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.

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.

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.

Humoral signalling compounds in remote ischaemic preconditioning of the kidney, a role for the opioid receptor.

BACKGROUND: Renal ischaemia-reperfusion injury (IRI) is a common clinical problem associated with significant mortality and morbidity. One strategy to reduce this damage is remote ischaemic preconditioning (RIPC), in which brief ischaemia of a limb protects the kidney against a prolonged ischaemic insult. The mechanism of renal RIPC has not yet been elucidated. Here, we address the gap in our understanding of renal RIPC signalling, using a rat model of renal IRI and RIPC by brief hind limb ischaemia. METHODS: Rats were treated with either no RIPC, RIPC+vehicle or RIPC+ an inhibitor or antagonist of one of the following candidate signalling molecules: noradrenalin, cannabinoids, glucocorticoids, inducible nitric oxide synthase, calcitonin gene-related peptide, ganglion-mediated signalling, haem oxygenase and free radicals. Subsequently, the animals underwent 25 min of renal ischaemia and 2 days of reperfusion, after which renal function and damage were assessed. RESULTS: RIPC by three 4 min cycles of hind limb ischaemia effectively reduced renal IRI. Pre-treatment with the opioid receptor antagonist naloxone completely blocked this protective effect, when compared with animals treated with RIPC+vehicle; serum creatinine and urea increased (307.8±43.7 versus 169.5±16.7 µmol/L and 42.2±4.9 versus 27.6±2.2 mmol/L, respectively), as did the renal histological damage (score 4.2±0.7 versus 2.8±0.5) and expression of kidney injury molecule-1 (KIM-1; relative-fold increase in mRNA expression 164±18 versus 304±33). All other antagonists were without effect. CONCLUSIONS: Renal RIPC by brief hind limb ischaemia may be the result of endorphin release from the hind limb. The importance of opioid signalling in renal RIPC provides vital clues for its successful translation to the clinical setting.

Subunit-specific incorporation efficiency and kinetics in mitochondrial complex I homeostasis.

Studies employing native PAGE suggest that most nDNA-encoded CI subunits form subassemblies before assembling into holo-CI. In addition, in vitro evidence suggests that some subunits can directly exchange in holo-CI. Presently, data on the kinetics of these two incorporation modes for individual CI subunits during CI maintenance are sparse. Here, we used inducible HEK293 cell lines stably expressing AcGFP1-tagged CI subunits and quantified the amount of tagged subunit in mitoplasts and holo-CI by non-native and native PAGE, respectively, to determine their CI incorporation efficiency. Analysis of time courses of induction revealed three subunit-specific patterns. A first pattern, represented by NDUFS1, showed overlapping time courses, indicating that imported subunits predominantly incorporate into holo-CI. A second pattern, represented by NDUFV1, consisted of parallel time courses, which were, however, not quantitatively overlapping, suggesting that imported subunits incorporate at similar rates into holo-CI and CI assembly intermediates. The third pattern, represented by NDUFS3 and NDUFA2, revealed a delayed incorporation into holo-CI, suggesting their prior appearance in CI assembly intermediates and/or as free monomers. Our analysis showed the same maximum incorporation into holo-CI for NDUFV1, NDUFV2, NDUFS1, NDUFS3, NDUFS4, NDUFA2, and NDUFA12 with nearly complete loss of endogenous subunit at 24 h of induction, indicative of an equimolar stoichiometry and unexpectedly rapid turnover. In conclusion, the results presented demonstrate that newly formed nDNA-encoded CI subunits rapidly incorporate into holo-CI in a subunit-specific manner.

Acute acetaminophen intoxication leads to hepatic iron loading by decreased hepcidin synthesis.

Acetaminophen (APAP), a major cause of acute liver injury in the Western world, is mediated by metabolism and oxidative stress. Recent studies have suggested a role for iron in potentiating APAP-induced liver injury although its regulatory mechanism is not completely understood. The current study was designed to unravel the iron-regulating pathways in mice after APAP-induced hepatotoxicity. Mice with severe injury showed a significant increase in liver iron concentration and oxidative stress. Concurrently, the plasma concentration of hepcidin, the key regulator in iron metabolism, and hepatic hepcidin antimicrobial peptide (Hamp) mRNA expression levels were significantly reduced. We showed that hepcidin transcription was inhibited via several hepcidin-regulating factors, including the bone morphogenetic protein/small mother against decapentaplegic (BMP/SMAD) pathway, CCAAT/enhancer-binding protein α (C/EBPα), and possibly also via erythropoietin (EPO). Downregulation of the BMP/SMAD signaling pathway was most likely caused by hypoxia-inducible factor 1α (HIF-1α), which was increased in mice with severe APAP-induced liver injury. HIF-1α stimulates cleaving of hemojuvelin, the cofactor of the BMP receptor, thereby blocking BMP-induced signaling. In addition, gene expression levels of C/ebpα were significantly reduced, and Epo mRNA expression levels were significantly increased after APAP intoxication. These factors are regulated through HIF-1α during oxidative stress and suggest that HIF-1α is a key modulator in reduced hepcidin transcription after APAP-induced hepatotoxicity. In conclusion, acute APAP-induced liver injury leads to activation of HIF-1α, which results in a downregulation in hepcidin expression through a BMP/SMAD signaling pathway and through C/EBPα inhibition. Eventually, this leads to hepatic iron loading associated with APAP cytotoxicity.

Regulation of P-glycoprotein in renal proximal tubule epithelial cells by LPS and TNF-alpha.

During endotoxemia, the ATP-dependent drug efflux pump P-glycoprotein (Abcb1/P-gp) is upregulated in kidney proximal tubule epithelial cells. The signaling pathway through which lipopolysaccharide (LPS) or tumor necrosis factor-alpha (TNF-alpha) regulates P-gp expression and activity was investigated further in the present study. Exposure of rat kidney proximal tubule cells to TNF-alpha alone or TNF-alpha and LPS increased P-gp gene and protein expression levels and efflux activity, suggesting de novo P-gp synthesis. Upon exposure to TNF-alpha in combination with LPS, P-gp activity in renal proximal tubule cells is increased under influence of nitric oxide (NO) produced by inducible NO synthase. Upon exposure to TNF-alpha alone, P-gp upregulation seems to involve TLR4 activation and nuclear factor kappaB (NF-kappaB) translocation, a pathway that is likely independent of NO. These findings indicate that at least two pathways regulate P-gp expression in the kidney during endotoxemia.

Characterization of P-glycoprotein and multidrug resistance proteins in rat kidney and intestinal cell lines.

The activity of P-glycoprotein (Pgp/MDR1/ABCB1) and multidrug resistance proteins (MRP/ABCC) influence the pharmacokinetics and bioavailability of many drugs. Few suitable cell lines for the study of drug transport exist. Additional non-human cell lines may help clarify species differences and contribute to the current knowledge of drug transport. The aim of the present study was to characterize three rat epithelial cell lines for transporter expression and activity. Transporter expression was assessed in intestinal IEC-6 and renal GERP and NRK-52E cells using RT-PCR and Western blot analysis. Pgp and Mrp transport activity were analyzed by measuring calcein accumulation and glutathione-S-bimane efflux, respectively. The three cell lines showed Pgp expression and Pgp-dependent transport, both decreasing with culture time after reaching confluency. Besides Pgp, cells expressed Mrp1, Mrp3, Mrp4, and Mrp5, while Mrp2 and Mrp6 were absent. In addition, they showed temperature- and Mrp-dependent efflux of glutathione-S-bimane. Exposure to a panel of different inhibitors showed that this efflux was probably mediated by Mrp4. In conclusion, the three rat epithelial cell lines investigated showed Pgp and Mrp expression and transport. Mrp dependent transport was most likely mediated by Mrp4. In future, these cell lines may be used as in vitro models to study drug transport.

Intravenously administered short interfering RNA accumulates in the kidney and selectively suppresses gene function in renal proximal tubules.

Different gene-silencing methods, like antisense and short interfering RNA (siRNA), are widely used as experimental tools to inhibit gene expression. In the present study, the in vivo behavior of siRNA in rats and siRNA-mediated silencing of genes in the renal proximal tubule were investigated. To study the biodistribution of siRNA, rats were injected i.v. with radiolabeled siRNA or radiolabel alone (control), and scintigraphic images were acquired at different time intervals postinjection. The siRNA preferentially accumulated in the kidneys and was excreted in the urine. One hour after injection, the amount of siRNA present in both kidneys (1.7 +/- 0.3% of injected dose/g tissue) was on average 40 times higher than in other tissues (liver, brain, intestine, muscle, lung, spleen, and blood). Besides the biodistribution, the effect of siRNA on multidrug resistance protein isoform 2 (Mrp2/Abcc2, siRNAMrp2) in renal proximal tubules was investigated. Mrp2 function was assessed by measuring the excretion of its fluorescent substrate calcein in the isolated perfused rat kidney. Four days after administration, siRNAMrp2 reduced the urinary calcein excretion rate significantly (35% inhibition over the period 80-150 min of perfusion). This down-regulation was specific because another siRNA sequence directed against a different transporter in the proximal tubule, Mrp4 (Abcc4, siRNAMrp4), did not alter the Mrp2-mediated excretion of calcein. In conclusion, siRNA accumulates spontaneously in the kidney after i.v. injection, where it selectively suppresses gene function in the proximal tubules. Therefore, i.v. administered siRNA provides a novel experimental and potential therapeutic tool for gene silencing in the kidney.

The MRP4/ABCC4 gene encodes a novel apical organic anion transporter in human kidney proximal tubules: putative efflux pump for urinary cAMP and cGMP.

The cyclic nucleotides cAMP and cGMP play key roles in cellular signaling and the extracellular regulation of fluid balance. In the kidney, cAMP is excreted across the apical proximal tubular membrane into urine, where it reduces phosphate reabsorption through a dipyridamole-sensitive mechanism that is not fully understood. It has long been known that this cAMP efflux pathway is dependent on ATP and is inhibited by probenecid. However, its identity and whether cGMP shares the same transporter have not been established. Here the expression, localization, and functional properties of human multidrug resistance protein 4 (MRP4) are reported. MRP4 is localized to the proximal tubule apical membrane of human kidney, and membrane vesicles from Sf9 cells expressing human MRP4 exhibit ATP-dependent transport of [(3)H]cAMP and [(3)H]cGMP. Both probenecid and dipyridamole are potent MRP4 inhibitors. ATP-dependent [(3)H]methotrexate and [(3)H]estradiol-17beta-D-glucuronide transport by MRP4 and interactions with the anionic conjugates S-(2,4-dinitrophenyl)-glutathione, N-acetyl-(2,4-dinitrophenyl)-cysteine, alpha-naphthyl-beta-D-glucuronide, and p-nitrophenyl-beta-D-glucuronide are also demonstrated. In kidneys of rats deficient in the apical anionic conjugate efflux pump Mrp2, Mrp4 expression is maintained at the same level. It is concluded that MRP4 is a novel apical organic anion transporter and the putative efflux pump for cAMP and cGMP in human kidney proximal tubules.