Visual Overview
Abstract
Autosomal dominant polycystic kidney disease (ADPKD) is a common form of inherited polycystic kidney disease (PKD) and is a leading cause of kidney failure. Fluid-filled cysts develop in the kidneys of patients with ADPKD, and cysts often form in their liver and other organs. Previous data have shown that bile acids are increased in the liver of polycystic kidney (PCK) rats, a rodent model of PKD; these changes may be associated with alterations in liver transporter expression and function. However, the impact of PKD on hepatic transporters has not been characterized. Therefore, this preclinical study was designed to investigate hepatic transporter expression and function in PCK compared with wild-type (WT) Sprague-Dawley rats. Transporter gene expression was measured by quantitative polymerase chain reaction, and protein levels were quantified by Western blot and liquid chromatography-tandem mass spectroscopy (LC-MS/MS)–based proteomic analysis in rat livers. Transporter function was assessed in isolated perfused livers (IPLs), and biliary and hepatic total glutathione content was measured. Protein expression of Mrp2 and Oatp1a4 was decreased 3.0-fold and 2.9-fold, respectively, in PCK rat livers based on Western blot analysis. Proteomic analysis confirmed a decrease in Mrp2 and a decrease in Oatp1a1 expression (PCK/WT ratios, 0.368 ± 0.098 and 0.563 ± 0.038, respectively; mean ± S.D.). The biliary excretion of 5(6)-carboxy-2′,7′-dichlorofluorescein, a substrate of Oatp1a1, Mrp2, and Mrp3, was decreased 28-fold in PCK compared with WT rat IPLs. Total glutathione was significantly reduced in the bile of PCK rats. Differences in hepatic transporter expression and function may contribute to altered disposition of Mrp2 and Oatp substrates in PKD.
Introduction
Polycystic kidney disease (PKD) is the fourth leading cause of kidney failure worldwide (Chebib and Torres, 2016). Autosomal dominant PKD (ADPKD) is caused by mutations in the polycystin-1 (PKD1) or polycystin-2 (PKD2) genes and is the most common form of PKD (prevalence of >1 in 1000 people). Autosomal recessive PKD (ARPKD) is a rare neonatal form of PKD (Halvorson et al., 2010) and is caused by mutations in the polycystic kidney and hepatic disease 1 (PKHD1) gene (Harris and Torres, 2009; Hartung and Guay-Woodford, 2014). Despite the genetic differences between ADPKD and ARPKD, both diseases are associated with fluid-filled cysts and renal impairment. By the age of 70 years, 50% of patients with ADPKD require dialysis or kidney transplantation (Halvorson et al., 2010). Hepatic cysts are the most common extrarenal manifestation in patients with ADPKD and occur in 75%–90% of patients with ADPKD. Hepatic cysts typically develop later than renal cysts (Chauveau et al., 2000; Harris and Torres, 2009; Cnossen and Drenth, 2014), and their presence is generally considered benign, although hepatic biochemical abnormalities have been reported (Everson et al., 1988; Qian et al., 2003; Hogan et al., 2015). Increased concentrations of serum γ-glutamyl transferase or alkaline phosphatase of approximately two to five times the upper limit of normal have occurred in some patients with large polycystic livers (Chauveau et al., 2000; Qian et al., 2003). Although the impact of PKD on the hepatobiliary disposition of drugs has not been evaluated in humans, a case report in a patient with ADPKD who exhibited increased hepatic retention of the multidrug resistance–associated protein (MRP) 2 probe substrate 99mTc-mebrofenin suggests that MRP2 function may be impaired (Salam and Keeffe, 1989).
Until recently, ADPKD therapy has been directed only toward symptomatic treatment. Tolvaptan, an oral, selective vasopressin V2 receptor antagonist, has been shown to slow disease progression in patients with ADPKD, and was approved in April 2018 by the Food and Drug Administration to slow kidney function decline in adults at risk for rapidly progressing ADPKD (https://www.accessdata.fda.gov/drugsatfda_docs/nda/2018/204441Orig1s000TOC.cfm). However, in two pivotal clinical trials (Torres et al., 2012, 2017), the incidence of alanine aminotransferase (ALT) elevations was ∼5% higher in patients receiving tolvaptan compared with placebo. Alternate causes for ALT elevations were ruled out, and, therefore, tolvaptan has been associated with liver injury in patients with ADPKD (Watkins et al., 2015). One possible explanation could be that tolvaptan interacts with hepatic transport proteins, especially bile acid transporters. In vitro studies indicated that tolvaptan is a substrate and competitive inhibitor of P-glycoprotein (MDR1/ABCB1) (Shoaf et al., 2011), whereas tolvaptan and the primary plasma metabolites DM-4103 and DM-4107 inhibit several hepatic transporters. For example, DM-4103 strongly inhibited the hepatic bile acid uptake transporter Na+-taurocholate cotransporting polypeptide (NTCP/SLC10A1), the canalicular bile salt export pump (BSEP/ABCB11), and the basolateral efflux transporter MRP4/ABCC4 (Slizgi et al., 2016). Interestingly, elevated liver enzyme levels have not been reported in patients without ADPKD who were treated with tolvaptan (Watkins et al., 2015). Concomitantly, PKD may alter the function of hepatic transport proteins.
Polycystic kidney (PCK) rats have the same genetic defect as in ARPKD (i.e., PKHD1) and exhibit similar hepatorenal abnormalities (e.g., congenital hepatic fibrosis and development of cysts) as patients with ARPKD or ADPKD (Katsuyama et al., 2000; Lager et al., 2001, Masyuk et al., 2004). Various bile acids that are substrates for hepatic transporters (Dawson et al., 2009) are increased in PCK rat serum and liver (Ruh et al., 2013; Munoz-Garrido et al., 2015; Brock et al., 2018). In addition, serum bilirubin concentrations are increased in PCK rats, suggesting impaired Mrp2 function (Mason et al., 2010; Shimomura et al., 2015). Furthermore, the biliary clearance of tolvaptan was significantly decreased in isolated perfused livers (IPLs) from PCK rats compared with wild-type (WT) rats (Beaudoin et al., 2019). The IPL is a physiologically relevant ex situ model (Brouwer and Thurman, 1996) that has been used to evaluate the hepatic transport and metabolic properties of various compounds (Chandra et al., 2005a; Miranda et al., 2008; Pfeifer et al., 2013).
This preclinical study was designed to elucidate possible mechanisms of altered hepatobiliary disposition of drugs and endogenous compounds in a rodent model of PKD. Hepatic transporter mRNA and protein expression were investigated in PCK and WT rat livers. Functional changes in organic anion transporting polypeptides (Oatps), Mrp2 and Mrp3 (Zamek-Gliszczynski et al., 2003) were measured in WT and PCK rat IPLs using 5(6)-carboxy-2′,7′-dichlorofluorescein (CDF), a fluorescent and metabolically stable probe substrate. In addition, hepatic and biliary total glutathione was quantified in WT and PCK rats.
Materials and Methods
Chemicals and Reagents.
CDF was obtained from Sigma-Aldrich (St. Louis, MO). All other chemicals and reagents were of analytical grade or higher and were readily available from commercial sources. The stable isotope–labeled peptides for Na+/K+ ATPase/Na+/K+ ATPase, Slco1a1/Oatp1a1, Slco1a4/Oatp1a4, Slco1b2/Oatp1b2, Slc10a1/Ntcp, Abcc2/Mrp2, Abcc3/Mrp3, and Abcb11/Bsep (Table 1) were generated by a peptide synthesis platform (PEPscreen, Custom Peptide Libraries; Sigma-Genosys).
Animals.
Male WT Sprague-Dawley or PCK (PCK/CrljCrl-Pkhd1pck/Crl) rats were purchased from Charles River Laboratories (Wilmington, MA). Rats were housed in a constant alternating 12-hour light/dark cycle, were allowed water and food ad libitum, and were acclimated for a minimum of 1 week prior to experimentation. All animal procedures complied with the guidelines of the Institutional Animal Care and Use Committee (University of North Carolina, Chapel Hill, NC). Single-pass IPL procedures were performed, and bile and liver tissue samples were collected at 16 weeks of age because the expected pathologic changes (e.g., hepatic cysts) in PCK rats are well defined at this age (Lager et al., 2001; Mason et al., 2010).
Quantitative Reverse-Transcription Polymerase Chain Reaction Analysis.
Total RNA from WT and PCK rat liver tissue samples were isolated using the RNeasy Plus Mini Kit (Qiagen GmbH, Hilden, Germany) according to the manufacturer instructions. The concentration and purity of isolated RNA were analyzed with a NanoDrop Spectrophotometer (ND-1000; Thermo Fisher Scientific, Waltham, MA). RNA was converted to cDNA, and cDNA was analyzed as described previously (Jackson et al., 2016). Briefly, reverse transcription was performed using a Life Technologies High Capacity cDNA Archive Kit, following the manufacturer instructions (Thermo Fisher Scientific, Carlsbad, CA). Abcc2 (Mrp2), Abcc3 (Mrp3), Abcb1b (Mdr1), Slco1a1 (Oatp1a1), Slco1a4 (Oatp1a4), Slco1b2 (Oatp1b2), Slc51a (Ostα), and Slc51b (Ostβ) cDNA were analyzed from each sample in duplicate or triplicate using gene-specific TaqMan® assays [Abcc2 (catalog number Rn00563231_m1); Abcc3 (catalog number Rn01452854); Abcb1b (catalog number Rn01529252); Slco1a1 (catalog number Rn00755148); Slco1a4 (catalog number Rn00756233); Slco1b2 (catalog number Rn01492635); Slc51a (catalog number Rn01763289_m1); and Slc51b (catalog number Rn01767005_m1); Thermo Fisher Scientific] and a Real-Time PCR System (Life Technologies QuantStudio 6 Flex; Thermo Fisher Scientific). The expression level of the studied genes was normalized against the selected reference gene glyceraldehyde 3-phosphate dehydrogenase (Gapdh; catalog number Rn01775763_g1; Thermo Fisher Scientific), and the relative quantification (2−ΔΔCt, fold difference in PCK relative to WT) was calculated against the calibrator sample (cDNA from WT rat liver sample). No template (water) control was present on each PCR plate.
Western Blot Analysis.
Membrane proteins from rat liver samples (from the median liver lobe) were extracted using a proteoExtract native membrane protein extraction kit (catalog number 444810; Calbiochem, San Diego, CA). The total protein was determined by a Pierce BCA Protein Assay Kit (Thermo Fisher Scientific) using the manufacturer protocol. Samples for Western blots were prepared according to the manufacturer instructions (Thermo Fisher Scientific); 35 µg was loaded onto Bis-Tris 4%–12% (Oatp1a1, Oatp1a4, Oatp2b1, Ostβ, Mdr1) or Tris Acetate 7% (Mrp2, Mrp3) gels. After electrophoresis, the transfer of proteins to polyvinylidene fluoride membranes according to standard procedures was performed. Each protein was analyzed separately on a different membrane, and Na+/K+ ATPase was probed on the same blot as the respective protein and used as the loading control. The membranes were incubated with antibody anti-Oatp1 (1:200; catalog number AB3670P; Millipore, Burlington, MA), anti-Oatp1a4 (1:500; catalog number AB3672P; Millipore), anti-Oatp1b2 (1:100; catalog number sc-376904; Santa Cruz Biotechnology, Dallas, TX), anti-Mrp3 (1:1000; catalog number 399095; Cell Signaling Technology, Danvers, MA), anti-Ostβ antibody (1:150; catalog number HPA008533; Sigma-Aldrich), anti-Mrp2 (1:20; catalog number MC-206; Kamiya Biochemical Company, Seattle, WA), anti-Mdr1b (1:100; catalog number sc-55510; Santa Cruz Biotechnology), or anti-N+/K+ ATPase (1:5000; catalog number sc-28800; Santa Cruz Biotechnology) overnight in diluted 5% (w/v) bovine serum albumin-Tris–buffered saline Tween 20. After washing, the blots were probed with horseradish peroxidase–conjugated anti-rabbit (for Oatp1a1, Oatp1a4, Ostβ, and Mrp3; 1:7000; catalog number sc-2030; Santa Cruz Biotechnology) or horseradish peroxidase–conjugated anti-mouse IgG secondary antibody (for Oatp1b2, Mdr1, and Na+/K+ ATPase; 1:10,000; catalog number 115-035-003; Jackson ImmunoResearch, West Grove, PA) in 5% milk for 1 hour at room temperature. Detection was enhanced using chemiluminescence reagents (ECLTM Select; GE Healthcare Bio-Sciences, Piscataway, NJ) and measured with ImageLab software version 4.1 (Bio-Rad, Hercules, CA). The data were normalized to loading control (Na+/K+ ATPase) using densitometric analysis to compare protein expression between WT and PCK rats with ImageJ version 1.51 (National Institutes of Health).
Protease Digestion and Solid Phase Extraction.
As described previously (Malinen et al., 2019), membrane protein samples, as prepared for Western blot, were diluted to a concentration of 100 µg membrane protein/100 µl and mixed in ammonium bicarbonate buffer (25 mM) containing 5% deoxycholate and 10 mM dithiothreitol. After incubation for 40 min at 56°C, iodoacetamide was added (final concentration of 15 mM), and the sample was incubated for 30 min at room temperature. For protease digestion, samples were diluted 5-fold with ammonium bicarbonate buffer and digested using Lys-C protease (Thermo Fisher Scientific) in a protein/protease ratio of 20:1. After incubation for 4 hours at 37°C, a protein/trypsin ratio of 20:1 was applied followed by incubation overnight at 37°C. Formic acid was added (final concentration, 2% v/v) to stop the protease reaction. The mixture of stable isotope-labeled peptides (Table 1) was used as internal standards. For solid-phase extraction, the cartridge (Oasis HLB; Waters Co., Milford, MA) was prewashed with methanol and equilibrated with 0.1% formic acid in water (v/v) before the sample was added. After washing the cartridge with 0.1% formic acid in water (v/v), the peptides were eluted by adding 0.1% formic acid in acetonitrile (v/v). A centrifugal evaporator (SpeedVac High-Capacity Concentrator; Thermo Fisher Scientific) was used to evaporate the eluate, and samples were reconstituted in 0.1% formic acid and 2% acetonitrile in water (v/v/v). After centrifugation (21,000g, 1 minute), the supernatant was analyzed by LC-MS/MS.
Signature Peptide Selection and Quantification by LC-MS/MS.
Signature peptides and internal standard peptides for each transporter (Li et al., 2008, 2009; Uchida et al., 2011; Prasad et al., 2014; Wang et al., 2015) (Table 1) were analyzed by LC-MS/MS [Thermo Scientific TSQ Quantum Ultra Triple Quadrupole Mass spectrometer (Thermo Fisher Scientific) with NanoAcquity UPLC (Waters Co.)] in the selected reaction monitoring (SRM) mode with three sets of transitions. As described previously (Malinen et al., 2019), samples were injected onto the Waters Acquity UPLC M-Class Symmetry C18 Trap Column (5, 180 µm × 20 mm; Waters Co.) in 98% mobile phase A (0.1% formic acid in water) and 2% mobile phase B (0.1% formic acid in acetonitrile) (flow rate of 5 μl/min for 3 min). The peptides were first trapped and then separated on an analytical column (Waters Acquity UPLC HSS T3 NanoAcquity Column, 1.8, 100 µm × 100 mm; Waters Co.) at a flow rate of 0.6 μl/min, and a linear gradient of mobile phase B concentration of 5% for 0–1 minute, 5%–35% for 1–25 min, and 35%–90% for 25–25.5 min, followed by the washing step using 90% mobile phase B for 4.5 min, and re-equilibrium for 10 min. Xcalibur software (Thermo Fisher Scientific) was used to process the data by integrating the peak areas generated from the reconstructed ion chromatograms for the analyte peptides and their respective internal standards. Transporters were considered to be expressed when analyte peaks were detected in three sets of transitions. Then, the peak area ratios of the analyte peptide to its internal standard were calculated for three sets of transitions. To compare the expression levels of transporters among samples, the PCK/WT ratio was calculated with the peak area mean from each transition (n = 3) and compared with Na+/K+ ATPase PCK/WT ratio, assuming that there was no change in the Na+/K+ ATPase protein in PCK compared with WT rat livers. The corresponding peptides, heavy labeled at [13C615N2]-lysine (K*) and [13C615N4]-arginine (R*) residues, were used as the internal standards.
Total Glutathione Determination.
Total glutathione concentrations [reduced glutathione (GSH) plus glutathione disulfide (GSSG)] were determined according to the manufacturer instructions (CS0260; Sigma-Aldrich). Briefly, 10 µl of blank bile (collected before IPLs) and 40–200 mg of liver tissue were diluted with 5% sulfosalicylic acid solution. The solutions were vigorously mixed by vortex and left for 10 min on ice prior to centrifugation (10,000g) for 10 min at 4°C. The supernatant volume was measured and samples were loaded on a 96-well plate and incubated for 5 min with the working solution containing 95 mM potassium phosphate, 0.95 mM EDTA, 0.031 mg/ml 5,5′-dithiobis(2-nitrobenzoic acid), 0.115 U/ml glutathione reductase from Baker’s yeast, and 0.24% 5-sulfosalicylic acid. After adding NADPH to a final concentration of 0.038 mg/ml and mixing, the samples were read at 412 nm at 1-minute intervals for 5 min with a spectrophotometer (PowerWave XS; Biotek, Winooski, VT). Concentrations were calculated according to the manufacturer instructions.
CDF Disposition in Rat IPLs.
WT and PCK rat livers were perfused as described previously (Brouwer and Thurman, 1996; Chandra et al., 2005a). Briefly, animals were anesthetized with ketamine/xylazine (60:12 mg/kg, i.p.), and the bile duct and portal vein were cannulated. After cannulation and the collection of blank bile, the livers were perfused ex situ in a single-pass manner with continuously oxygenated Krebs-Henseleit buffer (38 ml/min). First, livers were perfused with blank perfusate for a 15-minute equilibration period, then with CDF (∼1 µM) for 30 min followed by perfusion for 20 min with CDF-free buffer. Outflow perfusate and bile were collected continuously. Outflow perfusate was collected for 50 min by sampling directly from the perfusate flowing out of the vena cava; bile was collected over 5-minute intervals for 50 min, and each sample was weighed to determine the total volume assuming a specific gravity of 1.0. Liver viability was monitored by gross morphology, portal perfusion pressure (<15 cm H2O), bile flow (>2 μl/min) (Zhang et al., 2016), and by lactate dehydrogenase (LDH) levels in the outflow perfusate measured by the Pierce LDH Cytotoxicity Assay Kit (Thermo Fisher Scientific) per the manufacturer instructions.
CDF Sample Preparation.
Perfusate and bile samples were thawed and diluted with buffer and analyzed for CDF by fluorescence spectroscopy using a Cytation 3 Imaging Reader (BioTek) at wavelengths of 485 nm (excitation) and 518 nm (emission). Two standard curves were prepared by diluting CDF in perfusate buffer, and blank bile was diluted in perfusate buffer (1:100). The lower limit of quantitation was 0.1 nM for the perfusate buffer matrix and 10 nM for the diluted blank bile matrix.
Pharmacokinetic Analysis.
The hepatobiliary disposition of CDF was analyzed using a pharmacokinetic modeling approach. The compartmental model, consisting of a sinusoidal, a hepatocellular, and a bile compartment (scheme depicted in Fig. 1), was fit simultaneously to the average CDF outflow perfusate and biliary excretion rate data from rat IPLs. Data from WT and PCK rat IPLs were fit separately. A combined additive and multiplicative residual error model (Phoenix WinNonlin version 8.1; Certara USA, Inc., Princeton, NJ) was used to estimate the parameters listed in Table 2. The bile compartment for PCK rat IPLs consisted of five transit compartments to describe the delay in the detection of CDF in bile. The differential equations, assuming linear conditions and an initial concentration of zero in all compartments, are as follows:
WT and PCK sinusoidal compartment:(1)WT and PCK hepatocellular compartment:(2)WT and PCK concentration data in outflow perfusate expressed as :(3)WT concentration data in bile expressed as :(4)PCK transit compartment 1 after biliary excretion:(5)PCK transit compartments 2–5 after biliary excretion:(6)PCK concentration data in bile expressed as :(7)where represents the rate of change of CDF in a specific compartment with respect to time, and , , , , , and represent the CDF mass in the sinusoidal space, hepatocellular space, outflow perfusate, bile (in the absence of delayed biliary transit), nth biliary transit compartment, and bile in the presence of delayed biliary transit, respectively. Other parameters are defined as follows:
: inflow concentration.
: uptake clearance from the sinusoidal space to the hepatocellular space.
: basolateral efflux rate constant from the hepatocellular space to the sinusoidal space.
:biliary efflux rate constant from the hepatocellular space to bile.
: transit rate constant for the biliary excretion of CDF.
, calculated as represents the extracellular concentration in the sinusoidal compartment and was assumed to be equal to The sinusoidal space was assumed to be in equilibrium with the extracellular volume of the liver, , and was estimated as 20% of the total liver mass (Watanabe et al., 2009; Hobbs et al., 2012), which was converted to total liver volume () using the average density of rat liver (1.084 g/ml) (Pretlow and Pretlow, 1987). The observed outflow perfusate rate data were described by the perfusate flow rate multiplied by the concentration in the sinusoidal compartment (Q ⋅ CS), whereas the observed biliary excretion rate data were described by and for WT and PCK rat IPLs, respectively.
Data Analysis.
All data are presented as the mean and S.D. from n = 3 livers per group (WT and PCK). Statistical tests [unpaired, two-tailed t tests (α = 0.05) corrected (only for proteomics data) by using the Bonferroni-Dunn multiple-comparison method] were performed using GraphPad Prism 7.0b for macOS (GraphPad Software, La Jolla, CA). Final parameter estimates were significantly different if the 95% confidence intervals (CIs) between the WT and PCK groups did not overlap (Schenker and Gentleman, 2001).
Results
Transporter Gene Expression in WT Compared with PCK Rat Livers.
The relative transporter gene expression in WT compared with PCK rat livers, assessed by quantitative polymerase chain reaction, is shown in Fig. 2. The mRNA levels of both Slco1a1 (Oatp1a1) and Slco1a4 (Oatp1a4) were significantly decreased in PCK rat livers by 2.6-fold and 2.5-fold, respectively. Slc51a (Ostα), Slc51b (Ostβ), and Abcc3 (Mrp3) mRNA levels were significantly increased by 88-fold, 28-fold, and 9-fold, respectively, in livers from PCK compared with WT rats. Abcc2 (Mrp2) and Slco1b2 (Oatp1b2) mRNA levels were decreased by 2.4-fold and 1.5-fold, and Abcb1b (Mdr1b) showed a 71-fold increase in PCK compared with WT rat livers, but differences were not statistically significant.
Transporter Protein Expression in WT Compared with PCK Rat Livers.
Protein expression levels were determined using Western blot analysis (Fig. 3) and LC-MS/MS proteomic analysis (Fig. 4). Immunoblot analysis of liver tissue from WT and PCK rats revealed a significant decrease in Mrp2 and Oatp1a4 (3.0-fold and 2.9-fold, respectively) (Fig. 3) in PCK rat livers. A 1.4-fold, 2.0-fold, and 2.3-fold increase in protein levels were detected in PCK rat livers for Mrp3, Ostβ, and Mdr1b, respectively, but these differences were not statistically significant (Fig. 3). In addition, protein levels of Oatp1a1 and Oatp1b2 were decreased by 1.6-fold and 1.7-fold, respectively, in PCK rat livers, but differences were not significant (Fig. 3). Proteomics data showed a decrease in Oatp1a1, Ntcp, and Mrp2 with a PCK/WT ratio (mean ± S.D.) of 0.563 ± 0.038, 0.691 ± 0.097, and 0.368 ± 0.098, respectively, but only Oatp1a1 and Mrp2 were significantly different (Fig. 4). Bsep protein expression was unchanged in PCK compared with WT rat livers (Fig. 4). Oatb1b2, Oatp1a4, and Mrp3 could not be detected with the selected sequence peptides (Table 1).
Total Glutathione (GSH + GSSG) Concentrations in Bile and Livers from WT and PCK Rats.
Biliary total glutathione concentrations were reduced in PCK compared with WT rats (Fig. 5A). No significant changes in hepatic total glutathione concentrations were detected in PCK compared with WT rats (Fig. 5B).
Outflow Perfusate and Biliary Excretion Rates of CDF in Ex Situ IPLs from WT and PCK Rats.
LDH release from IPLs into outflow perfusate was negligible (<2% cytotoxicity; data not shown). To investigate whether changes in transporter expression in PCK rat livers affected substrate transport at a functional level, the hepatobiliary disposition of CDF was measured in ex situ IPLs. Although the body weight of WT and PCK rats was similar (458 ± 34 and 495 ± 3.6 g, respectively), PCK rat livers (28.1 ± 9.1 g) were approximately 2-fold larger than WT rat livers (15.4 ± 1.0 g). CDF outflow perfusate excretion rate versus time data were similar between WT and PCK rat IPLs, but the biliary excretion rates were markedly reduced in PCK IPLs (Fig. 6). The first four data points for the biliary excretion rates in PCK rats were below the limit of quantitation and are, therefore, not shown in Fig. 6.
Pharmacokinetic Modeling.
The WT and PCK outflow perfusate and biliary excretion rate versus time data were well described by a model consisting of a single hepatocellular compartment (Figs. 1 and 6). A lag time for the movement of CDF from the hepatocellular space into bile was incorporated only in the model describing the PCK rat IPL data. The CDF KBile was significantly decreased in PCK IPLs by more than an order of magnitude (Table 2). The CLUP was significantly decreased (∼2 fold) and the KBL of CDF was significantly increased (∼2 fold) in PCK IPLs.
Discussion
Previous studies suggest that the hepatobiliary disposition of endogenous and exogenous substrates may be altered in rodents with PKD. For example, various bile acids including unconjugated chenodeoxycholic acid (CDCA), deoxycholic acid (DCA), and lithocholic acid, and the glycine and taurine conjugates of CDCA and DCA are significantly increased in the serum and liver of PCK rats (Munoz-Garrido et al., 2015; Brock et al., 2018), a rodent model of PKD (Katsuyama et al., 2000; Masyuk et al., 2004). Although no significant increases in ALT, aspartate aminotransferase, or mRNA of the bile acid synthesis enzyme Cyp7a1 were detected in PCK rats (Munoz-Garrido et al., 2015), serum bilirubin concentrations were increased in PCK rats, which is suggestive of impaired Mrp2 function (Mason et al., 2010; Shimomura et al., 2015). Previous published data have shown that liver disease can affect hepatic transporter expression and function (Thakkar et al., 2017). The present study is the first to report altered protein expression and function of hepatic transporters in PCK rat livers.
The significant decrease in protein levels of Oatps in PCK rat livers was notable; Oatp1a4 measured by immunoblot and Oatp1a1 measured by immunoblot and LC-MS/MS were both decreased (Figs. 3 and 4). Decreased Oatp protein levels in PCK rat livers could have a significant impact on the hepatic disposition of Oatp substrates in this rodent model. The clinical implications of decreased hepatic OATP protein levels could lead to altered efficacy or toxicity of some drugs, particularly in patients with ADPKD. This warrants further investigation. Downregulation of Abcc2 gene expression and a 3.0-fold decrease in Mrp2 protein levels were observed in PCK compared with WT rat livers (Figs. 2–4). In contrast, Abcc3 gene expression was significantly increased in PCK rat livers. However, due to variability in protein levels, Mrp3 protein was not significantly different (Figs. 2 and 3).
Biliary concentrations of endogenous total glutathione (GSH + GSSG) were markedly reduced in PCK rats (Fig. 5A), consistent with decreased Mrp2 protein levels. Similar results were observed in Mrp2-deficient TR− rats (Elferink et al., 1989). Interestingly, hepatic total glutathione concentrations were not significantly different in PCK compared with WT rats. Liver weights were greater in PCK rats, which might be due, in part, to the hepatic cysts that amount to 3.92 ml, on average, in 16-week-old PCK rats (Brock et al., 2018), or up to 15% of the liver tissue (Munoz-Garrido et al., 2015).
Altered hepatobiliary disposition of CDF in PCK IPLs clearly demonstrated that changes in protein expression translated to functional changes. CDF is a fluorescent anion that is excreted unchanged into bile by Mrp2 in rats (Zamek-Gliszczynski et al., 2003). CDF is also a substrate for Oatp1a1-mediated hepatic uptake and undergoes basolateral efflux by Mrp3 when Mrp2 function is impaired (Xiong et al., 2000; Zamek-Gliszczynski et al., 2003; Chandra et al., 2005b; Nezasa et al., 2006). The concentration of CDF (∼1 µM) used in the IPL studies was well below the reported Km (Lineweaver-Burk constant) value (22 ± 10 µM) for uptake in rat hepatocytes (Zamek-Gliszczynski et al., 2003). A pharmacokinetic model assuming linear disposition was used to evaluate changes in the hepatic uptake, biliary excretion, and basolateral efflux of CDF. In general, the model adequately described the data with low coefficient of variation (CV%) (<50%). Pharmacokinetic modeling revealed that the hepatic uptake clearance and biliary excretion rate constant of CDF were decreased by 1.9-fold and 28-fold, respectively, consistent with the observed decrease in Oatp1a1 and Mrp2 protein levels in PCK rat livers (Fig 3 and 4; Table 2). Interestingly, incorporation of a lag time for the movement of CDF from the liver to bile was required only in the PCK rat livers to adequately describe the data. One possible explanation for the lag time may be the dilated cystic bile ducts in PCK rats (Masyuk et al., 2004). In addition, the basolateral efflux rate constant of CDF, presumably mediated by Mrp3, was increased by 2-fold. Mrp3 serves as a compensatory route for the excretion of Mrp2 substrates when biliary excretion is impaired ((König et al., 1999); Ogawa et al., 2000; Chandra and Brouwer, 2004).
These results reveal that protein levels and the function of hepatic drug transporters are altered in PCK rats. If similar alterations in hepatic transporters occur in humans with PKD, these patients may be predisposed to liver injury by drugs that inhibit hepatic transporters, such as tolvaptan (Slizgi et al., 2016). Patients with ADPKD treated with tolvaptan have an increased risk of drug-induced liver injury and may have altered drug disposition compared with the population without ADPKD (Torres et al., 2012, 2017).
In conclusion, these novel findings indicate that significant alterations in hepatic transporter expression and function in PCK rat livers lead to decreased hepatic uptake and impaired biliary excretion of CDF, a probe substrate of organic anion transporters. Impaired Oatp-mediated hepatic uptake and Mrp2-mediated biliary excretion of drugs and metabolites have important implications for the use of this rodent model in drug development investigations. Furthermore, altered hepatic transporter function can influence the hepatobiliary disposition of endogenous (e.g., bile acids) and exogenous compounds, and may be a predisposing risk factor in drug-induced liver injury.
Acknowledgments
The authors acknowledge Certara for providing academic research licenses for Phoenix software as part of the Center of Excellence program for educational institutions. Liver tissues from WT and PCK rats (16 weeks) were obtained from experiments conducted by Dr. Jason R. Slizgi. We acknowledge the support provided by the Biomarkers Mass Spectrometry sub-core supported in part by NIEHS grant P30ES010126 (J. Swenberg). We thank Dr. Noora Sjöstedt for her support with the visual abstract.
Authorship Contributions
Participated in research design: Bezençon, Beaudoin, Roth, Brock, and Brouwer.
Conducted experiments: Bezençon, Beaudoin, and Ito.
Performed data analysis: Bezençon, Beaudoin, Ito, Fu, Roth, Brock, and Brouwer.
Wrote or contributed to the writing of the manuscript: Bezençon, Beaudoin, and Brouwer.
Footnotes
- Received February 26, 2019.
- Accepted May 30, 2019.
This work was supported, in part, by Otsuka Pharmaceutical Development & Commercialization, Inc., and the National Institutes of Health National Institute of General Medical Sciences [Grants R01 GM041935 and R35 GM122576].
This work was presented, in part, at the 10th Biomedical Transporter Conference, the American Association of Pharmaceutical Scientists Transporter Workshop 2018, and the 2018 Drug Metabolism Gordon Research Conference. Any opinions, findings, conclusions, or recommendations expressed in this publication are those of the authors and do not necessarily reflect the views of the Otsuka Pharmaceutical Group or the National Institutes of Health.
Abbreviations
- ADPKD
- autosomal dominant polycystic kidney disease
- ALT
- alanine aminotransferase
- ARPKD
- autosomal recessive polycystic kidney disease
- Bsep
- bile salt export pump
- CDCA
- chenodeoxycholic acid
- CDF
- 5(6)-carboxy-2′,7′-dichlorofluorescein
- CLUP
- uptake clearance
- CV
- coefficient of variation
- DCA
- deoxycholic acid
- Gapdh
- glyceraldehyde 3-phosphate dehydrogenase
- GSH
- reduced glutathione
- GSSG
- glutathione disulfide
- IPL
- isolated perfused liver
- KBile
- biliary efflux rate constant
- KBL
- basolateral efflux rate constant
- KLag,Bile
- transit rate constant for biliary excretion
- LC-MS/MS
- liquid chromatography-tandem mass spectroscopy
- LDH
- lactate dehydrogenase
- MDR/Mdr
- multidrug resistance
- MRP/Mrp
- multidrug resistance–associated protein
- NTCP/Ntcp
- Na+-taurocholate cotransporting polypeptide
- OATP/Oatp
- organic anion transporting polypeptide
- Ost
- organic solute transporter
- PCK
- polycystic kidney
- PKD
- polycystic kidney disease
- PKD1/2
- Polycystic Kidney Disease 1/2
- PKHD1
- Polycystic Kidney and Hepatic Disease 1
- SRM
- selected reaction monitoring
- WT
- wild-type
- Copyright © 2019 by The American Society for Pharmacology and Experimental Therapeutics