Polycystic liver disease (PLD) is a hereditary liver disease characterized by progressive enlargement of fluid-filled cysts in the liver. Liver volume increases up to 3.7% annually (1) and causes severe symptoms and complications such as hepatomegaly, back pain, gastro-esophageal reflex, dyspnea, bleeding, infection, and/or rupture of the cysts (2), which worsen quality of life (1). PLD has two forms: autosomal dominant PLD and autosomal dominant polycystic kidney disease (ADPKD) complicated by liver cysts (3). ADPKD and autosomal recessive polycystic kidney disease are classified as liver fibropolycystic disease because they are associated with liver fibrosis (2,3). PLD surgery has only short-term effects, and recurrence and complication rates are high (3). Although total liver volume was decreased by the treatment of somatostatin analog in patients with severe PLD, the volume increased after treatment cessation (4). Therefore, effective treatments for PLD have not been established.
Long-term exercise is known to have beneficial effects in several liver disease models. Moderate-intensity treadmill exercise for 12 wk improved the liver fibrosis and inflammation in nonalcoholic fatty liver disease model rats (5). Prevoluntary wheel running for 45 d prevented tumor necrosis factor–mediated acute liver injury (6). Moderate-intensity treadmill exercise for 32 wk suppressed carcinogenesis and cell proliferation of hepatocellular carcinoma in mice deficient in phosphatase and tensin homolog deleted from chromosome 10 (PTEN) (7). In addition, decreased exercise capacity is closely related to a poor prognosis in liver cirrhosis and liver transplant patients (8,9), and moderate-intensity cycle ergometer for 8 wk improves quality of life in patients with liver cirrhosis (10). However, the effects of long-term exercise on PLD have not been reported.
Polycystic kidney (PCK) rat is a spontaneous mutant animal model derived from a colony of Sprague–Dawley (SD) rats that shows the autosomal recessive pattern. PCK rat is not only a PCK disease model but also a PLD model because PCK rat always has polycystic liver (11,12). Cystic growth is caused by the abnormal proliferation of the cyst lining epithelium and fluid secretion into cyst lumens (13). Proliferation and fluid secretion are mediated through mitogen-activated protein kinase and mammalian target of rapamycin (mTOR) pathways (14,15). Many pharmacotherapeutic approaches have been tested, some of which ameliorate PLD progression in PCK rats (2,3,15–17). However, the effects of exercise on PLD have not been reported in PCK rats. Then we investigated whether the exercise could inhibit liver cyst formation and fibrosis in PCK rats.
All animal experiments were approved by the Tohoku University Committee for Animal Experiments and were performed in accordance with the Guidelines for Animal Experiments and Related Activities of Tohoku University and the guiding principles of the Physiological Society of Japan and the U.S. National Institutes of Health.
Five-week-old male PCK (n = 20) and SD rats (n = 10; 130–170 g) were obtained from Charles River Laboratories Japan Inc. (Kanagawa, Japan). Rats were housed in the animal care facility at Tohoku University Graduate School of Medicine under controlled temperature (24°C) and a 12-h light–12-h dark cycle. All rats had free access to standard laboratory chow and water. PCK rats were randomized into a sedentary (Sed-PCK) or long-term exercise (Ex-PCK) group (n = 10 per group). SD rats were set as a control (Con-SD) group (n = 10). Ex-PCK group underwent forced treadmill exercise by using an electrode positioned behind the treadmill (KN-73; Natsume Industries, Tokyo, Japan) for 12 wk (28 m·min−1, 60 min·d−1, 5 d·wk−1) (18). Rats were exercised for 10 min·d−1 at an initial treadmill speed of 20 m·min−1 up a 0% grade. The treadmill speed was increased gradually to 28 m·min−1, and the duration of exercise was increased to 60 min·d−1 for 1 wk. A pilot study confirmed that oxygen consumption (V[Combining Dot Above]O2) when rats were running at a speed of 28 m·min−1 corresponded to approximately 65% of the V˙O2max, which is assumed to be moderate-intensity aerobic exercise. V˙O2max and total running distance in cardiopulmonary exercise test were measured using an oxygen–carbon dioxide metabolism measuring system with a sealed chamber treadmill (Model MK-5000; Muromachikikai, Tokyo, Japan) on the first and final day of the exercise protocol (19).
Plasma parameters and blood pressure measurements
Systolic blood pressure was measured using an indirect tail-cuff method (CODA High-Throughput System Version 4.1; Kent Scientific Corporation, Torrington, CT). Five days after the last exercise session, all rats were euthanized with sodium pentobarbitone (100 mg·kg−1 ip), and blood samples were collected from the ventral aorta. Blood samples were centrifuged for 15 min at 1500g, and the supernatant was collected and stored at −80°C. Triglyceride, total cholesterol, free fatty acid, total bilirubin, aspartate aminotransferase (AST), and alanine aminotransferase (ALT) were determined by standard autoanalysis techniques (SRL, Tokyo, Japan).
Preparation of tissue samples
The liver was removed and prepared for histological and immunoblotting assessment. For histological assessment, the liver was fixed in 10% formalin. For immunoblotting, the liver was homogenized in phosphate-buffered saline containing 0.1 mmol·L−1 phenylmethylsulfonyl fluoride. Samples were stored at −80°C. Protein concentration was measured by using the Bradford methods, with bovine γ globulin as the standard.
Cyst index and fibrosis index
Histological analysis was performed using published methods (16). The cystic area was measured from five random fields (×100 magnification) of hematoxylin and eosin–stained liver sections from all rats. The fibrosis area was measured from five random fields (×100 magnification) of picrosirius red–stained sections of liver sections. Cystic and fibrotic indexes (%total field) were measured and calculated using Image J (National Institutes of Health, Bethesda, MD).
Immunohistochemistry was conducted, and the labeling index of Ki-67, phosphorylated (p-) extracellular signal–regulated kinase (ERK) and mTOR, which indicates cell proliferation of cholangiocytes, was evaluated as previously described (17,18). The liver tissues were fixed with 10% paraformaldehyde and embedded in paraffin. Tissue sections were deparaffinized in xylene, rehydrated in graded ethanol, and then rinsed in phosphate-buffered saline. To block endogenous peroxidase activity, rehydrated sections were treated with 0.3% H2O2 in absolute ethanol for 5 min then processed for immunostaining with antibodies against Ki-67 (diluted 1:300; Nichirei Biosciences, Tokyo, Japan), p-ERK (diluted 1:100; Cell Signaling Technology, Danvers, MA), p-mTOR (diluted 1:100; Santa Cruz Biotechnology, Dallas, TX), and Histofine Simple Stain Max PO kits (Nichirei Biosciences) according to the manufacturer’s instructions. After washing with phosphate-buffered saline, a chromogen solution (diaminobenzidine and H2O2) was applied to the sections. The slides were counterstained with hematoxylin for 30 s. At least 500 randomly chosen cholangiocytes were surveyed for each specimen, and the percentage of positive cells were expressed as the Ki-67 labeling index.
Western blot analysis
Sample proteins were separated by electrophoresis on a sodium dodecyl sulfate polyacrylamide gel for 1.5 h at 150 V. The proteins were transferred electrophoretically to a nitrocellulose membrane at 100 V for 1 h. The membrane was blocked overnight at 4°C by immersion into a TBST-20. The membrane was then incubated for 2 h with primary antibodies raised against ERK, p-ERK, mTOR, p-mTOR, AMP-activated protein kinase (AMPK), p-AMPK, S6, p-S6, cystic fibrosis transmembrane conductance regulator (CFTR) (Cell signaling Technology), aquaporin (AQP) 1 (Sigma, USA), type 1 collagen (COL1) (Cosmo Bio, Japan), and transforming growth factor β (TGF-β) (Santa Cruz Biotechnology). The membrane was rinsed several times with TBST-20 buffer and incubated with a secondary antibody (Santa Cruz Biotechnology) for 1 h. Excess secondary antibody was removed by three to four washes in TBST-20, and the immunoblots were developed using a chemical luminescence substrate (Super Signal; Thermo Fisher Scientific, Waltham, MA). The relative intensities of the bands were quantified using Image J software (version 1.40; National Institutes of Health). The intensities of each band were normalized to those for β-actin as an internal standard or total protein density, and the intensity of the band in the Con-SD group was assigned a value of 1.
For comparisons of exercise capacity data, repeated-measures ANOVA with group–time interaction was used. Comparisons between the groups were performed using one-way ANOVA. If the ANOVA showed a significant effect, further post hoc analysis was performed using Tukey’s method for comparisons. All statistical tests were performed using SPSS version 21.0 (IBM Corp., Tokyo, Japan). Two-tailed P < 0.05 was considered significant. Data are presented as the mean ± SEM.
Effects of exercise on body weight, blood pressure, and biochemical parameters
The characteristics of three groups at the end of experiment are shown in Table 1. Body weight, liver weight, and plasma levels of total cholesterol, AST, and ALT were significantly higher in the Sed-PCK group than in the Con-SD group (P < 0.01, respectively). Body weight, triglycerides (P < 0.05, respectively), total cholesterol, and liver weight (P < 0.01, respectively) were significantly lower in the Ex-PCK group than in the Sed-PCK group. There was no significant difference in blood pressure, AST, or ALT between the Sed-PCK and the Ex-PCK groups.
Effects on exercise capacity
Significant interactions between groups and time were observed in exercise capacity. At the start of experiment, total running distance and V˙O2max were significantly lower in the Sed-PCK and Ex-PCK groups than in the Con-SD group (P < 0.01, respectively) (Figs. 1A and 1B). After 12 wk, total running distance was significantly greater in the Ex-PCK group than in the Con-SD and Sed-PCK groups (P < 0.01, respectively). V˙O2max was significantly greater in the Ex-PCK group than in the Sed-PCK groups (P < 0.01).
Effects on histological changes
Figures 2A–2I show a representative picture of liver cysts and fibrosis in each group. Liver cysts were formed diffusely in PCK rats (Figs. 2A–2F), and collagen fibers were highly expressed around the cyst (Figs. 2G–2I). The cyst index was significantly higher in the Sed-PCK and Ex-PCK groups than in the Con-SD group, and it was significantly lower in the Ex-PCK group than in the Sed-PCK group (P < 0.01) (Fig. 2J). The fibrosis index was significantly higher in the Sed-PCK and Ex-PCK groups than in the Con-SD group, and it was significantly lower in the Ex-PCK group than in the Sed-PCK group (P < 0.01) (Fig. 2K). Figures 3A–3I show representative pictures of immunostained liver against anti-Ki-67, p-ERK, and p-mTOR in each group. Similar to liver fibrosis, positive cells for Ki-67, p-ERK, and p-mTOR were highly expressed around the cyst of PCK rats. The Ki-67 labeling index was significantly higher in the Sed-PCK and Ex-PCK groups than in the Con-SD group, and it was significantly lower in the Ex-PCK group than in the Sed-PCK group (P < 0.01) (Fig. 3J). The expressions of p-ERK and p-mTOR in cholangiocytes of PCK rats were lower in the Ex-PCK group than in the Sed-PCK group.
Effects of exercise on protein expression and phosphorylation
Concerning pathways of cell proliferation, the expressions of p-ERK, p-mTOR, and p-S6 were significantly higher in the Sed-PCK group than in the Con-SD group (P < 0.05, P < 0.05, and P < 0.01, respectively) (Figs. 4B–4D), and the expression of p-AMPK was significantly lower in the Sed-PCK group than in the Con-SD group (P < 0.05) (Fig. 4E). The expressions of p-ERK, p-mTOR, and p-S6 were significantly lower in the Ex-PCK group than in the Sed-PCK group (P < 0.05, P < 0.01, and P < 0.05, respectively) (Figs. 4B–4D). The expression of p-AMPK was significantly higher in the Ex-PCK group than in the Sed-PCK group (P < 0.01) (Fig. 4E). These results suggest that the exercise for 12 wk inhibited the increased ERK, mTOR, and S6 activities and activated the decreased AMPK activity in the liver of PCK rats.
Concerning altered secretion of cholangiocytes and liver fibrosis, the expressions of CFTR, AQP1, COL1, and TGF-β were significantly higher in the Sed-PCK group than in the Con-Sed group (P < 0.05, respectively) (Figs. 5B–E). The expressions of CFTR, AQP1, COL1, and TGF-β were significantly lower in the Ex-PCK group than in the Sed-PCK group (P < 0.05, respectively) (Figs. 5B–5E).
Long-term exercise is known to has beneficial effects in several liver disease models (5–7,19,20), but the effects of long-term exercise on PLD have not been reported. We previously reported the beneficial effects of long-term moderate-intensity exercise in various disease models (21–23), and the present study is the first article to reveal the beneficial effects of long-term exercise in a PLD model. Moderate-intensity exercise for 12 wk attenuated liver cysts formation and fibrosis with inhibition of excessive cell proliferation around cysts in PCK rat.
The excessive cell proliferation of cholangiocytes is one of mechanisms of liver cyst formation (2). Mitogen-activated protein kinase and mTOR pathways are involved in excessive cell proliferation in the cholangiocytes of PCK rats (17,24). ERK was phosphorylated and activated in cholangiocytes of PCK rats, and pioglitazone, a PPAR-γ activator, inhibited ERK phosphorylation and liver cyst formation (15). Everolimus, an mTOR inhibitor, inhibited liver cyst formation (25). In addition, AMPK activation by metformin slows renal cystogenesis with inhibiting mTOR pathway in ADPKD model mice (26). The present results indicated that the exercise inhibited ERK and mTOR pathways and activated AMPK in the liver of PCK rats. Swimming for 8 wk inhibited ERK pathway in the liver of obese model mice (27). Moderate-intensity treadmill exercise for 12 wk activated AMPK in the liver of Wistar rats (28). Moderate-intensity treadmill exercise for 32 wk suppressed carcinogenesis and cell proliferation of hepatocellular carcinoma with activating AMPK and inhibiting mTOR pathway in PTEN-deficient mice (7). Moderate-intensity treadmill exercise for 4 wk also suppressed carcinogenesis in tumor implantation rats (29).
Altered secretion of cholangiocytes is another mechanism of liver cyst formation (30). CFTR and AQP1, which are expressed on the apical membrane of cholangiocytes, mediate the passive movement of water molecules into liver cysts (31). Fluid secretion into liver cysts promotes stretching of the epithelium lining the cyst, which releases cytokines, which then accelerate cyst growth (32). CFTR and AQP1 are overexpressed in cholangiocytes of PCK rats (30,31). The present results indicate that the exercise reduced the overexpressed CFTR and AQP1 in the liver of PCK rats, which suggests that long-term exercise may decrease water secretion into liver cysts. CFTR is negatively controlled by AMPK (33). The exercise-activated AMPK may be involved in changes in fluid secretion into liver cysts.
Although liver fibrosis is another pathogenesis for PLD, the mechanism is not completely clear. TGF-β, which is a mediator of liver fibrosis (16), increased collagen contents of cholangiocytes in PCK rats (34). Telmisartan, an angiotensin II receptor blocker and partial PPAR-γ agonist, attenuated excessive TGF-β expression and inhibited liver fibrosis in PCK rats (16). The present results indicated that the exercise decreased TGF-β expression and inhibited liver fibrosis. Consisted with the present results, moderate-intensity treadmill exercise for 16 wk decreased TGF-β expression and liver fibrosis in nonalcoholic fatty liver disease model mice, and swimming for 9 wk decreased TGF-β expression in liver cancer model mice (35,36).
Decreased exercise capacity and poor prognosis are closely related in patients with liver disease (8,9). Moderate-intensity aerobic exercise has been shown to improve exercise capacity in patients with liver cirrhosis (37). Moderate-intensity exercise is recommended for patients with liver disease (38), and aerobic exercise (30 min·d−1, 5 d·wk−1) is recommended for patients with ADPKD (39). However, there is little clinical evidence to support these recommendations. Exercise capacity is reduced in ADPKD patients compared with healthy subjects (40). The present results indicate that exercise capacity was lower in PCK rats than in control SD rats, and the exercise improved exercise capacity in PCK rats. Long-term moderate-intensity exercise may improve exercise capacity and prognosis in PLD patients.
The present study has several limitations. First, it was not clear whether the inhibition of liver cyst formation and fibrosis was a direct effect of long-term exercise. In this regard, we confirmed that the exercise for 12 wk significantly inhibited cyst formation in the liver and kidney, but the exercise for 8 wk significantly inhibited cyst formation only in the liver (data not shown). These results suggested that long-term exercise might inhibit cyst formation in the liver prior to the kidney. Second, the present study does not clarify the precise mechanism of long-term exercise-inhibited cystic formation by blocking kinases associated with liver cyst formation. However, we examined the effects of 12-wk treatment of metformin, an AMPK activator on liver cyst formation in PCK rats, and confirmed that metformin prevented liver cyst formation with stimulation of AMPK phosphorylation and inhibition of mTOR, ERK phosphorylation, and CFTR expression. Because the effects of long-term exercise and metformin on kinases associated with liver cyst formation were identical, the inhibitory effects of long-term exercise on liver cyst formation might be mediated mainly by an activation of AMPK. Third, the levels of long-term exercise-induced AMPK phosphorylation in cholangiocytes were not clear because long-term exercise induces the AMPK phosphorylation not only in cholangiocytes but also in hepatocytes (28).
In conclusion, long-term exercise improves exercise capacity and inhibits liver cyst formation and fibrosis with the inhibition of signaling cascades responsible for cellular proliferation and fibrosis in PCK rats. Although the results of present study may not be directly applicable to humans, long-term moderate-intensity exercise has beneficial effects and may be a new therapeutic approach for preventing of liver cyst formation in PLD patients.
This work was supported by Grants-in-Aid for Scientific Research (JSPS KAKENHI nos. 15K12573 and 17H02119). The authors greatly appreciate technical support from the Biomedical Research Unit of Tohoku University Hospital in histopathological analysis.
The authors declare no conflicts of interest associated with this manuscript. They thank Mark Cleasby, Ph.D., from Edanz Group (www.edanzediting.com/ac) for editing a draft of this manuscript.
The results of the study are presented clearly, honestly, and without fabrication or inappropriate data manipulation. The results of the present study do not constitute endorsement by the American College of Sports Medicine.
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