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. Author manuscript; available in PMC: 2019 Mar 1.
Published in final edited form as: Int J Toxicol. 2018 Mar-Apr;37(2):144–154. doi: 10.1177/1091581818760746

Bile Acids as Potential Biomarkers to Assess Liver Impairment in Polycystic Kidney Disease

William J Brock 1, James J Beaudoin 2, Jason R Slizgi 2, Mingming Su 3, Wei Jia 3, Sharin E Roth 4, Kim L R Brouwer 2,*
PMCID: PMC5877312  NIHMSID: NIHMS939548  PMID: 29587557

Abstract

Polycystic kidney disease is characterized by the progressive development of kidney cysts and declining renal function with frequent development of cysts in other organs including the liver. The polycystic kidney (PCK) rat is a rodent model of polycystic liver disease that has been used to study hepatorenal disease progression and evaluate pharmacotherapeutic interventions. Biomarkers that describe the cyst progression, liver impairment, and/or hepatic cyst burden could provide clinical utility for this disease. In the present study, hepatic cyst volume was measured by magnetic resonance imaging in PCK rats at 12, 16, and 20 weeks. After 20 weeks, Sprague-Dawley (n=4) and PCK (n=4) rats were sacrificed and 42 bile acids were analyzed in the liver, bile, serum, and urine by LC-MS/MS. Bile acid profiling revealed significant increases in total bile acids (molar sum of all measured bile acids) in the liver (13-fold), serum (6-fold) and urine (3-fold) in PCK rats, including those speciated bile acids usually associated with hepatotoxicity. Total serum bile acids correlated with markers of liver impairment [liver weight, total liver bile acids, total hepatotoxic liver bile acids, and cyst volume (r > 0.75; p < 0.05)]. Based on these data, serum bile acids may be useful biomarkers of liver impairment in polycystic hepatorenal disease.

Keywords: polycystic kidney disease, bile acids, hepatotoxicity, biomarkers

Introduction

Polycystic kidney disease (PKD) is a class of ciliopathic disorders that arise from abnormalities in the primary cilium in many epithelial organs that lead to the development of fluid-filled cysts.1 Autosomal dominant polycystic kidney disease (ADPKD) is the most common form of PKD.2–4 Although, the destruction/displacement of renal tubules as a result of cyst growth is central to the morbidity and mortality in patients with ADPKD, cysts may occur in other epithelial organs including the spleen, pancreas, lungs, and liver.5–8 The liver is the more frequently affected extrarenal organ in ADPKD.9 However, as with many liver diseases, typical biochemical measures of liver impairment are not always sufficiently sensitive or specific to characterize the degree of liver dysfunction.10–13 Over the last decade, there has been considerable effort within the pharmaceutical industry, in cooperation with regulatory authorities, to identify more sensitive and/or specific molecular biomarker(s) of liver dysfunction. Several of those biomarkers continue to be examined in preclinical studies including miR-122, Cytokeratin 18 (CK-18), total and hyperacetylated high mobility group protein B1 (HMGB1), osteopontin, macrophage colony-stimulating factor 1 receptor (CSF1R), and speciated bile acid profiles.14–16

Several rodent models of PKD have been described with some animal models displaying isolated renal disease whereas other animal models exhibit hepatic abnormalities in addition to renal disease.17,18 In 2000, a spontaneous mutation in a colony of Sprague-Dawley rats displaying bilateral renal and hepatic cysts, now known as the polycystic kidney (PCK) rat, was described by Katsuyama et al.19 Although the genetic inheritance of PKD in PCK rats is autosomal recessive, the renal and hepatic histopathological features resemble human ADPKD.20 For example, the kidney in PCK rats and human ADPKD are normal at birth but exhibit slow, progressive renal cyst growth. Furthermore, liver ductal plate abnormalities, biliary dysgenesis, and hepatic cyst development in PCK rats are consistent with the natural history of human ADPKD.20–22 Hence, the PCK rat has been a valuable tool to understand the progression of ADPKD as well as to evaluate therapeutic interventions.20,23,24 Therefore, the PCK rat model was selected in the present study to evaluate the association between bile acids as potential biomarkers and liver impairment in PKD.

Bile acids are a diverse, but a structurally similar group of cholesterol-derived acidic steroids important in digestion/absorption and normal hepatic function. Bile acids also act as signaling molecules and play a critical role in the regulation of lipid metabolism and inflammation.25 Bile acids have been evaluated as biomarkers in various liver diseases such as cirrhosis, hepatocellular carcinoma, and nonalcoholic steatohepatitis.26–28 The purpose of the experiments described in the present study was to examine bile acids as a potential marker of liver injury and investigate the association between bile acid concentrations and hepatic cyst involvement in the PCK rodent model of PKD.

Materials and Methods

Materials

All bile acid standards were obtained from Steraloids Inc. (Newport, RI) and TRC Chemicals (Toronto, ON, Canada). Nine stable isotope-labeled standards were obtained from C/D/N Isotopes Inc. (Quebec, Canada) and Steraloids Inc. (Newport, RI). Methanol (Optima LC-MS), acetonitrile (Optima LC-MS) and formic acid (Optima LC-MS) were purchased from Thermo-Fisher Scientific (FairLawn, NJ). Ultrapure water was produced by a Mill-Q Reference system equipped with a LC-MS Pak filter (Millipore, Billerica, MA).

Animals

Male Sprague-Dawley or polycystic kidney disease (PCK/Crlj Crl-Pkhd1pck/Crl) rats were purchased from Charles River Laboratories (Wilmington, MA). Rats were approximately 5-6 weeks of age at receipt and were housed in a constant alternating 12-hr light/dark cycle and allowed water and food ad libitum and acclimated for a minimum of one 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).

Overview of study design

The livers of Sprague-Dawley and PCK rats were scanned by magnetic resonance imaging at 12, 16 and 20 weeks of age. At 72 hr following the scan of 20-week old rats, samples of liver, bile and blood were collected for bile acid profiling as detailed below (n=4 rats/cohort except n=3 for bile collection in Sprague-Dawley rats).

Magnetic resonance imaging (MRI)

Images were acquired with a 9.4T Bruker Biospec 94/30USR (Bruker BioSpin, Billerica, MA) controlled with ParaVision software (v5.1. Billerica, MA). Rats were anesthetized with 2% isoflurane in an air/oxygen mix. Respiratory-gated axial images were acquired with a 2D FLASH sequence with a repetition time of 533ms and an echo time of 10ms. Liver or cyst volumes were calculated by segmenting and measuring the areas of each region of interest on each slice using ImageJ 1.48v (National Institutes of Health, USA), then summing the areas and multiplying by the slice thickness. Axial slices covered a 6×6 cm2 field of view with a 256×256 matrix at a separation of 2 mm and utilized two averages. To cover the entire liver, 24 to 30 slices were acquired.

Sample collection

At 72 hr following the last MRI at 20 weeks, the rats were fasted overnight and anesthetized with ketamine:xylazine (60:12 mg/kg, intraperitoneal injection). Following anesthesia, bile was collected by cannulating the bile duct using PE-10 tubing, and urine was collected from the bladder by cystocentesis. Subsequently, the rats were sacrificed and liver samples were collected from the median lobe. Whole blood was collected and centrifuged at 1500g for 10 min to collect serum. All samples were immediately frozen at −80°C until analysis.

Sample preparation and bioanalysis

Sample preparation and bile acid quantification methodology were adapted from Xie et al.29 Briefly, an internal standard solution containing nine internal standards was added to serum, urine, bile, liver homogenate, or standard solution and centrifuged. The internal standards consisted of 100 nM for d4-glycocholic acid (GCA), d4-taurocholic acid (TCA), d9-taurochenodeoxycholic acid (TCDCA), d4-ursodeoxycholic acid (UDCA), d4-cholic acid (CA), d4-glycochenodeoxycholic acid (GCDCA), d4-glycodeoxycholic acid (GDCA), d4-deoxycholic acid (DCA), and 200 nM for d4-lithocholic acid (LCA) (see Table 1 for bile acid abbreviations). Liver tissue was homogenized with 100 μL of 50% methanol using a Bullet Blender Tissue Homogenizer (Next Advance, Inc., Averill Park, NY). After centrifugation, the supernatant was evaporated to dryness using a freeze dryer system (Labconco, Kansas City, MO). The residue was reconstituted in 1:1 (v/v) mobile phase B (95:5, v/v, acetonitrile/methanol) and mobile phase A (water with formic acid, pH=3.25), and centrifuged at 13,500g (4°C) for 20 min. The supernatant was transferred to a 96-well plate for analysis. An ultra-performance liquid chromatography coupled to tandem mass spectrometry (UPLC-MS/MS) system (ACQUITY UPLC-Xevo TQ-S, Waters Corp., Milford, MA) was used to quantify bile acids in the rat bile, serum, urine, and liver homogenate samples. Calibration solutions containing all analytes were prepared as a series of concentrations in pooled naïve serum and urine depleted of bile acids with activated charcoal, and were utilized to generate standard curves using TargetLynx application manager (Waters Corp, Milford, MA). The lower limit of quantification (LLOQ) was 1.024 nM for all bile acid species, except for an LLOQ of 2.56 nM for 3β-cholic acid (bCA), murocholic acid (muroCA), GDCA, GCDCA, UDCA, hyodeoxycholic acid (HDCA), 6-ketoLCA, tauroursodeoxycholic acid (TUDCA), taurohyodeoxycholic acid (THDCA) and chenodeoxycholic acid 24-acyl-β-D-glucuronide (CDCA-24G). The LLOQ for tauro-α-muricholic acid (TαMCA), tauro-β-muricholic acid (TβMCA), tauro-ω-muricholic acid (TωMCA) and TCA was 12.8 nM.

Table 1.

Abbreviations of Bile Acids and Bile Acid Species

Abbreviation Bile acid Species
12-ketoLCA 12-ketolithocholic acid DCA species
3-dehydroCA 3-dehydrocholic acid CA species
6-ketoLCA 6-ketolithocholic acid HDCA species
7-dehydroCA 7-ketodeoxycholic acid CA species
7-ketoLCA 7-ketolithocholic acid CDCA species
alloCA Allocholic acid CA species
alloLCA Allolithocholic acid LCA species
bCA 3β-cholic acid CA species
CA Cholic acid CA species
CDCA Chenodeoxycholic acid CDCA species
CDCA-24G Chenodeoxycholic acid 24-acyl-β-D-glucuronide CDCA species
CDCA-3G Chenodeoxycholic acid-3-β-D-glucuronide CDCA species
DCA Deoxycholic acid DCA species
dehydroLCA Dehydrolithocholic acid LCA species
GCA Glycocholic acid CA species
GCDCA Glycochenodeoxycholic acid CDCA species
GDCA Glycodeoxycholic acid DCA species
GHCA Glycohyocholic acid MCA species
GHDCA Glycohyodeoxycholic acid HDCA species
GLCA Glycolithocholic acid LCA species
GUDCA Glycoursodeoxycholic acid UDCA species
HCA Hyocholic acid MCA species
HDCA Hyodeoxycholic acid HDCA species
LCA Lithocholic acid LCA species
LCA-S Lithocholic acid 3-sulfate LCA species
muroCA Murocholic acid HDCA species
norCA 23-norcholic acid CA species
norDCA 23-nordeoxycholic acid DCA species
TCA Taurocholic acid CA species
TCDCA Taurochenodeoxycholic acid CDCA species
TDCA Taurodeoxycholic acid DCA species
THDCA Taurohyodeoxycholic acid HDCA species
TLCA Taurolithocholic acid LCA species
TUDCA Tauroursodeoxycholic acid UDCA species
TαMCA Tauro-α-muricholic acid MCA species
TβMCA Tauro-β-muricholic acid MCA species
TωMCA Tauro-ω-muricholic acid MCA species
UCA Ursocholic acid CA species
UDCA Ursodeoxycholic acid UDCA species
αMCA α-muricholic acid MCA species
βMCA β-muricholic acid MCA species
ωMCA ω-muricholic acid MCA species
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CA, CDCA, GCA, GCDCA, GHCA, HCA, TCA, TCDCA, TαMCA, TβMCA, αMCA and βMCA are primary bile acid species. The remaining bile acids are secondary bile acids.

Data and statistical analysis

The molar sum of all measured bile acids is reported as total bile acids. Total unconjugated and glycine- and taurine-conjugated bile acids were calculated as the sum of all unconjugated or glycine- or taurine-conjugated species of CA, CDCA, HDCA, muroCA, DCA, LCA, UDCA and hyocholic acid (HCA). The relative abundance of each group of bile acid species was calculated as the total molar concentration for that particular species divided by the total bile acid molar concentration. Measured bile acid concentrations below the LLOQ, but above the limit of detection (LOD), were imputed as one-half of the LLOQ. Concentrations measured below the LOD were imputed as zero. Statistical significance was determined using the nonparametric Mann-Whitney U test (α=0.05) to compare PCK versus Sprague-Dawley rats. Principal component analysis (PCA) was performed to visualize the bile acid metabolome in PCK compared to Sprague-Dawley rats (SIMCA 14, Umetrics, Umea, Sweden). Spearman correlation coefficients (two-tailed) were determined using GraphPad Prism (v.6.0 GraphPad Software, La Jolla, CA) where p-values ≤ 0.05 were considered statistically significant.

Results

Hepatic Characteristics in Sprague-Dawley and PCK Rats

Hepatic cyst volume was measured at 12, 16, and 20 weeks in PCK rats to monitor liver cyst progression over time. These time frames are consistent with the presentation of hepatic cysts and the progression of cystic disease and liver dysfunction in PCK rats.19,20 Furthermore, mortality in PCK rats is high after 20 weeks of age due to progression of the disease.19

The cyst volume, determined from the MRI images in PCK rats, increased approximately 85% from week 12 to week 20 (Table 2). Representative MRI images at 20 weeks are shown in Fig. 1. At 20 weeks, liver and total kidney weights were significantly increased in PCK rats compared to Sprague-Dawley rats (Table 2).

Table 2.

Body Weight, Organ Weight, and Estimated Cyst Volume in Sprague Dawley and PCK Rats at 20 Weeks of Age

Parameter Sprague-Dawley PCK
Body weight (g) 409 ± 87 501 ± 21
Liver weight (g) 10.1 ± 1.9 20.2 ± 3.5*
Liver weight/ body weight (%) 2.47 ± 0.17 4.03 ± 0.74*
Total kidney weight (g) 2.50 ± 0.30 6.97 ± 0.45*
Total kidney weight/ body weight (%) 0.611 ± 0.069 1.39 ± 0.18*
Estimated cyst volume a (mL) N/A 5.20 ± 2.61
Week 12 N/A 2.83 ± 1.42
Week 16 N/A 3.92 ± 1.98
Week 20 N/A 5.20 ± 2.61
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Data represent the mean ± standard deviation (n=4/cohort).

*

p < 0.05, Sprague Dawley vs. PCK rats. N/A: no liver cysts were apparent in Sprague Dawley rats.

a

Estimated using MRI as described in Materials and Methods.

Figure 1.

Figure 1

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Representative magnetic resonance images of the hepatic parenchyma and cysts (white) in (A) Sprague-Dawley and (B-D) three different polycystic kidney (PCK) rats at 20 weeks of age.

The greater body weights, liver weights and kidney weights observed in the PCK rat are considered to be associated with the progression of the liver cysts. Although Fig. 1 is representative of the MRI images in the PCK rat at 20 weeks, there was some variation in the occurrence of liver cysts (total cyst burden) observed in each of the PCK rats at the same timepoint as measured by MRI. Regardless, there was progression (increased cyst burden) in all PCK rats. An increase in liver cyst burden with clinical disease progression and age is consistent with patients that present with polycystic liver and kidney disease.30–32

Bile Acid Composition

After overnight fasting conditions, 42 bile acids were measured in the liver, bile, serum, and urine of 20-week old Sprague-Dawley and PCK rats. Total liver and serum bile acids were significantly increased in PCK rats by ~13- and ~6-fold compared to Sprague-Dawley rats, respectively (p < 0.05; Fig. 2A and 2C). Total urine bile acids also were increased ~3-fold in PCK rats, but the difference did not reach statistical significance (Fig. 2D). There was no apparent difference in total biliary bile acids (Fig. 2B). Statistically significant differences using the nonparametric Mann-Whitney U test were not evaluated for bile samples due to the small sample size (n=3). As shown, the difference in liver bile acids was driven by an approximate 8-fold or greater increase in unconjugated, taurine- and glycine-conjugated bile acids (p < 0.05; Fig. 3A). Bile acids in the serum of PCK rats (Fig. 3C) were significantly increased (p < 0.05). Also, unconjugated bile acids appeared to increase to a greater extent (~7-fold) than taurine and glycine conjugates (~4- and ~6-fold). In urine, taurine and glycine conjugates increased by ~7- and ~6-fold, respectively, compared to an approximate 3-fold increase in unconjugated bile acids in PCK rats (Fig. 3D). Interestingly, no apparent differences in unconjugated or glycine conjugated bile acids were observed in the bile of Sprague-Dawley and PCK rats.

Figure 2.

Figure 2

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Total bile acid concentrations in the (A) liver, (B) bile, (C) serum, and (D) urine in Sprague-Dawley (white) and polycystic kidney (PCK) (grey) rats. Data represent the mean, first and third quartiles, and the maximum and minimum values (n=4/cohort except n=3 in bile of Sprague-Dawley). Individual values are shown (black circles); * p < 0.05, Sprague-Dawley vs. PCK rats.

Figure 3.

Figure 3

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Unconjugated and conjugated bile acids in the (A) liver, (B) bile, (C) serum, and (D) urine of Sprague-Dawley (white) and polycystic kidney (PCK) (grey) rats. Data represent the mean, first and third quartiles, and the maximum and minimum values (n=4/cohort except n=3 in bile of Sprague-Dawley). Individual values are shown (black circles); * p < 0.05, Sprague-Dawley vs. PCK rats.

Hepatic sulfate (i.e., lithocholic acid 3-sulfate) and glucuronide [i.e., CDCA-24G and CDCA 3-acyl-β-D-glucuronide (CDCA-3G)] bile acid conjugates also were significantly increased in PCK rats by ~5-fold and ~7-fold, respectively (p < 0.05; Supplementary Table 1). Serum and urine glucuronide conjugates also were significantly increased by ~12-fold and ~5-fold (p < 0.05), respectively, in PCK rats. Serum and urine sulfate conjugates appeared to increase in PCK rats but differences did not reach statistical significance. No significant differences were apparent in biliary sulfate or glucuronide bile acid conjugates.

The relative abundance of total unconjugated and conjugated bile acids for each of the following bile acid species in each matrix was compared between Sprague-Dawley and PCK rats (expressed as a percentage of the total molar concentration): CA, CDCA, DCA, HDCA, LCA, MCA and UDCA (Table 1). A few notable differences between Sprague-Dawley and PCK rats were observed (Table 3). For example, the relative abundance of CDCA, DCA and LCA species identified in Table 1 decreased in the liver of PCK rats, whereas HDCA species increased ~13-fold (p < 0.05; Table 3). The relative abundance of CDCA and LCA species also decreased significantly in the serum of PCK rats, whereas HDCA species increased ~24-fold (p < 0.05). In urine, the relative abundance of CA species significantly decreased in PCK rats (p < 0.05). Secondary bile acids [e.g., glycohyodeoxycholic acid (GHDCA), HDCA] were among the bile acid species that displayed the greatest fold change in PCK compared to Sprague-Dawley rats (Supplementary Table 2).

Table 3.

Relative Abundance of Bile Acids in Sprague Dawley and PCK Rats as a Percentage of Total Bile Acid Concentrations in Each Matrix

Matrix Bile Acid Species Sprague-Dawley
(% of total)		 PCK
(% of total)
Liver CA species 55.111 ± 10.543 44.845 ± 5.685
CDCA species 9.531 ± 3.947 5.802 ± 0.695*
DCA species 10.743 ± 4.258 5.433 ± 1.897*
HDCA species 1.392 ± 0.323 17.628 ± 3.433*
LCA species 1.788 ± 0.224 0.398 ± 0.160*
MCA species 19.829 ± 4.429 24.822 ± 7.839
UDCA species 1.605 ± 0.571 1.071 ± 0.166
Bile CA species 64.959 ± 6.887 48.736 ± 4.633
CDCA species 11.399 ± 3.933 5.893 ± 1.102
DCA species 6.387 ± 2.253 5.671 ± 1.100
HDCA species 1.203 ± 0.054 17.167 ± 4.000
LCA species 0.656 ± 0.131 0.262 ± 0.052
MCA species 14.171 ± 4.470 21.234 ± 5.652
UDCA species 1.225 ± 0.372 1.036 ± 0.039
Serum CA species 56.131 ± 13.481 35.281 ± 6.634
CDCA species 11.211 ± 9.440 3.297 ± 0.898*
DCA species 6.310 ± 0.740 5.178 ± 0.897
HDCA species 1.397 ± 0.258 34.089 ± 6.913*
LCA species 0.470 ± 0.111 0.166 ± 0.040*
MCA species 22.905 ± 4.910 20.673 ± 4.204
UDCA species 1.577 ± 0.538 1.317 ± 0.117
Urine CA species 57.001 ± 5.754 37.826 ± 2.860*
CDCA species 2.696 ± 1.737 2.865 ± 1.477
DCA species 2.149 ± 1.329 1.584 ± 0.342
HDCA species 11.003 ± 8.165 20.814 ± 3.796
LCA species 1.204 ± 0.675 0.621 ± 0.070
MCA species 24.317 ± 10.581 35.433 ± 6.151
UDCA species 1.630 ± 1.384 0.857 ± 0.203
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Data are presented as mean ± standard deviation (n=4/cohort except n=3 in bile of Sprague-Dawley rats).

*

p < 0.05, Sprague-Dawley vs. PCK rats. See Table 1 for abbreviations of bile acids.

Bile Acids Associated with Hepatotoxicity

Due to the detergent-like effects, bile acid hydrophobicity has been associated with liver injury.33 Generally recognized hepatotoxic bile acids include DCA and CDCA.34 Unconjugated and glycine- and taurine-conjugated DCA and CDCA species are relatively hydrophobic,35 but it is unclear whether each conjugated species is hepatotoxic. However, hepatotoxicity of glycine-conjugated DCA and CDCA has been shown.36,37 In addition, the hydrophobic bile acid LCA has been reported to be hepatotoxic.38,39 When bile acids associated with hepatotoxicity (i.e., DCA, GDCA, CDCA, GCDCA, and LCA] were analyzed separately (Supplementary Table 3), total toxic bile acids were significantly increased in PCK rat liver, serum and urine by ~5-fold, 3-fold and 6-fold, respectively (Fig.4A, 4C and 4D). GDCA, GCDCA and LCA were significantly increased by ~11-fold, 23-fold and 3-fold in the liver of PCK rats, respectively, whereas in serum, DCA, GDCA, CDCA, GCDCA, and LCA were significantly increased (i.e., 2-to 6-fold). In urine, DCA was significantly increased by ~30-fold. No notable differences were observed in biliary hepatotoxic bile acids (Fig. 4B).

Figure 4.

Figure 4

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Bile acids associated with hepatotoxicity [i.e., deoxycholic acid (DCA), glycodeoxycholic acid (GDCA), chenodeoxycholic acid (CDCA), glycochenodeoxycholic (GCDCA), lithocholic acid (LCA)] in the (A) liver, (B) bile, (C) serum, and (D) urine of Sprague-Dawley (white) and polycystic kidney (PCK) (grey) rats. Data represent the mean, first and third quartiles, and the maximum and minimum values (n=4/cohort except n=3 in bile of Sprague-Dawley). Individual values are shown (black circles); * p < 0.05, Sprague-Dawley vs. PCK rats.

Principal Component Analysis (PCA)

Separate PCA score plots for bile acids in each matrix were used to evaluate differences in the profile of individual bile acid species in Sprague-Dawley and PCK rats. PCA score plots of bile acid profiles indicated that PCK rats were clearly different from Sprague-Dawley rats (liver cumulative r2 = 0.874; bile cumulative r2 = 0.707; serum cumulative r2 = 0.804; urine cumulative r2 = 0.652) under fasting conditions (Fig. 5).

Figure 5.

Figure 5

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Score plots of the Principal Component Analysis models of bile acid profiles in the (A) liver, (B) bile, (C) serum, and (D) urine of Sprague-Dawley (black circles) and polycystic kidney (PCK) (open circles) rats.

Correlation Analysis

Correlation analysis was used to evaluate the relationship between serum bile acids and liver weight, total liver bile acids, total toxic liver bile acids, and cyst volume to test the hypothesis that serum bile acids correlated with markers of liver impairment (α = 0.05). Serum bile acids were positively correlated with liver weight (r = 0.92), total liver bile acids (r = 0.98), total toxic liver bile acids (r = 0.76), and cyst volume (r = 0.94) (Fig. 6); p-values were < 0.05. Total serum bile acids associated with hepatotoxicity were also positively correlated with liver weight (r = 0.96) and cyst volume (r = 0.94) (Fig. 6); p-values were < 0.01. After adjusting using the Bonferroni correction, p-values remained statistically significant, except for the correlation between total serum bile acids and total toxic liver bile acids.

Figure 6.

Figure 6

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Correlation between (A) liver weight, (B) total liver bile acids, (C) total liver bile acids associated with hepatotoxicity, (D) cyst volume and total serum bile acids; and between (E) liver weight, (F) cyst volume and total serum bile acids associated with hepatotoxicity in Sprague-Dawley (black circles) and polycystic kidney (PCK) (open circles) rats (n=4/cohort). The best-fit line is represented by the solid black line. Cyst volume was assumed to be zero in Sprague-Dawley rats.

Discussion

Total serum bile acid concentrations have been used as a clinical index of liver pathology although the current trend is to use speciated bile acid concentrations as a marker for possible hepatotoxicity. In this manuscript, we examined the relationship between speciated serum and liver bile acids, including those associated with hepatotoxicity, as possible biomarkers of liver impairment in a rodent model of PKD. As shown, total liver bile acids were increased by ~13-fold in PCK rats compared to Sprague-Dawley rats (Fig. 2A). Munoz-Garrido et al.40 reported that total hepatic bile acids were elevated in PCK rats, a finding that is in agreement with the current data. Elevated total hepatic bile acids are consistent with cholestasis in PCK rats, including observations of dilated bile ducts in the current study and in previous publications.21,40,41 Total serum and urine bile acids were increased by ~6- and ~3-fold, respectively, in PCK rats (Fig.2C and 2D), suggesting basolateral excretion of bile acids from hepatocytes into the systemic circulation followed by urinary elimination. Cyp7a1, the rate-limiting step in bile acid synthesis, was not altered in PCK rats.40 Thus, it is unlikely that increased bile acid synthesis could account for elevated hepatic bile acid concentrations in PCK rats. Other enzymes involved in bile acid metabolism (e.g., Cyp27A1, Cyp8B1, bile acid– coenzyme A amino acid N-acyltransferase)42,43 may be dysregulated in PCK rats, and should be investigated in future studies.

High concentrations of bile acids can be cytotoxic, which may lead to hepatocellular damage and acute liver injury.44 Because of the positive relationship between hydrophobicity and detergent effects, hydrophobic bile acids (e.g. LCA, DCA, CDCA) are considered the most cytotoxic.33 Mechanistic studies indicate that hydrophobic bile acids can disrupt cell membranes, promote the generation of reactive oxygen species, and cause mitochondrial and endoplasmic reticulum stress triggering hepatocyte apoptosis and necrosis.34 Therefore, the disposition of hepatotoxic bile acids in PCK rats was evaluated. Although the relative abundance of bile acid species was generally similar between Sprague-Dawley and PCK rats (Table 3), concentrations of total bile acids associated with hepatotoxicity (i.e., DCA, GDCA, CDCA, GCDCA and LCA) were increased by ~5-, 3 and 6-fold in the liver, serum and urine of PCK rats, respectively. In accordance with increased hepatotoxic bile acids, liver biochemical abnormalities are generally observed in PCK rats. For example, gamma-glutamyl transpeptidase (GGT) and glutamate dehydrogenase (GLDH), markers of hepatocellular damage, were increased in PCK rats.45 Additionally, elevated levels of bilirubin and transaminases also may indicate hepatocellular damage.21,41 Contrasting observations of increased or unchanged levels of alkaline phosphatase (ALP), a marker of bile duct obstruction and cholestasis, have been reported in PCK rats.21,40,41

Although liver tests (e.g., transaminases, bilirubin, ALP) are used routinely in clinical practice to assess liver function, these measures are generally not sensitive, specific, and/or may display large interindividual variability in the presence of liver impairment. Therefore, we sought to determine whether serum bile acids were correlated with total liver bile acids, total hepatotoxic liver bile acids, liver weight, or cyst volume. In addition to bile acids, liver weight was chosen as a marker of liver impairment because hepatomegaly may indicate vascular swelling, inflammation, and portal hypertension, which can lead to cirrhosis.46 Cyst volume also was used as a marker of liver impairment because cysts are associated with infection, hemorrhaging, liver cancer, and in some cases, cholangitis.46–50 As shown, serum bile acids correlated with liver weight, total liver bile acids, total hepatotoxic liver bile acids, and cyst volume (r > 0.75; p < 0.05; Fig. 6). An association between serum bile acids and hepatic cyst bile acid concentrations was reported in patients with polycystic liver disease.40 The present data indicate that elevated serum bile acid concentrations are associated with elevated liver bile acid concentrations in rats. Given the similar hepatorenal abnormalities observed between PCK rats and human ADPKD, bile acids also may be increased in ADPKD patients. Bile acid profiling in ADPKD patients will be necessary to test this hypothesis.

Overall, the results of these studies indicate that bile acids are increased in the liver and the serum of PCK rats, including bile acids usually associated with hepatotoxicity. Total serum bile acids were positively correlated with liver weight, total liver bile acids, total hepatotoxic liver bile acids, and cyst volume, which suggests that serum bile acids may be useful biomarkers of hepatic impairment in PKD. Measuring bile acid species, in addition to total bile acids, in the serum of patients with suspected liver disease may add to the “tool box” for clinical diagnosis.

Supplementary Material

Acknowledgments

We thank the Small Animal Imaging Core facility at the UNC Biomedical Imaging Research Center for providing the MRI service; the imaging core is supported in part by an NCI Cancer Center grant #P30-CA016086-35-37.

Funding Information

This research was supported by Otsuka Pharmaceutical Development and Commercialization Inc., and, in part, by the National Institute of General Medical Sciences of the National Institutes of Health (NIH) under Award Number R01 GM041935 and R35 GM122576 (K.L.R.B.). The content is solely the responsibility of the authors and does not necessarily represent the official views of Otsuka or the NIH.

Abbreviations

ADPKD

autosomal dominant polycystic kidney disease

ARPKD

autosomal recessive polycystic kidney disease

BSEP

bile salt export pump

DILI

drug-induced liver injury

IHC

immunohistochemistry

LC-MS/MS

liquid chromatography coupled to tandem mass spectrometry

MRI

magnetic resonance imaging

PCA

Principal Component Analysis

PCK

polycystic kidney

PKD

polycystic kidney disease

see Table 1

bile acid abbreviations

Footnotes

This work was submitted to the Graduate School of the University of North Carolina at Chapel Hill in partial fulfillment of requirements for the Doctor of Philosophy degree in Pharmaceutical Sciences (J.R.S.).

The data in this manuscript were presented, in part, at the 37th (2016) Annual Meeting of the American College of Toxicology

Conflict of Interest

The authors declare that there are no conflicts of interest with respect to the research, authorship or the publication of this article.

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