of other renal drug transporters is elusive. Here, cisplatin-induced toxicity was evaluated in human-derived conditionally immortalized proximal tubule epithelial cells (ciPTEC) expressing renal drug transporters, including OCT2 and organic anion transporters 1 (OAT1) or 3 (OAT3). Parent ciPTEC demonstrated OCT2-dependent cisplatin toxicity (TC 50 34 6 1 mM after 24-hour exposure), as determined by cell viability. Overexpression of OAT1 and OAT3 resulted in reduced sensitivity to cisplatin (TC 50 45 6 6 and 64 6 11 mM after 24-hour exposure, respectively). This effect was independent of OAT-mediated transport, as the OAT substrates probenecid and diclofenac did not influence cytotoxicity. Decreased cisplatin sensitivity in OAT-expressing cells was associated directly with a trend toward reduced intracellular cisplatin accumulation, explained by reduced OCT2 gene expression and activity. This was evaluated by V max of the OCT2-model substrate ASP + (23.5 6 0.1, 13.1 6 0.3, and 21.6 6 0.6 minutes 21 in ciPTEC-parent, ciPTEC-OAT1, and ciPTEC-OAT3, respectively). Although gene expression of cisplatin efflux transporter multidrug and toxin extrusion 1 (MATE1) was 16.2 6 0.3-fold upregulated in ciPTEC-OAT1 and 6.1 6 0.7-fold in ciPTEC-OAT3, toxicity was unaffected by the MATE substrate pyrimethamine, suggesting that MATE1 does not play a role in the current experimental set-up. In conclusion, OAT expression results in reduced cisplatin sensitivity in renal proximal tubule cells, explained by reduced OCT2-mediated uptake capacity. In vitro drug-induced toxicity studies should consider models that express both OCT and OAT drug transporters.