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.

Implementation of a Human Renal Proximal Tubule on a Chip for Nephrotoxicity and Drug Interaction Studies.

Proximal tubule epithelial cells (PTEC) are susceptible to drug-induced kidney injury (DIKI). Cell-based, two-dimensional (2D) in vitro PTEC models are often poor predictors of DIKI, probably due to the lack of physiological architecture and flow. Here, we assessed a high throughput, 3D microfluidic platform (Nephroscreen) for the detection of DIKI in pharmaceutical development. This system was established with four model nephrotoxic drugs (cisplatin, tenofovir, tobramycin and cyclosporin A) and tested with eight pharmaceutical compounds. Measured parameters included cell viability, release of lactate dehydrogenase (LDH) and N-acetyl-ß-d-glucosaminidase (NAG), barrier integrity, release of specific miRNAs, and gene expression of toxicity markers. Drug-transporter interactions for P-gp and MRP2/4 were also determined. The most predictive read outs for DIKI were a combination of cell viability, LDH and miRNA release. In conclusion, Nephroscreen detected DIKI in a robust manner, is compatible with automated pipetting, proved to be amenable to long-term experiments, and was easily transferred between laboratories. This proof-of-concept-study demonstrated the usability and reproducibility of Nephroscreen for the detection of DIKI and drug-transporter interactions. Nephroscreen it represents a valuable tool towards replacing animal testing and supporting the 3Rs (Reduce, Refine and Replace animal experimentation).

Flow stimulates drug transport in a human kidney proximal tubule-on-a-chip independent of primary cilia.

BACKGROUND: Kidney disease modeling and assessment of drug-induced kidney injury can be advanced using three-dimensional (3D) microfluidic models that recapitulate in vivo characteristics. Fluid shear stress (FSS) has been depicted as main modulator improving in vitro physiology in proximal tubule epithelial cells (PTECs). We aimed to elucidate the role of FSS and primary cilia on transport activity and morphology in PTECs. METHODS: Human conditionally immortalized PTEC (ciPTEC-parent) was cultured in a microfluidic 3D device, the OrganoPlate, under a physiological peak FSS of 2.0 dyne/cm2 or low peak FSS of 0.5 dyne/cm2. Upon a 9-day exposure to FSS, albumin-FITC uptake, activity of P-glycoprotein (P-gp) and multidrug resistance-associated proteins 2/4 (MRP2/4), cytotoxicity and cell morphology were determined. RESULTS: A primary cilium knock-out cell model, ciPTEC-KIF3α-/-, was successfully established via CRISPR-Cas9 genome editing. Under physiological peak FSS, albumin-FITC uptake (p = .04) and P-gp efflux (p = .002) were increased as compared to low FSS. Remarkably, a higher albumin-FITC uptake (p = .03) and similar trends in activity of P-gp and MRP2/4 were observed in ciPTEC-KIF3α-/-. FSS induced cell elongation corresponding with the direction of flow in both cell models, but had no effect on cyclosporine A-induced cytotoxicity. CONCLUSIONS: FSS increased albumin uptake, P-gp efflux and cell elongation, but this was not attributed to a mechanosensitive mechanism related to primary cilia in PTECs, but likely to microvilli present at the apical membrane. GENERAL SIGNIFICANCE: FSS-induced improvements in biological characteristics and activity in PTECs was not mediated through a primary cilium-related mechanism.

Therapy with 2'-O-Me Phosphorothioate Antisense Oligonucleotides Causes Reversible Proteinuria by Inhibiting Renal Protein Reabsorption.

Antisense oligonucleotide therapy has been reported to be associated with renal injury. Here, the mechanism of reversible proteinuria was investigated by combining clinical, pre-clinical, and in vitro data. Urine samples were obtained from Duchenne muscular dystrophy (DMD) patients treated with drisapersen, a modified 2'O-methyl phosphorothioate antisense oligonucleotide (6 mg/kg). Urine and kidney tissue samples were collected from cynomolgus monkeys (Macaca fascicularis) dosed with drisapersen (39 weeks). Cell viability and protein uptake were evaluated in vitro using human conditionally immortalized proximal tubule epithelial cells (ciPTECs). Oligonucleotide treatment in DMD patients was associated with an increase in urinary alpha-1-microglobulin (A1M), which returned to baseline following treatment interruptions. In monkeys, increased urinary A1M correlated with dose-dependent accumulation of oligonucleotide in kidney tissue without evidence of tubular damage. Furthermore, oligonucleotides accumulated in the lysosomes of ciPTECs and reduced the absorption of A1M, albumin, and receptor-associated protein, but did not affect cell viability when incubated for up to 7 days. In conclusion, phosphorothioate oligonucleotides appear to directly compete for receptor-mediated endocytosis in proximal tubules. We postulate that oligonucleotide-induced low molecular weight proteinuria in patients is therefore a transient functional change and not indicative of tubular damage.

Organic anion transporters 1 and 3 influence cellular energy metabolism in renal proximal tubule cells.

Organic anion transporters (OATs) 1 and 3 are, besides being uptake transporters, key in several cellular metabolic pathways. The underlying mechanisms are largely unknown. Hence, we used human conditionally immortalized proximal tubule epithelial cells (ciPTEC) overexpressing OAT1 or OAT3 to gain insight into these mechanisms. In ciPTEC-OAT1 and -OAT3, extracellular lactate levels were decreased (by 77% and 71%, respectively), while intracellular ATP levels remained unchanged, suggesting a shift towards an oxidative phenotype upon OAT1 or OAT3 overexpression. This was confirmed by increased respiration of ciPTEC-OAT1 and -OAT3 (1.4-fold), a decreased sensitivity to respiratory inhibition, and characterized by a higher demand on mitochondrial oxidative capacity. In-depth profiling of tricarboxylic acid (TCA) cycle metabolites revealed reduced levels of intermediates converging into α-ketoglutarate in ciPTEC-OAT1 and -OAT3, which via 2-hydroxyglutarate metabolism explains the increased respiration. These interactions with TCA cycle metabolites were in agreement with metabolomic network modeling studies published earlier. Further studies using OAT or oxidative phosphorylation (OXPHOS) inhibitors confirmed our idea that OATs are responsible for increased use and synthesis of α-ketoglutarate. In conclusion, our results indicate an increased α-ketoglutarate efflux by OAT1 and OAT3, resulting in a metabolic shift towards an oxidative phenotype.

A novel multi-parametric high content screening assay in ciPTEC-OAT1 to predict drug-induced nephrotoxicity during drug discovery.

Drug-induced nephrotoxicity is a major concern in the clinic and hampers the use of available treatments as well as the development of innovative medicines. It is typically discovered late during drug development, which reflects a lack of in vitro nephrotoxicity assays available that can be employed readily in early drug discovery, to identify and hence steer away from the risk. Here, we report the development of a high content screening assay in ciPTEC-OAT1, a proximal tubular cell line that expresses several relevant renal transporters, using five fluorescent dyes to quantify cell health parameters. We used a validation set of 62 drugs, tested across a relevant concentration range compared to their exposure in humans, to develop a model that integrates multi-parametric data and drug exposure information, which identified most proximal tubular toxic drugs tested (sensitivity 75%) without any false positives (specificity 100%). Due to the relatively high throughput (straight-forward assay protocol, 96-well format, cost-effective) the assay is compatible with the needs in the early drug discovery setting to enable identification, quantification and subsequent mitigation of the risk for nephrotoxicity.

Recellularized Native Kidney Scaffolds as a Novel Tool in Nephrotoxicity Screening.

Drug-induced kidney injury in medicinal compound development accounts for over 20% of clinical trial failures and involves damage to different nephron segments, mostly the proximal tubule. Yet, currently applied cell models fail to reliably predict nephrotoxicity; neither are such models easy to establish. Here, we developed a novel three-dimensional (3D) nephrotoxicity platform on the basis of decellularized rat kidney scaffolds (DS) recellularized with conditionally immortalized human renal proximal tubule epithelial cells overexpressing the organic anion transporter 1 (ciPTEC-OAT1). A 5-day SDS-based decellularization protocol was used to generate DS, of which 100-µm slices were cut and used for cell seeding. After 8 days of culturing, recellularized scaffolds (RS) demonstrated 3D-tubule formation along with tubular epithelial characteristics, including drug transporter function. 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. This was most probably attributable 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. Compared with 2D cultures, RS showed an increased sensitivity to cisplatin and tenofovir toxicity after 24-hour exposure (9 and 2.2 fold, respectively). In conclusion, we developed a physiologically relevant 3D nephrotoxicity screening platform that could be a novel tool in drug development.

Screening of Drug-Transporter Interactions in a 3D Microfluidic Renal Proximal Tubule on a Chip.

Drug-transporter interactions could impact renal drug clearance and should ideally be detected in early stages of drug development to avoid toxicity-related withdrawals in later stages. This requires reliable and robust assays for which current high-throughput screenings have, however, poor predictability. Kidney-on-a-chip platforms have the potential to improve predictability, but often lack compatibility with high-content detection platforms. Here, we combined conditionally immortalized proximal tubule epithelial cells overexpressing organic anion transporter 1 (ciPTEC-OAT1) with the microfluidic titer plate OrganoPlate to develop a screenings assay for renal drug-transporter interactions. In this platform, apical localization of F-actin and intracellular tight-junction protein zonula occludens-1 (ZO-1) indicated appropriate cell polarization. Gene expression levels of the drug transporters organic anion transporter 1 (OAT1; SLC22A6), organic cation transporter 2 (OCT2; SLC22A2), P-glycoprotein (P-gp; ABCB1), and multidrug resistance-associated protein 2 and 4 (MRP2/4; ABCC2/4) were similar levels to 2D static cultures. Functionality of the efflux transporters P-gp and MRP2/4 was studied as proof-of-concept for 3D assays using calcein-AM and 5-chloromethylfluorescein-diacetate (CMFDA), respectively. Confocal imaging demonstrated a 4.4 ± 0.2-fold increase in calcein accumulation upon P-gp inhibition using PSC833. For MRP2/4, a 3.0 ± 0.2-fold increased accumulation of glutathione-methylfluorescein (GS-MF) was observed upon inhibition with a combination of PSC833, MK571, and KO143. Semi-quantitative image processing methods for P-gp and MRP2/4 was demonstrated with corresponding Z'-factors of 0.1 ± 0.3 and 0.4 ± 0.1, respectively. In conclusion, we demonstrate a 3D microfluidic PTEC model valuable for screening of drug-transporter interactions that further allows multiplexing of endpoint read-outs for drug-transporter interactions and toxicity.

Expression of Organic Anion Transporter 1 or 3 in Human Kidney Proximal Tubule Cells Reduces Cisplatin Sensitivity.

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 (TC50 34 ± 1 µM after 24-hour exposure), as determined by cell viability. Overexpression of OAT1 and OAT3 resulted in reduced sensitivity to cisplatin (TC50 45 ± 6 and 64 ± 11 µM 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 Vmax of the OCT2-model substrate ASP+ (23.5 ± 0.1, 13.1 ± 0.3, and 21.6 ± 0.6 minutes-1 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 ± 0.3-fold upregulated in ciPTEC-OAT1 and 6.1 ± 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.

Allostimulatory capacity of conditionally immortalized proximal tubule cell lines for bioartificial kidney application.

Novel renal replacement therapies, such as a bioartificial kidney (BAK), are needed to improve current hemodialysis treatment of end-stage renal disease (ESRD) patients. As BAK applications may reveal safety concerns, we assessed the alloimmunization and related safety aspects of readily available conditionally immortalized human proximal tubule epithelial cell (ciPTEC) lines to be used in BAK. Two ciPTEC lines, originally derived from urine and kidney tissue, were characterized for the expression and secretion of relevant molecules involved in alloimmunization and inflammatory responses, such as HLA class-I, HLA-DR, CD40, CD80, CD86, as wells as soluble HLA class I and proinflammatory cytokines (IL-6, IL-8 and TNF-α). A lack of direct immunogenic effect of ciPTEC was shown in co-culture experiments with peripheral blood mononuclear cells (PBMC), after appropriate stimulation of ciPTEC. Tight epithelial cell monolayer formation on polyethersulfone flat membranes was confirmed by zonula occludens-1 (ZO-1) expression in the ciPTEC tight junctions, and by restricted inulin-FITC diffusion. Co-culture with (activated) PBMC did not jeopardize the transepithelial barrier function of ciPTEC. In conclusion, the absence of allostimulatory effects and the stability of ciPTEC monolayers show that these unique cells could represent a safe option for BAK engineering application.

Cetuximab Prevents Methotrexate-Induced Cytotoxicity in Vitro through Epidermal Growth Factor Dependent Regulation of Renal Drug Transporters.

The combination of methotrexate with epidermal growth factor receptor (EGFR) recombinant antibody, cetuximab, is currently being investigated in treatment of head and neck carcinoma. As methotrexate is cleared by renal excretion, we studied the effect of cetuximab on renal methotrexate handling. We used human conditionally immortalized proximal tubule epithelial cells overexpressing either organic anion transporter 1 or 3 (ciPTEC-OAT1/ciPTEC-OAT3) to examine OAT1 and OAT3, and the efflux pumps breast cancer resistance protein (BCRP), multidrug resistance protein 4 (MRP4), and P-glycoprotein (P-gp) in methotrexate handling upon EGF or cetuximab treatment. Protein kinase microarrays and knowledge-based pathway analysis were used to predict EGFR-mediated transporter regulation. Cytotoxic effects of methotrexate were evaluated using the dimethylthiazol bromide (MTT) viability assay. Methotrexate inhibited OAT-mediated fluorescein uptake and decreased efflux of Hoechst33342 and glutathione-methylfluorescein (GS-MF), which suggested involvement of OAT1/3, BCRP, and MRP4 in transepithelial transport, respectively. Cetuximab reversed the EGF-increased expression of OAT1 and BCRP as well as their membrane expressions and transport activities, while MRP4 and P-gp were increased. Pathway analysis predicted cetuximab-induced modulation of PKC and PI3K pathways downstream EGFR/ERBB2/PLCg. Pharmacological inhibition of ERK decreased expression of OAT1 and BCRP, while P-gp and MRP4 were increased. AKT inhibition reduced all transporters. Exposure to methotrexate for 24 h led to a decreased viability, an effect that was reversed by cetuximab. In conclusion, cetuximab downregulates OAT1 and BCRP while upregulating P-gp and MRP4 through an EGFR-mediated regulation of PI3K-AKT and MAPKK-ERK pathways. Consequently, cetuximab attenuates methotrexate-induced cytotoxicity, which opens possibilities for further research into nephroprotective comedication therapies.

Kidney-on-a-chip technology for renal proximal tubule tissue reconstruction.

The renal proximal tubule epithelium is responsible for active secretion of endogenous and exogenous waste products from the body and simultaneous reabsorption of vital compounds from the glomerular filtrate. The complexity of this transport machinery makes investigation of processes such as tubular drug secretion a continuous challenge for researchers. Currently available renal cell culture models often lack sufficient physiological relevance and reliability. Introducing complex biological culture systems in a 3D microfluidic design improves the physiological relevance of in vitro renal proximal tubule epithelium models. Organ-on-a-chip technology provides a promising alternative, as it allows the reconstruction of a renal tubule structure. These microfluidic systems mimic the in vivo microenvironment including multi-compartmentalization and exposure to fluid shear stress. Increasing data supports that fluid shear stress impacts the phenotype and functionality of proximal tubule cultures, for which we provide an extensive background. In this review, we discuss recent developments of kidney-on-a-chip platforms with current and future applications. The improved proximal tubule functionality using 3D microfluidic systems is placed in perspective of investigating cellular signalling that can elucidate mechanistic aberrations involved in drug-induced kidney toxicity.

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 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.

A Human Renal Proximal Tubule Cell Line with Stable Organic Anion Transporter 1 and 3 Expression Predictive for Antiviral-Induced Toxicity.

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.

Heterogeneous transport of digitalis-like compounds by P-glycoprotein in vesicular and cellular assays.

Digitalis-like compounds (DLCs), the ancient medication of heart failure and Na,K-ATPase inhibitors, are characterized by their toxicity. Drug-drug interactions (DDIs) at absorption and excretion levels play a key role in their toxicity, hence, knowledge about the transporters involved might prevent these unwanted interactions. In the present study, the transport of fourteen DLCs with human P-glycoprotein (P-gp; ABCB1) was studied using a liquid chromatography-mass spectrometry (LC-MS) quantification method. DLC transport by P-gp overexpressing Madin-Darby canine kidney (MDCK) and immortalized human renal cells (ciPTEC) was compared to vesicular DLC transport. Previously, we identified convallatoxin as a substrate using membrane vesicles overexpressing P-gp; however, we could not measure transport of other DLCs in this assay (Gozalpour et al., 2014a). Here, we showed that lipophilic digitoxin, digoxigenin, strophanthidin and proscillaridin A are P-gp substrates in cellular accumulation assays, whereas the less lipophilic convallatoxin was not. P-gp function in the cellular accumulation assays depends on the entrance of lipophilic compounds by passive diffusion, whereas the vesicular transport assay is more appropriate for hydrophilic substrates. In conclusion, we identified digitoxin, digoxigenin, strophanthidin and proscillaridin A as P-gp substrates using cellular accumulation assays and recognized lipophilicity as an important factor in selecting a suitable transport assay.

Kidney-on-a-Chip Technology for Drug-Induced Nephrotoxicity Screening.

Improved model systems to predict drug efficacy, interactions, and drug-induced kidney injury (DIKI) are crucially needed in drug development. Organ-on-a-chip technology is a suitable in vitro system because it reproduces the 3D microenvironment. A kidney-on-a-chip can mimic the structural, mechanical, transport, absorptive, and physiological properties of the human kidney. In this review we address the application of state-of-the-art microfluidic culturing techniques, with a focus on culturing kidney proximal tubules, that are promising for the detection of biomarkers that predict drug interactions and DIKI. We also discuss high-throughput screening and the challenges for in vitro to in vivo extrapolation (IVIVE) that will need to be overcome for successful implementation.

Proximal tubular efflux transporters involved in renal excretion of p-cresyl sulfate and p-cresyl glucuronide: Implications for chronic kidney disease pathophysiology.

The uremic solutes p-cresyl sulfate (pCS) and p-cresyl glucuronide (pCG) accumulate in patients with chronic kidney disease (CKD), and might contribute to disease progression. Moreover, retention of these solutes may directly be related to renal tubular function. Here, we investigated the role of the efflux transporters Multidrug Resistance Protein 4 (MRP4) and Breast Cancer Resistance Protein (BCRP) in pCS and pCG excretion, and studied the impact of both solutes on the phenotype of human conditionally immortalized renal proximal tubule epithelial cells (ciPTEC). Our results show that p-cresol metabolites accumulate during CKD, with a shift from sulfation to glucuronidation upon progression. Moreover, pCS inhibited the activity of MRP4 by 40% and BCRP by 25%, whereas pCG only reduced MRP4 activity by 75%. Moreover, BCRP-mediated transport of both solutes was demonstrated. Exposure of ciPTEC to pCG caused epithelial-to-mesenchymal transition, indicated by increased expression of vimentin and Bcl-2, and diminished E-cadherin. This was associated with altered expression of key tubular transporters. In conclusion, BCRP is likely involved in the renal excretion of both solutes, and pCG promotes phenotypical changes in ciPTEC, supporting the notion that uremic toxins may be involved in CKD progression by negatively affecting renal tubule cell phenotype and functionality.

Development of a living membrane comprising a functional human renal proximal tubule cell monolayer on polyethersulfone polymeric membrane.

The need for improved renal replacement therapies has stimulated innovative research for the development of a cell-based renal assist device. A key requirement for such a device is the formation of a "living membrane", consisting of a tight kidney cell monolayer with preserved functional organic ion transporters on a suitable artificial membrane surface. In this work, we applied a unique conditionally immortalized proximal tubule epithelial cell (ciPTEC) line with an optimized coating strategy on polyethersulfone (PES) membranes to develop a living membrane with a functional proximal tubule epithelial cell layer. PES membranes were coated with combinations of 3,4-dihydroxy-l-phenylalanine and human collagen IV (Coll IV). The optimal coating time and concentrations were determined to achieve retention of vital blood components while preserving high water transport and optimal ciPTEC adhesion. The ciPTEC monolayers obtained were examined through immunocytochemistry to detect zona occludens 1 tight junction proteins. Reproducible monolayers were formed when using a combination of 2 mg ml(-1) 3,4-dihydroxy-l-phenylalanine (4 min coating, 1h dissolution) and 25 µg ml(-1) Coll IV (4 min coating). The successful transport of (14)C-creatinine through the developed living membrane system was used as an indication for organic cation transporter functionality. The addition of metformin or cimetidine significantly reduced the creatinine transepithelial flux, indicating active creatinine uptake in ciPTECs, most likely mediated by the organic cation transporter, OCT2 (SLC22A2). In conclusion, this study shows the successful development of a living membrane consisting of a reproducible ciPTEC monolayer on PES membranes, an important step towards the development of a bioartificial kidney.

Polycystin-1 but not polycystin-2 deficiency causes upregulation of the mTOR pathway and can be synergistically targeted with rapamycin and metformin.

Autosomal dominant polycystic kidney disease (ADPKD) is caused by loss-of-function mutations in either PKD1 or PKD2 genes, which encode polycystin-1 (TRPP1) and polycystin-2 (TRPP2), respectively. Increased activity of the mammalian target of rapamycin (mTOR) pathway has been shown in PKD1 mutants but is less documented for PKD2 mutants. Clinical trials using mTOR inhibitors were disappointing, while the AMP-activated kinase (AMPK) activator, metformin is not yet tested in patients. Here, we studied the mTOR activity and its upstream pathways in several human and mouse renal cell models with either siRNA or stable knockdown and with overexpression of TRPP2. Our data reveal for the first time differences between TRPP1 and TRPP2 deficiency. In contrast to TRPP1 deficiency, TRPP2-deficient cells did neither display excessive activation of the mTOR-kinase complex nor inhibition of AMPK activity, while ERK1/2 and Akt activity were similarly affected among TRPP1- and TRPP2-deficient cells. Furthermore, cell proliferation was more pronounced in TRPP1 than in TRPP2-deficient cells. Interestingly, combining low concentrations of rapamycin and metformin was more effective for inhibiting mTOR complex 1 activity in TRPP1-deficient cells than either drug alone. Our results demonstrate a synergistic effect of a combination of low concentrations of drugs suppressing the increased mTOR activity in TRPP1-deficient cells. This novel insight can be exploited in future clinical trials to optimize the efficiency and avoiding side effects of drugs in the treatment of ADPKD patients with PKD1 mutations. Furthermore, as TRPP2 deficiency by itself did not affect mTOR signaling, this may underlie the differences in phenotype, and genetic testing has to be considered for selecting patients for the ongoing trials.

Optimized metabolomic approach to identify uremic solutes in plasma of stage 3-4 chronic kidney disease patients.

BACKGROUND: Chronic kidney disease (CKD) is characterized by the progressive accumulation of various potential toxic solutes. Furthermore, uremic plasma is a complex mixture hampering accurate determination of uremic toxin levels and the identification of novel uremic solutes. METHODS: In this study, we applied (1)H-nuclear magnetic resonance (NMR) spectroscopy, following three distinct deproteinization strategies, to determine differences in the plasma metabolic status of stage 3-4 CKD patients and healthy controls. Moreover, the human renal proximal tubule cell line (ciPTEC) was used to study the influence of newly indentified uremic solutes on renal phenotype and functionality. RESULTS: Protein removal via ultrafiltration and acetonitrile precipitation are complementary techniques and both are required to obtain a clear metabolome profile. This new approach, revealed that a total of 14 metabolites were elevated in uremic plasma. In addition to confirming the retention of several previously identified uremic toxins, including p-cresyl sulphate, two novel uremic retentions solutes were detected, namely dimethyl sulphone (DMSO2) and 2-hydroxyisobutyric acid (2-HIBA). Our results show that these metabolites accumulate in non-dialysis CKD patients from 9±7 µM (control) to 51±29 µM and from 7 (0-9) µM (control) to 32±15 µM, respectively. Furthermore, exposure of ciPTEC to clinically relevant concentrations of both solutes resulted in an increased protein expression of the mesenchymal marker vimentin with more than 10% (p<0.05). Moreover, the loss of epithelial characteristics significantly correlated with a loss of glucuronidation activity (Pearson r = -0.63; p<0.05). In addition, both solutes did not affect cell viability nor mitochondrial activity. CONCLUSIONS: This study demonstrates the importance of sample preparation techniques in the identification of uremic retention solutes using (1)H-NMR spectroscopy, and provide insight into the negative impact of DMSO2 and 2-HIBA on ciPTEC, which could aid in understanding the progressive nature of renal disease.