Development of a Highly Differentiated Human Primary Proximal Tubule MPS Model (aProximate MPS Flow).

The kidney proximal tubule (PT) mediates renal drug elimination in vivo and is a major site of drug-induced toxicity. To reliably assess drug efficacy, it is crucial to construct a model in which PT functions are replicated. Current animal studies have proven poorly predictive of human outcome. To address this, we developed a physiologically relevant micro-physiological system (MPS) model of the human PT, the aProximate MPS Flow platform (Patent No: G001336.GB). In this model, primary human PT cells (hPTCs) are subjected to fluidic media flow and a shear stress of 0.01-0.2 Pa. We observe that these cells replicate the polarity of hPTCs and exhibit a higher expression of all the key transporters of SLC22A6 (OAT1), SLC22A8 (OAT3), SLC22A2 (OCT2), SLC47A1 (MATE1), SLC22A12 (URAT1), SLC2A9 (GLUT9), ABCB1 (MDR1), ABCC2 (MRP2), LRP2 (megalin), CUBN (cubilin), compared with cells grown under static conditions. Immunofluorescence microscopy confirmed an increase in OAT1, OAT3, and cilia protein expression. Increased sensitivity to nephrotoxic protein cisplatin was observed; creatinine and FITC-albumin uptake was significantly increased under fluidic shear stress conditions. Taken together, these data suggest that growing human PT cells under media flow significantly improves the phenotype and function of hPTC monolayers and has benefits to the utility and near-physiology of the model.

c-Rel orchestrates energy-dependent epithelial and macrophage reprogramming in fibrosis.

Fibrosis is a common pathological feature of chronic disease. Deletion of the NF-κB subunit c-Rel limits fibrosis in multiple organs, although the mechanistic nature of this protection is unresolved. Using cell-specific gene-targeting manipulations in mice undergoing liver damage, we elucidate a critical role for c-Rel in controlling metabolic changes required for inflammatory and fibrogenic activities of hepatocytes and macrophages and identify Pfkfb3 as the key downstream metabolic mediator of this response. Independent deletions of Rel in hepatocytes or macrophages suppressed liver fibrosis induced by carbon tetrachloride, while combined deletion had an additive anti-fibrogenic effect. In transforming growth factor-ß1-induced hepatocytes, c-Rel regulates expression of a pro-fibrogenic secretome comprising inflammatory molecules and connective tissue growth factor, the latter promoting collagen secretion from HMs. Macrophages lacking c-Rel fail to polarize to M1 or M2 states, explaining reduced fibrosis in RelΔLysM mice. Pharmacological inhibition of c-Rel attenuated multi-organ fibrosis in both murine and human fibrosis. In conclusion, activation of c-Rel/Pfkfb3 in damaged tissue instigates a paracrine signalling network among epithelial, myeloid and mesenchymal cells to stimulate fibrogenesis. Targeting the c-Rel-Pfkfb3 axis has potential for therapeutic applications in fibrotic disease.

The Importance of Incorporating OCT2 Plasma Membrane Expression and Membrane Potential in IVIVE of Metformin Renal Secretory Clearance.

Transporter expression, determined by quantitative proteomics, together with PBPK models is a promising approach for in vitro-to-in vivo extrapolation (IVIVE) of transporter-mediated drug clearance. OCT2-expressing HEK293 and MDCKII cells were used to predict in vivo renal secretory clearance (CLr,sec) of metformin. [14C]-Metformin uptake clearance in OCT2-expressing cells was determined and scaled to in vivo CLr,sec by using OCT2 expression in the cells versus the human kidney cortex. Through quantitative targeted proteomics, the total expression of OCT2 in HEK293, MDCKII cells, and human kidney cortex was 369.4 ± 26.8, 19 ± 1.1, and 7.6 ± 3.8 pmol/mg cellular protein, respectively. The expression of OCT2 in the plasma membrane of HEK293 and MDCKII cells, measured using an optimized biotinylation method followed by quantitative proteomics, was 30.2% and 51.6%, respectively. After correcting for percent of OCT2 expressed in the plasma membrane and the resting membrane potential (millivolts) difference between the OCT2-expressing cells and the renal epithelial cells, the predicted CLr,sec of metformin was 250.7 ml/min, a value within the range of the observed CLr,sec of metformin. These data demonstrate the promise of using quantitative proteomics for IVIVE of transporter-mediated drug clearance and highlight the importance of quantifying plasma membrane expression of transporters and utilizing cells that mimic the in vivo mechanism(s) of transport of drugs.

Microsomal and Cytosolic Scaling Factors in Dog and Human Kidney Cortex and Application for In Vitro-In Vivo Extrapolation of Renal Metabolic Clearance.

In vitro-in vivo extrapolation of drug metabolism data obtained in enriched preparations of subcellular fractions rely on robust estimates of physiologically relevant scaling factors for the prediction of clearance in vivo. The purpose of the current study was to measure the microsomal and cytosolic protein per gram of kidney (MPPGK and CPPGK) in dog and human kidney cortex using appropriate protein recovery marker and evaluate functional activity of human cortex microsomes. Cytochrome P450 (CYP) content and glucose-6-phosphatase (G6Pase) activity were used as microsomal protein markers, whereas glutathione-S-transferase activity was a cytosolic marker. Functional activity of human microsomal samples was assessed by measuring mycophenolic acid glucuronidation. MPPGK was 33.9 and 44.0 mg/g in dog kidney cortex, and 41.1 and 63.6 mg/g in dog liver (n = 17), using P450 content and G6Pase activity, respectively. No trends were noted between kidney, liver, and intestinal scalars from the same animals. Species differences were evident, as human MPPGK and CPPGK were 26.2 and 53.3 mg/g in kidney cortex (n = 38), respectively. MPPGK was 2-fold greater than the commonly used in vitro-in vivo extrapolation scalar; this difference was attributed mainly to tissue source (mixed kidney regions versus cortex). Robust human MPPGK and CPPGK scalars were measured for the first time. The work emphasized the importance of regional differences (cortex versus whole kidney-specific MPPGK, tissue weight, and blood flow) and a need to account for these to improve assessment of renal metabolic clearance and its extrapolation to in vivo.

Abundance of Drug Transporters in the Human Kidney Cortex as Quantified by Quantitative Targeted Proteomics.

Protein expression of renal uptake and efflux transporters was quantified by quantitative targeted proteomics using the surrogate peptide approach. Renal uptake transporters assessed in this study included organic anion transporters (OAT1-OAT4), organic cation transporter 2 (OCT2), organic/carnitine cation transporters (OCTN1 and OCTN2), and sodium-glucose transporter 2 (SGLT2); efflux transporters included P-glycoprotein, breast cancer resistance protein, multidrug resistance proteins (MRP2 and MRP4), and multidrug and toxin extrusion proteins (MATE1 and MATE2-K). Total membrane was isolated from the cortex of human kidneys (N = 41). The isolated membranes were digested by trypsin and the digest was subjected to liquid chromatography-tandem mass spectrometry analysis. The mean expression of surrogate peptides was as follows (given with the standard deviation, in picomoles per milligram of total membrane protein): OAT1 (5.3 ± 1.9), OAT2 (0.9 ± 0.3), OAT3 (3.5 ± 1.6), OAT4 (0.5 ± 0.2), OCT2 (7.4 ± 2.8), OCTN1 (1.3 ± 0.6), OCTN2 (0.6 ± 0.2), P-glycoprotein (2.1 ± 0.8), MRP2 (1.4 ± 0.6), MRP4 (0.9 ± 0.6), MATE1 (5.1 ± 2.3), and SGLT2 (3.7 ± 1.8). Breast cancer resistance protein (BCRP) and MATE2-K proteins were detectable but were below the lower limit of quantification. Interestingly, the protein expression of OAT1 and OAT3 was significantly correlated (r > 0.8). A significant correlation was also observed between expression of multiple other drug transporters, such as OATs/OCT2 or OCTN1/OCTN2, and SGLT2/OCTNs, OCT, OATs, and MRP2. These renal transporter data should be useful in deriving in vitro to in vivo scaling factors to accurately predict renal clearance and kidney epithelial cell exposure to drugs or their metabolites.

Characterization of multidrug transporter-mediated efflux of avermectins in human and mouse neuroblastoma cell lines.

ABC transporters play an important role in the disposition of avermectins in several animal species. In this study the interactions of three key avermectins, abamectin, emamectin and ivermectin, with human and mouse homologues of MDR1 (ABCB1/Abcb1a) and MRP (ABCC/Abcc), transporters endogenously expressed by human SH-SY5Y and mouse N2a neuroblastoma cells were investigated. In both cell lines, retention of the fluorescent dye H33342 was found to be significantly increased in the presence of avermectins and cyclosporin A. These effects were shown to be unresponsive to the BCRP inhibitor Ko-143 and therefore MDR1/Mdr1-dependent. Avermectins inhibited MDR1/Mdr1a-mediated H33342 dye efflux, with apparent Ki values of 0.24±0.08 and 0.18±0.02µM (ivermectin); 0.60±0.07 and 0.56±0.02µM (emamectin) and 0.95±0.08 and 0.77±0.25µM (abamectin) in SH-SY5Y and N2a cells, respectively. There were some apparent affinity differences for MDR1 and Mdr1a within each cell line (affinity for ivermectin>emamectin≥abamectin, P<0.05 by One-Way ANOVA), but importantly, the Ki values for individual avermectins for human MDR1 or mouse Mdr1a were not significantly different. MK571-sensitive retention of GSMF confirmed the expression of MRP/Mrp efflux transporters in both cell lines. Avermectins inhibited MRP/Mrp-mediated dye efflux with IC50 values of 1.58±0.51 and 1.94±0.72µM (ivermectin); 1.87±0.57 and 2.74±1.01µM (emamectin) and 2.25±0.01 and 1.68±0.63µM (abamectin) in SH-SY5Y and N2a cells, respectively. There were no significant differences in IC50 values between individual avermectins or between human MRP and mouse Mrp. Kinetic data for endogenous human MDR1/MRP isoforms in SH-SY5Y cells and mouse Mdr1a/b/Mrp isoforms in N2a cells are comparable for the selected avermectins. All are effluxed at concentrations well above 0.05-0.1µM ivermectin detected in plasma (Ottesen and Campbell, 1994; Ottesen and Campbell, 1994) This is an important finding in the light of toxicity seen in the Mdr1-deficient animal models CF-1 mice, Mdr1ab (-/-) double knockout mice and Collie dogs. We also confirm MRP/Mrp-mediated avermectin transport in both N2a and SH-SY5Y cell lines.

Utility of B-13 progenitor-derived hepatocytes in hepatotoxicity and genotoxicity studies.

AR42J-B-13 (B-13) cells form hepatocyte-like (B-13/H) cells in response to glucocorticoid treatment. To establish its utility in toxicity and genotoxicity screening, cytochrome P450 (CYP) induction, susceptibility to toxins, and transporter gene expression were examined. Conversion to B-13/H cells resulted in expression of male-specific CYP2C11 and sensitivity to methapyrilene. B-13/H cells constitutively expressed CYP1A, induced expression in response to an aryl hydrocarbon receptor agonist, and activated benzo[α]pyrene to a DNA-damaging species. Functional CYP1A2 was not expressed due to deletions in the Cyp1a2 gene. A B-13 cell line stably expressing the human CYP1A2 was therefore engineered (B-13(-TR/h1A2)) and the derived B-13/H cells expressed metabolically functional CYP1A2. Treatment with the cooked food mutagen 2-amino-1-methyl-6-phenylimidazo(4,5-b)pyridine resulted in a dose-dependent increase in DNA damage. B-13/H cells expressed constitutive androstane receptor (CAR) and induced CYP2B1 mRNA levels in response to classical CAR activators. However, translation to functional CYP2B1 protein was low and increased minimally by CAR activator treatment. B-13/H cells expressed high levels of pregnane X-receptor (PXR) and induced CYP3A1 in response to classical PXR activators. CYP3A genes were inducible, functional, and activated aflatoxin B1 to a DNA-damaging species. All 23 major hepatic transporters were induced when B-13 cells were converted to B-13/H cells, although in many cases, levels remained below those present in adult rat liver. However, bile salt export pump, Abcb1b, multidrug resistance-associated protein, and breast cancer resistance protein transporters were functional in B-13/H cells. These data demonstrate that the B-13 cell generates hepatocyte-like cells with functional drug metabolism and transporter activities, which can alone--or in a humanized form--be used to screen for hepatotoxic and genotoxic endpoints in vitro.

Renal differentiation from adult spermatogonial stem cells.

There is considerable interest in the use of multi-potent stem cells in kidney tissue regeneration. We studied if spermatogonial stem cells have the ability to undergo kidney differentiation. Spermatogonial stem cell differentiation was induced using in vitro and ex vivo co-culture techniques. Conditioned media from human kidney fibroblasts induced the expression of epithelial and endothelial lineages in spermatogonial stem cells, consistent with nephrogenesis. Furthermore, we showed that these cells up-regulated renal tubular-specific markers alkaline phosphatase, mineralocorticoid receptor, renal epithelial sodium channel and sodium-glucose transporter-2 (p<0.05). GFP-labeled spermatogonial stem cells were engrafted into metanephric kidney organ cultures harvested from E12.5 mouse embryos. After 5 days of organ culture, focal anti-GFP staining was detectable in all inoculated kidneys demonstrating integration of spermatogonial stem cells into the developing kidney (p<0.01). Histological assessment showed early nephron-like architecture. In summary, we show that spermatogonial stem cells have the potential to generate renal tissue and lay the foundations for further investigations into a novel therapeutic approach for renal insufficiency.

Characterisation of the roles of ABCB1, ABCC1, ABCC2 and ABCG2 in the transport and pharmacokinetics of actinomycin D in vitro and in vivo.

Actinomycin D plays a key role in the successful treatment of Wilms tumour. However, associated liver toxicities remain a drawback to potentially curative treatment. We have used MDCKII cells over-expressing ABCB1, ABCC1, ABCC2 and ABCG2, alongside knockout mouse models to characterise actinomycin D transport and its impact on pharmacokinetics. Growth inhibition, intracellular accumulation and cellular efflux assays were utilised. A 59-fold difference in GI(50) was observed between MDCKII-WT and MDCKII-ABCB1 cells (12.7 nM vs. 745 nM, p<0.0001). Reduced sensitivity was also seen in MDCKII-ABCC1 and ABCC2 cells (GI(50) 25.7 and 40.4 nM respectively, p<0.0001). Lower intracellular accumulation of actinomycin D was observed in MDCKII-ABCB1 cells as compared to MDCKII-WT (0.98 nM vs. 0.1 nM, p<0.0001), which was reversed upon ABCB1 inhibition. Lower accumulation was also seen in MDCKII-ABCC1 and ABCC2 cells. Actinomycin D efflux over 2 h was most pronounced in MDCKII-ABCB1 cells, with 5.5-fold lower intracellular levels compared to WT. In vivo studies showed that actinomycin D plasma concentrations were significantly higher in Abcb1a/1b(-/-) as compared to WT mice following administration of 0.5 mg/kg actinomycin D (AUC(0-6 h) 242 vs. 152 µg/Lh respectively). While comparable actinomycin D concentrations were observed in the kidneys and livers of Abcb1a/1b(-/-) and Abcc2(-/-) mice, concentrations in the brain were significantly higher at 6h following drug administration in Abcb1a/1b(-/-) mice compared to WT. Results confirm actinomycin D as a substrate for ABCB1, ABCC1 and ABCC2, and indicate that Abcb1a/1b and Abcc2 can influence the in vivo disposition of actinomycin D. These data have implications for ongoing clinical pharmacology trials involving children treated with actinomycin D.

The limitations of renal epithelial cell line HK-2 as a model of drug transporter expression and function in the proximal tubule.

Acquiring a mechanistic understanding of the processes underlying the renal clearance of drug molecules in man has been hampered by a lack of robust in vitro models of human proximal tubules. Several human renal epithelial cell lines derived from the renal cortex are available, but few have been characterised in detail in terms of transporter expression. This includes the HK-2 proximal tubule cell line, which has been used extensively as a model of nephrotoxicity. The aim of this study was to investigate the expression and function of drug transporters in HK-2 cells and their suitability as an in vitro model of the human proximal tubule. qPCR showed no mRNA expression of the SLC22 transporter family (OAT1, OAT3, OCT2) in HK-2 cells compared to renal cortex samples. In contrast, SLC16A1 (MCT1), which is important in the uptake of monocarboxylates, and SLCO4C1 (OATP4C1) were expressed in HK-2 cells. The functional expression of these transporters was confirmed by uptake studies using radiolabelled prototypic substrates DL-lactate and digoxin, respectively. The mRNA expression of apical membrane efflux transporters ABCB1 (MDR1) and several members of the ABCC family (multidrug resistance proteins, MRPs) was shown by qPCR. ABCG1 (BCRP) was not detected. The efflux of Hoechst 33342, a substrate for MDR1, was blocked by MDR1 inhibitor cyclosporin A, suggesting the functional expression of this transporter. Similarly, the efflux of the MRP-specific fluorescent dye glutathione methylfluorescein was inhibited by the MRP inhibitor MK571. Taken together, the results of this study suggest that HK-2 cells are of limited value as an in vitro model of drug transporter expression in the human proximal tubule.

Characterisation of human tubular cell monolayers as a model of proximal tubular xenobiotic handling.

The aim of this study was to determine whether primary human tubular cell monolayers could provide a powerful tool with which to investigate the renal proximal tubular handling of xenobiotics. Human proximal and distal tubule/collecting duct cells were grown as monolayers on permeable filter supports. After 10 days in culture, proximal tubule cells remained differentiated and expressed a wide palette of transporters at the mRNA level including NaPi-IIa, SGLT1, SGLT2, OCT2, OCTN2, OAT1, OAT3, OAT4, MDR1, MRP2 and BCRP. At the protein level, the expression of a subset of transporters including NaPi-IIa, OAT1 and OAT3 was demonstrated using immunohistochemistry. Analysis of the expression of the ATP binding cassette efflux pumps MDR1, MRP2 and BCRP confirmed their apical membrane localisation. At the functional level, tubule cell monolayers retain the necessary machinery to mediate the net secretion of the prototypic substrates; PAH and creatinine. PAH secretion across the monolayer consisted of the uptake of PAH across the basolateral membrane by OAT1 and OAT3 and the apical exit of PAH by a probenecid and MK571-sensitive route consistent with actions of MRP2 or MRP4. Creatinine secretion was by OCT2-mediated uptake at the basolateral membrane and via MDR1 at the apical membrane. Functional expression of MDR1 and BCRP at the apical membrane was also demonstrated using a Hoechst 33342 dye. Similarly, measurement of calcein efflux demonstrated the functional expression of MRP2 at the apical membrane of cell monolayers. In conclusion, human tubular cell monolayers provide a powerful tool to investigate renal xenobiotic handling.

Human proximal tubular epithelium actively secretes but does not retain rosuvastatin.

Rosuvastatin is a potent HMG-CoA reductase inhibitor that has proven to be effective in the treatment of dyslipidemia. Rosuvastatin is cleared from the body by both biliary and renal clearance, the latter believed to be due to active tubular secretion. Whereas the mechanisms of hepatic clearance of rosuvastatin are well documented, those of renal clearance are not. Because rosuvastatin (and other statins) may alter proximal tubular function, this study aimed to characterize the mechanisms of tubular rosuvastatin secretion to define the factors that could influence the presence/concentration of rosuvastatin in proximal tubular cells. Hereto, polarized monolayers of primary human tubular cells were used. We found rosuvastatin net secretion across proximal tubule cells, which was saturable (K50=20.4+/-4.1 microM). The basolateral uptake step was rate-limiting and mediated by OAT3. Rosuvastatin efflux at the apical membrane was mediated by MRP2/4 and ABCG2 together with a small contribution from MDR1 or P-glycoprotein. These data, obtained in an intact human tubule cell model, provide a detailed insight into rosuvastatin's renal handling and the possible factors influencing it.

Rosuvastatin pharmacokinetics in heart transplant recipients administered an antirejection regimen including cyclosporine.

BACKGROUND: Cyclosporine (INN, ciclosporin) increases the systemic exposure of all statins. Therefore rosuvastatin pharmacokinetic parameters were assessed in an open-label trial involving stable heart transplant recipients (> or =6 months after transplant) on an antirejection regimen including cyclosporine. Rosuvastatin has been shown to be a substrate for the human liver transporter organic anion transporting polypeptide C (OATP-C). Inhibition of this transporter could increase plasma concentrations of rosuvastatin. Therefore the effect of cyclosporine on rosuvastatin uptake by cells expressing OATP-C was also examined. METHODS: Ten subjects were assessed while taking 10 mg rosuvastatin for 10 days; 5 of these were then assessed while taking 20 mg rosuvastatin for 10 days. Rosuvastatin steady-state area under the plasma concentration-time curve from time 0 to 24 hours [AUC(0-24)] and maximum observed plasma concentration (Cmax) were compared with values in controls (historical data from 21 healthy volunteers taking 10 mg rosuvastatin). Rosuvastatin uptake by OATP-C-transfected Xenopus oocytes was also studied by use of radiolabeled rosuvastatin with and without cyclosporine. RESULTS: In transplant recipients taking 10 mg rosuvastatin, geometric mean values and percent coefficient of variation for steady-state AUC(0-24) and Cmax were 284 ng. h/mL (31.3%) and 48.7 ng/mL (47.2%), respectively. In controls, these values were 40.1 ng. h/mL (39.4%) and 4.58 ng/mL (46.9%), respectively. Compared with control values, AUC(0-24) and Cmax were increased 7.1-fold and 10.6-fold, respectively, in transplant recipients. In transplant recipients taking 20 mg rosuvastatin, these parameters increased less than dose-proportionally. Rosuvastatin had no effect on cyclosporine blood concentrations. The in vitro results demonstrate that rosuvastatin is a good substrate for OATP-C-mediated hepatic uptake (association constant, 8.5 +/- 1.1 micromol/L) and that cyclosporine is an effective inhibitor of this process (50% inhibition constant, 2.2 +/- 0.4 micromol/L when the rosuvastatin concentration was 5 micromol/L). CONCLUSIONS: Rosuvastatin exposure was significantly increased in transplant recipients on an antirejection regimen including cyclosporine. Cyclosporine inhibition of OATP-C-mediated rosuvastatin hepatic uptake may be the mechanism of the drug-drug interaction. Coadministration of rosuvastatin with cyclosporine needs to be undertaken with caution.

The effect of gemfibrozil on the pharmacokinetics of rosuvastatin.

BACKGROUND: Coadministration of statins and gemfibrozil is associated with an increased risk for myopathy, which may be due in part to a pharmacokinetic interaction. Therefore the effect of gemfibrozil on rosuvastatin pharmacokinetics was assessed in healthy volunteers. Rosuvastatin has been shown to be a substrate for the human hepatic uptake transporter organic anion transporter 2 (OATP2). Inhibition of this transporter could increase plasma concentrations of rosuvastatin. The effect of gemfibrozil on rosuvastatin uptake by cells expressing OATP2 was also examined. METHODS: In a randomized, double-blind, 2-period crossover trial, 20 healthy volunteers were given oral doses of gemfibrozil, 600 mg, or placebo twice daily for 7 days. On the fourth morning of each dosing period, a single oral dose of rosuvastatin, 80 mg, was coadministered. Plasma concentrations of rosuvastatin, N-desmethyl rosuvastatin, and rosuvastatin-lactone were measured. In addition, the effect of gemfibrozil on the uptake of radiolabeled rosuvastatin by OATP2-transfected Xenopus oocytes was studied. RESULTS: Gemfibrozil increased the rosuvastatin area under the plasma concentration-time curve from time 0 to the time of the last quantifiable concentration [AUC(0-t)] 1.88-fold (90% confidence interval, 1.60-2.21) and the maximum observed rosuvastatin plasma concentration (C(max)) 2.21-fold (90% confidence interval, 1.81-2.69) compared with placebo. N-desmethyl rosuvastatin AUC(0-t) and C(max) decreased by 48% and 39%, respectively. Pharmacokinetics of rosuvastatin-lactone was unchanged. The in vitro results indicate that the maximum gemfibrozil inhibition of rosuvastatin OATP2-mediated uptake was 50%; the inhibition constant for the inhibitory process was 4.0 +/- 1.3 micromol/L. CONCLUSIONS: Gemfibrozil increased rosuvastatin plasma concentrations approximately 2-fold, which is similar to the effect of gemfibrozil on pravastatin, simvastatin acid, and lovastatin acid plasma concentrations and substantially less than the effect observed for cerivastatin. Gemfibrozil inhibition of OATP2-mediated rosuvastatin hepatic uptake may contribute to the mechanism of the drug-drug interaction. Care is warranted when gemfibrozil is coadministered with rosuvastatin and other statins.