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Cannabinoid-2 receptor activation ameliorates hepatorenal syndrome
Journal Pre-proof Cannabinoid-2 receptor activation ameliorates hepatorenal syndrome Eszter Trojnar, Katalin Erdelyi, Csaba Matyas, Suxian Zhao, Janos Paloczi, Partha Mukhopadhyay, Zoltan V. Varga, Gyorgy Hasko, Pal Pacher PII:
S0891-5849(19)31745-9
DOI:
https://doi.org/10.1016/j.freeradbiomed.2019.11.027
Reference:
FRB 14498
To appear in:
Free Radical Biology and Medicine
Received Date: 15 October 2019 Accepted Date: 21 November 2019
Please cite this article as: E. Trojnar, K. Erdelyi, C. Matyas, S. Zhao, J. Paloczi, P. Mukhopadhyay, Z.V. Varga, G. Hasko, P. Pacher, Cannabinoid-2 receptor activation ameliorates hepatorenal syndrome, Free Radical Biology and Medicine (2019), doi: https://doi.org/10.1016/j.freeradbiomed.2019.11.027. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Inc.
Cannabinoid-2 receptor activation
Inflammation
Inflammation
Bile duct ligation
Oxidative stress
Oxidative stress
Fibrosis
Fibrosis
Microcirculation
ROS
Liver failure
Paracrine mediators (cytokines, chemokines, oxidants)
Activation
Endothelium
ROS
Hepatorenal syndrome
1
Cannabinoid-2 receptor activation ameliorates hepatorenal syndrome
2 3
Eszter Trojnar*1, Katalin Erdelyi*1, Csaba Matyas1, Suxian Zhao1, Janos Paloczi1, Partha
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Mukhopadhyay1, Zoltan V. Varga1, Gyorgy Hasko2, and Pal Pacher1
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*These authors contributed equally to this work
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Affiliations:
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1
Laboratory of Cardiovascular Physiology and Tissue Injury, National Institute on Alcohol
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Abuse and Alcoholism (NIAAA), 5625 Fishers Lane, 20852 Rockville, MD, USA
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2
Department of Anesthesiology, Columbia University, New York, NY 10032, USA
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Corresponding author:
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Pal Pacher, MD, PhD, FAHA, FACC
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Laboratory of Cardiovascular Physiology and Tissue Injury
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5625 Fishers Lane, Room 2N-17; Bethesda, MD 20892-9413
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Phone: (301)443-4830
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Email: [email protected]
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Email addresses of each author:
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[email protected]
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[email protected]
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[email protected]
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[email protected]
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[email protected]
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[email protected]
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[email protected]
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[email protected]
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[email protected]
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Conflict of interest disclosure: The authors have no conflict of interest to disclose.
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Keywords: Hepatorenal syndrome, Cannabinoid-2 receptor, HU-910, bile-duct ligation,
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endocannabinoid system
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34 35
Highlights
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• Bile duct ligation (BDL) causes hepatorenal syndrome (HRS)
37
• Oxidative damage/inflammation drives liver and kidney injury following BDL
38
• Cannabinoid-2 receptor (CB2-R) activation attenuates hepatic damage in BDL
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• CB2-R activation mitigates the renal inflammation and oxidative damage in BDL
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• CB2-R activation attenuates renal microcirculatory dysfunction in BDL
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Abstract
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Study rationale: Hepatorenal syndrome (HRS) is a life-threatening complication of end-stage
44
liver disease characterized by the rapid decline of kidney function. Herein, we explored the
45
therapeutic potential of targeting the cannabinoid 2 receptor (CB2-R) utilizing a commonly used
46
mouse model of liver fibrosis and hepatorenal syndrome (HRS), induced by bile duct ligation
47
(BDL).
48
Methods: Gene expression analysis, histological evaluation, determination of serum levels of
49
renal injury-biomarkers were used to characterize the BDL-induced organ injury; laser speckle
50
analysis to measure microcirculation in the kidneys.
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Key results: We found that liver injury triggered marked inflammation and oxidative stress also
52
in the kidneys of BDL-operated mice. We detected pronounced histopathological alterations with
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tubular injury paralleled with increased inflammation, oxidative/nitrative stress and fibrotic
54
remodeling both in hepatic and renal tissues as well as endothelial activation and markedly
55
impaired renal microcirculation. This was accompanied by increased CB2-R expression in both
56
liver and the kidney tissues of diseased animals. A selective CB2-R agonist, HU-910, markedly
57
decreased numerous markers of inflammation, oxidative stress and fibrosis both in the liver and
58
in the kidneys. HU-910 also attenuated markers of kidney injury and improved the impaired
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renal microcirculation in BDL-operated mice.
60
Conclusions: Our results suggest that oxidative stress, inflammation and microvascular
61
dysfunction are key events in the pathogenesis of BDL-associated renal failure. Furthermore, we
62
demonstrate that targeting the CB2-R by selective agonists may represent a promising new
63
avenue to treat HRS by attenuating tissue and vascular inflammation, oxidative stress, fibrosis
64
and consequent microcirculatory dysfunction in the kidneys.
65
66
Abbreviations
67
4-HNE – 4-hydroxy-2-nonenal
68
BDL – bile duct ligation
69
BUN – blood urea nitrogen
70
CB1-R – cannabinoid-1 receptor
71
CB2-R – cannabinoid-2 receptor
72
DAB – 3,3'-diaminobenzidine
73
ddPCR – droplet digital PCR
74
ECS – endocannabinoid system
75
FFPE – formalin-fixed and paraffin-embedded
76
HRP – horseradish peroxidase
77
HRS – hepatorenal syndrome
78
ICAM-1 – intercellular-adhesion molecule 1
79
KIM-1 – kidney injury molecule-1
80
MDA – malondialdehyde
81
NADPH oxidase– nicotinamide adenine dinucleotide phosphate oxidase
82
NGAL – neutrophil gelatinase-associated lipocalin
83
NO – nitrogen monoxide
84
OPN – osteopontin
85
3-NT – 3-nitrotyrosine
86
iNOS – inducible nitric-oxide synthase
87
88
Introduction
89
End-stage liver disease has been associated with increasing mortality rate during the past
90
two decades representing a major epidemiological burden in developed countries [1].
91
Independent of the etiology of liver cirrhosis, its association with kidney function decline, and
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subsequent development of extrahepatic complications is a major determinant of the clinical
93
outcome [2]. Up to 50% of hospitalized patients with liver cirrhosis may develop renal
94
dysfunction [1, 2], 20% of which fulfills the diagnostic criteria of HRS [1]. HRS is associated
95
with a poor prognosis and high mortality [3], with more than half of the patients with
96
concomitant cirrhosis and renal failure dying within one month of diagnosis [4]. Despite the
97
significant advancement in the understanding of the pathophysiology of HRS, still no specific
98
pharmacological approaches are available to treat this devastating condition. The exact molecular
99
steps leading to kidney function decline during chronic liver diseases/cirrhosis are poorly
100
understood, necessitating further investigations to identify novel therapeutic approaches for the
101
management of this condition.
102
Bile duct ligation (BDL) is an established animal model of cholestatic liver damage and
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fibrosis [5]. Following BDL, due to exhaustive bile flow blockade, increased levels of
104
intrahepatic and circulatory bile acids provoke an extensive inflammatory response and oxidative
105
stress with subsequent production of free radicals that ultimately lead to cytotoxicity and tissue
106
fibrosis in the hepatic tissue [6]. Time-dependent features of progressive renal dysfunction
107
mimicking HRS in humans associated with BDL in mice have been recently described [7],
108
promoting this model as a valuable in vivo tool to study the HRS pathogenesis and management.
109
The cannabinoid-2 receptor (CB2-R), primarily expressed in inflammatory cells and
110
activated endothelium, is a G-protein coupled receptor. The activation of this receptor by
111
endocannabinoids or synthetic ligands has been reported to exert anti-inflammatory effects in
112
models of atherosclerosis, stroke, myocardial, hepatic, and renal injury [8-13], [14-16].
113
In this study, we explored the potential role of inflammation and the disruption of renal
114
redox homeostasis in the evolvement of BDL-associated HRS. We also demonstrate by using a
115
selective CB2-R agonist HU-910 [17, 18] that targeting CB2-R attenuates not only liver injury,
116
but also kidney inflammation, oxidative/nitrative stress, and microcirculatory collapse in BDL,
117
suggesting a potential clinical utility of selective CB2-R agonists in HRS in humans.
118
119
Methods
120
Study design and animals
121
All animal experiments reported in this manuscript complied with the National Institutes of
122
Health “Guide for the Care and Use of Laboratory Animals” (NIH publication 86-23 revised
123
1985) and were approved by the Institutional Animal Care and Use Committee of the National
124
Institute on Alcohol Abuse and Alcoholism (Bethesda, MD). Animals were kept in the institute`s
125
designated animal facility under constant temperature (22±2°C) and humidity, with 12-hour
126
alternating light and dark cycles. For all experiments listed, 12-16 week-old C57BL/6J male
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mice were used (The Jackson Laboratory, Bar Harbor, ME, USA) and bile duct ligation was
128
performed as described earlier [7, 19], with Sham-operated mice used as controls. Following
129
surgery, the animals were randomly assigned to one of four groups: Sham (vehicle-treated),
130
Sham (HU-910-treated), BDL (vehicle-treated) and BDL (HU-910-treated). Starting on post-
131
operative day 1, all animals received a daily intraperitoneal injection of treatment or vehicle
132
only, consisting of 10mg/kg of the CB2-R agonist HU-910 (Institute for Drug Research, The
133
Hebrew University of Jerusalem, Israel), dissolved in dimethyl sulfoxide, Tween-80 and distilled
134
water in a ratio of 1:1:8. Animals were sacrificed on post-operative day 14, and respective tissues
135
were harvested following exsanguination and perfusion with sterile, cold phosphate buffered
136
saline.
137 138
Microvascular flow determination
139
On the post-operative day 14, kidney microcirculation was assessed by the application of the
140
laser speckle contrast method. Animals were anesthetized with 1-2% isoflurane, while their body
141
temperature was maintained at 37ºC. After opening the abdominal cavity, the left kidney was
142
exposed and scanned with MoorFLPI-2 blood flow imager (Moor Instruments, Wilmington, DE,
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USA). Microcirculation was quantified and expressed as flux arbitrary units with the analysis
144
software provided by Moor Instruments.
145 146
Histological evaluation of organ impairment
147
For pathological evaluation of structural alterations, 5 µm thick liver and kidney sections were
148
cut from formalin-fixed and paraffin-embedded (FFPE) tissues, and stained for hematoxylin &
149
eosin, periodic acid-Schiff, Masson’s trichrome (Thermo Fisher Scientific, Waltham, MA, USA)
150
and Sirius red (Electron Microscopy Sciences, PA, USA) using conventional staining methods
151
following the manufacturer’s instructions.
152
Evaluation of fibrosis was performed on Sirius red-stained liver and Masson`s trichrome-stained
153
kidney tissue sections. Quantification of the Sirius-red stained area and staining intensity was
154
performed by a blinded investigator using the following scoring system: staining intensity: 1: no
155
staining, 2: weak staining, 3: moderate staining, 4: strong staining; area positivity: 1: 0-25%, 2:
156
25-50%, 3: 50-75%, 4: 75-100%. Area*intensity score values were used to compare intergroup
157
differences. Intensity of the Masson`s-trichrome staining was evaluated by Image J.
158
Histopathological alterations in the kidneys were assessed on periodic acid-Schiff-stained tissue
159
sections. A blinded investigator evaluated five randomly selected areas under 100x magnification
160
and scored the extent of tubular dilatation, brush border loss, intraluminal cast formation, the
161
presence of interstitial edema and inflammatory cell infiltration on a scale of zero to five (0: 0-
162
10%, 1: 11-25%, 2: 25-50%, 3: 50-75%, 4: 75-100%). The thus obtained values were added up
163
and the average summary scores of each study group are presented in the respective figure panel.
164 165
Immunohistochemistry
166
Evaluation of oxidative and nitrative organ damage and endothelial activation was performed on
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FFPE tissue sections using the Mouse on Mouse (M.O.M.®) ImmPRESS® HRP (Peroxidase)
168
Polymer Kit or the ImmPRESS® Excel Amplified HRP Polymer Staining Kit (Anti-Rabbit IgG)
169
(Vector Laboratories, Burlingame, CA, USA), respectively. Following deparaffination and
170
antigen retrieval with 1x citrate buffer (95ºC for 15 minutes), endogenous peroxidase activity
171
was blocked by BloxAll reagent (Vector Laboratories), while the aspecific binding sites were
172
blocked with 2.5% normal-horse serum (Vector Laboratories). The below primary antibodies
173
were applied overnight at 4ºC: anti-4-hydroxy-2-nonenal (4-HNE) monoclonal antibody (1:200,
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Japan Institute for the control of Aging # MHN-100P, Japan), anti-malondialdehyde (MDA)
175
monoclonal antibody (1:100, Japan Institute for the control of Aging #MMD-030n, Japan), anti-
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3-nitrotyrosine (3-NT) polyclonal antibody (1:500, Cayman Chemicals #10189540, Ann Arbor,
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MI, USA) and anti ICAM-1 monoclonal antibody (1:4000, SinoBiological #50440-R020,
178
Wayne, PA, USA). Horseradish peroxidase (HRP)-conjugated secondary antibodies were used
179
conforming to the antibody host and the manufacturer's instructions. Signal development was
180
carried out with 3,3'-diaminobenzidine (DAB), followed by hematoxylin counter-staining.
181
For the assessment of inflammatory cell infiltration staining of F4/80 positive macrophages on
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FFPE tissue sections was performed using the ImmPRESS® HRP Anti-Rat IgG (Peroxidase)
183
Polymer Detection Kit, made in Goat (Vector Laboratories). Following dehydration of the tissue
184
sections, antigen retrieval was performed via Proteinase K-based digestion in Tris-EDTA buffer
185
(pH: 8). The primary antibody, F4/80 (1:50, BioRad #MCA497, Hercules, CA, USA) was
186
applied overnight at 4oC and signal development was performed with an HRP-conjugated
187
secondary antibody and DAB.
188
Quantification of the stained area and staining intensity was performed by two blinded
189
investigators using the following scoring system: staining intensity: 1: no staining, 2: weak
190
staining, 3: moderate staining, 4: strong staining; area positivity: 1: 0-25%, 2: 25-50%, 3: 50-
191
75%, 4: 75-100%. Area*intensity score values were used to compare intergroup differences.
192 193
Serum chemistry analysis
194
Laboratory markers of kidney function were assessed from sera of the animals collected on post-
195
operative day 14. Creatinine and blood urea nitrogen (BUN) levels were determined via the
196
Idexx VetTest 8008 (Idexx Laboratories, Westbrook, ME) system, while Kidney Injury
197
Molecule-1 (KIM-1), Neutrophil Gelatinase-Associated Lipocalin (NGAL) and Osteopontin
198
(OPN) levels were determined using commercially available ELISA assays (R&D Systems,
199
Minneapolis, MN, USA).
200 201
Gene expression analysis by quantitative real-time PCR and droplet digital PCR
202
RNA isolation for gene expression analysis was performed from hepatic and renal tissues using
203
the Direct-zol RNA Miniprep Kit (Zymo Research, Irvine, CA, USA) according to the
204
manufacturer’s instructions, following the homogenization of 15-20 mg tissue pieces in TRIzol
205
reagent (Thermo Fisher Scientific, Waltham, MA, USA) and separation of the RNA-rich
206
aqueous layer with chloroform. From each sample, the isolated RNA was reverse transcribed
207
using the High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific). cDNA
208
samples were then used for subsequent quantitative real-time PCR experiments with SyberSelect
209
PCR Master Mix (Thermo Fisher Scientific) on an ABI 7900HT Realtime PCR Instrument
210
(Applied Biosystems, Foster City, CA, USA) with each reaction performed in duplicates.
211
Relative quantification of target gene expression was calculated with the comparative CT
212
method. The average value of the housekeeping gene encoding the 18S ribosomal RNA (Rn18s)
213
was used as reference. Expression levels of the following target genes were analyzed with primer
214
pairs listed in Table 1.: CD11b, CD68, CB2-R (Cnr2), collagen, type I, alpha 1 (Col1a1),
215
collagen, type III, alpha 1 (Col3a1), F4/80, monocyte chemoattractant protein 1 (MCP1),
216
macrophage inflammatory protein 1α (MIP1α), chemokine (C-X-C motif) ligand 2 (MIP2),
217
NADPH oxidase 4 (NOX4), p22phox, gp91phox, p40phox, p47phox, p67phox, P-selectin,
218
superoxide dismutase 1-2 (SOD1, SOD2), transforming growth factor beta (Tgfβ), tumor necrosis
219
factor alpha (TNFα), vascular adhesion molecule 1 (Vcam-1), Catalase, inducible nitric-oxide
220
synthase (iNOS).
221
Expression of the CB2-R in the hepatic and renal tissues was further assessed by droplet digital
222
PCR (ddPCR) analysis using the QX200 ddPCR System (Bio-Rad, Hercules, CA, USA). For the
223
absolute quantification of the Cnr2 copy numbers EvaGreen-based chemistry (Bio-Rad) was
224
applied and equal amounts of cDNA, isolated from livers and kidneys of BDL and Sham
225
operated animals were loaded into separate assay wells. Following droplet generation and
226
thermal cycling, the Cnr2 copy number was determined using the QX200 Droplet Reader and the
227
QuantaSoft Software (Bio-Rad), with the same threshold applied in the corresponding study
228
groups for the determination of positive droplet count.
229 230
Statistical analysis
231
For statistical comparison of datasets, the Graph Pad Prism software version 6.00 was used
232
(GraphPad Software, La Jolla, CA, www.graphpad.com). In all figures, group mean values with
233
the standard error of mean are presented with the respective statistical test indicated in each
234
figure legend and significance level set at p<0.05.
235
236
Results
237
The BDL-induced hepatic inflammatory response, oxidative damage and liver fibrosis in mice is
238
attenuated by a CB2-R agonist
239
14 days post-operation peculiar structural alterations evolved in the livers of BDL-operated mice.
240
Prominent dilatation of the intrahepatic bile ducts was observed and signs of parenchymal
241
fibrosis was apparent on the Sirius red-stained tissue sections (Figure 1A-B). Massive
242
inflammatory cell infiltration of the portal areas was detected and confirmed by
243
immunohistochemistry using a specific cell surface marker of macrophages (F4/80) (Figure 1A,
244
middle panels). Inflammation and liver injury were accompanied by signs of oxidative damage in
245
BDL, as evidenced by the increased staining intensity of the lipid peroxidation marker 4-HNE in
246
the hepatic tissues (Figure 1A, right panels).
247
Histological features of fibrosis were paralleled by significantly elevated expression levels of
248
multiple markers of fibrotic remodeling such as Col1a1 and Col3a1 mRNA in BDL compared to
249
Sham (Figure 1C). Inflammatory cell infiltration was accompanied by a substantially increased
250
expression level of various proinflammatory cytokines and chemokines as well as a notable
251
increase in the expression level of inflammatory cell markers in the hepatic tissue of BDL-
252
operated animals in comparison to Sham-operated ones (Figure 1D-E). In agreement with
253
increased lipid peroxidation, we detected a markedly elevated expression level of multiple
254
isoforms and subunits of the phagocytic reactive oxygen species generating NADPH oxidases
255
(p40phox, p47phox, gp91phox, p67phox, p22phox), in BDL versus Sham mice (Figure 1F).
256
Chronic treatment with HU-910 attenuated the BDL-induced hepatic damage. CB2-R activation
257
alleviated the observed fibrotic changes (Figure 1A-C), exerted an anti-inflammatory effect via
258
both mitigation of the inflammatory cell infiltration (Figure 1A) and significant reduction of the
259
expression levels of inflammatory cell markers, cytokines and chemokines (Figure 1D-E). In line
260
with the attenuation of the inflammatory response, treatment with HU-910 in BDL also
261
effectively decreased the extent of oxidative injury, as shown by the reduced hepatic 4-HNE
262
staining intensity (Figure 1A), and moderation of the BDL-induced increment in gene expression
263
of NADPH oxidase isoforms (Figure 1F). The treatment by itself had no effect on the above-
264
mentioned variables in Sham-operated animals.
265
The BDL-induced extensive kidney injury is attenuated by a CB2-R agonist
266
As an extrahepatic complication of liver fibrosis, a progressive kidney function decline following
267
BDL was observed in mice (Figure 2). Classical laboratory signs of renal dysfunction (BUN and
268
creatinine levels) in BDL- compared to Sham-operated mice indicated reduction of renal
269
filtration capacity (Figure 2A). In addition to that, significantly increased KIM-1, OPN and
270
NGAL serum levels were measured, suggesting the presence of both proximal and distal tubular
271
damage in the kidneys (Figure 2A). Consistently with prior observations, histological signs of
272
marked renal tubular injury were evident by 14 days post-operation [7] (Figure 2B-C). In
273
vehicle-treated BDL mice we detected extensive tubular dilatation with signs of epithelial cell
274
necrosis, leukocyte infiltration and nuclear enlargement or loss of nuclear staining, respectively
275
(Figure 2B). In some areas flattening of the epithelial cells and the loss of cellular architecture
276
indicated atrophy of the tubuli. BDL was also associated with visible signs of interstitial fibrosis
277
in the kidneys of mice. These included a notable expansion of the collagenous area on Masson’s
278
trichrome-stained tissue sections (Figure 2B, D), corroborated by the increased expression level
279
of various indicators of fibrotic remodeling (Col1a1, Col3a1, TGFβ mRNA) in the renal tissue
280
(Figure 2E).
281
Similar to the BDL-induced hepatic injury, histological signs of kidney damage were
282
significantly attenuated by HU-910 treatment. HU-910 treatment in BDL attenuated tubular
283
dilatation with better preservation of the cellular architecture in large parts of the cortical area
284
(Figure 2B). Laboratory signs of kidney dysfunction were also ameliorated in the treatment
285
group, suggesting a partial rescue of the reduced renal filtration capacity and tubular injury
286
(Figure 2A). Furthermore, HU-910 also improved both the histological and genetic signs of renal
287
fibrosis (Figure 2B, D), while the drug by itself exerted no effect in Sham-operated animals.
288 289
The BDL-induced renal inflammation and oxidative/nitrative stress is attenuated by a CB2-R
290
agonist
291
To explore the molecular background of the observed organ impairment, we evaluated the extent
292
of inflammation and oxidative damage in the kidneys. We found a notable inflammatory cell
293
infiltration of the cortical area in BDL, shown by the increased F4/80 staining intensity
294
compared to the lack of F4/80 positive cells in the control groups (Figure 3A, left and middle
295
panels, Figure 3B). We also detected an increased staining intensity of the intercellular adhesion
296
molecule ICAM-1 in BDL compared to Sham-operated as well as HU-910-treated BDL animals
297
(Figure 3A, right panels, Figure 3E). The histological signs of inflammatory cell infiltration were
298
corroborated by increases in the expression levels of inflammatory cell markers (CD68 and
299
CD11b) (Figure 3C), as well as inflammatory cytokines and chemokines (MCP1, MIP2, TNFα)
300
(Figure 3D) in the kidneys of BDL-mice compared to Sham-operated ones.
301 302
The extensive renal inflammatory response was accompanied by signs of markedly enhanced
303
oxidative damage in the kidneys (Figure 4). An increased intensity of the lipid peroxidation
304
markers 4-HNE and MDA, as well as an enhanced renal 3-NT staining intensity were detected in
305
BDL (Figure 4A). The histological signs of oxidative and nitrative damage were further
306
supported by the increased renal expression of the mRNAs of p40phox, p47phox, p67phox,
307
gp91phox and NOX4 (Figure 4B) and an elevated iNOS expression level (Figure 4C), in parallel
308
with a significant reduction in the renal expression level of mRNA of cellular antioxidant
309
enzymes (Catalase, SOD1, SOD2) (Figure 4D).
310 311
HU-910 treatment in BDL not only attenuated the inflammatory cell infiltration (Figure 3), but it
312
also significantly abrogated the renal oxidative/nitrative stress, as evidenced by the reduced
313
staining intensity of the 4-HNE, MDA and 3-NT stainings, and the significant reduction of iNOS
314
and NADPH oxidase isoforms mRNA expression with partial recovery of the expression levels
315
of cellular antioxidant enzymes (Figure 4).
316 317
Nephropathy in BDL is associated with endothelial activation/inflammatory response and
318
decline of the renal microvascular flow, which is attenuated by CB2-R activation
319
In addition to the prominent inflammatory response and oxidative damage observed in BDL, the
320
progressive renal injury also involved notable endothelial activation and the reduction of the
321
microvascular flow in the kidneys, as confirmed by the reduced laser speckle intensity on the
322
surface of the renal tissue (Figure 5A-B). The reduction of the microvascular flow was
323
associated with a significant increment in the expression level of the endothelial
324
activation/inflammation markers Vcam-1 and P-selectin (Figure 5C). All of these pathological
325
alterations were partially restored in BDL-animals by HU-910 treatment.
326 327
Enhanced CB2-R expression in BDL is attenuated by CB2-R agonist
328
With the application of droplet digital PCR analysis we determined the absolute copy number of
329
the Cnr2 gene in both the hepatic and renal tissues of Sham and BDL-operated animals and
330
found that the CB2-R expression was significantly elevated in BDL, in both the liver and the
331
kidneys, whereas its levels remained very low in the control group (Figure 5D). We also
332
analyzed in detail the gene expression level of the Cnr2 gene in the kidneys and liver of BDL
333
and Sham operated mice following the application of the CB2-R agonist, HU-910. Consistently
334
with the attenuation of inflammatory cell infiltration and vascular inflammation (inflammatory
335
cells and activated endothelium express CB2-R) we found that chronic treatment with HU-910
336
significantly reduced the Cnr2 expression levels both in the liver and kidneys of BDL-operated
337
mice.
338
339
Discussion
340 341
In this study we confirm that BDL leads to progressive renal failure on the basis of liver
342
inflammation, oxidative stress and fibrosis, resembling characteristic features of HRS. We
343
conclude that tissue inflammation and oxidative damage are major driving forces of kidney
344
injury in BDL, leading to endothelial activation/inflammatory response and collapse of the renal
345
microcirculation. We also show increased expression of CB2-R in the liver and kidneys of
346
animals exposed to BDL and that a selective CB2-R agonist, HU-910, by attenuating tissue and
347
vascular inflammation, oxidative/nitrative stress and fibrosis significantly improves the HRS in
348
the mouse model of BDL.
349
Under physiological conditions CB1- and CB2-R expression are low in the hepatic tissue
350
[20-22]. However, dysregulation of the endocannabinoid system (involving CB1/2 receptors,
351
endocannabinoids and their metabolizing and synthetic enzymes) have been shown to play an
352
important role in the pathogenesis of liver cirrhosis and its complications [11, 22]. Activation of
353
the CB1-R on parenchymal and inflammatory cells and the vascular endothelium was reported to
354
stimulate hepatic inflammation and fibrogenesis [11, 20, 23], as well as to promote vasodilation
355
and cardiac dysfunction thus contributing to the decompensation of liver cirrhosis and
356
development of cirrhotic cardiomyopathy [24].
357
In normal liver CB2-R is primarily expressed in Kupffer cells (resident macrophages of
358
the liver) [25, 26]. Increment of hepatic CB2-R expression under pathological conditions is
359
mostly attributed to influx/activation of inflammatory cells and activated sinusoidal endothelial
360
cells [25, 27, 28]. In contrast to the CB1-R mediated effects, signaling through CB2-R has been
361
shown to exert anti-inflammatory and anti-fibrogenic effects in numerous pathological
362
conditions and in vivo as well as in vitro disease models of hepatic injury [18, 23, 28-31].
363
Activation of the CB2-R has also been shown to be beneficial in cisplatin- and diabetes-induced
364
nephropathy models [14, 16, 23, 32, 33].
365
We detected a notable elevation of the CB2-R expression not only in the liver, but also in
366
the kidneys of BDL-operated animals. Therefore, we hypothesized that its activation may exert
367
an anti-inflammatory effect in BDL-induced HRS by limiting infiltration of CB2-R positive
368
immune cells and by attenuating the vascular inflammatory response. To test this hypothesis we
369
used HU-910, one of the most selective CB2-R agonists with proven specificity and negligible
370
off-target activity in vivo [17], as a chronic therapy in the BDL-induced HRS model. We
371
observed that chronic treatment with HU-910 significantly attenuated inflammatory cell
372
infiltration and endothelial activation in both the liver and kidneys of mice subjected to BDL.
373
Inflammatory cell infiltration was accompanied by direct signs of oxidative/nitrative
374
damage in our mouse model of HRS. Overproduction of superoxide radicals in both the liver and
375
kidneys has been previously described in BDL [34, 35], likewise increased nitric oxide
376
production, in part attributed to the infiltrating inflammatory cells in the kidneys of rats subjected
377
to BDL [36]. Our observations are in agreement with these reports, since we detected a
378
significantly increased iNOS expression in the kidneys which could have contributed to the
379
enhanced peroxynitrite generation from the diffusion limited reaction of superoxide anion and
380
iNOS-derived nitric oxide, consequent protein nitration [37] observed as increment in the renal
381
3-NT staining intensity, as well as to increased lipid peroxidation. Elevation of multiple markers
382
of oxidative stress such as MDA and 4-HNE have also been described in patients with end-stage
383
renal disease [38], [39], with decreased levels following hemodialysis [40], [41]. Moreover,
384
MDA and 4-HNE were shown to correlate with the levels of proinflammatory cytokines [42] and
385
were reported to have direct proinflammatory effects in vitro [43] [44-46].
386
In addition to evident signs of oxidative damage and inflammation, we also detected
387
profound microvascular dysfunction in the kidneys, conforming clinical observations of vascular
388
alterations in renal biopsies of cirrhotic patients [47] or patients with obstructive hepatic damage
389
[48]. This microvascular dysfunction can be the consequence of decreased nitrogen monoxide
390
(NO) bioavailability as a result of increased peroxynitrite generation from superoxide and NO,
391
and also a consequence of increased endothelial activation and vascular inflammation.
392
Altogether,
the
observed
renal
inflammation,
oxidative/nitrative
damage
and
393
microvascular dysfunction might have led to tubular damage and kidney dysfunction [49, 50],
394
which suggest the decline of renal filtration capacity and the presence of both proximal and distal
395
tubular injury in BDL-induced HRS. Since treatment with HU-910 significantly decreased the
396
serum levels of all laboratory indicators of renal impairment, our results indicate that CB2-R
397
activation indeed attenuated the observed renal damage and possibly contributed to the
398
improvement of fluid balance.
399
Development of hepatic fibrosis is a hallmark of the BDL mouse model, which closely
400
resembles the features of end-stage liver disease in human patients [51]. The sustained
401
proinflammatory response induced by cholestasis in BDL significantly contributes to the fibrotic
402
remodeling of the affected organs. Herein, besides the confirmation of liver fibrosis, we detected
403
histological signs of tubulointerstitial fibrosis and a significantly elevated mRNA expression of
404
multiple markers of fibrotic remodeling in the kidneys, histological alterations that have been
405
described in patients with cholestatic liver diseases, too [52, 53]. Treatment with HU-910
406
significantly decreased the signs of fibrosis both in the hepatic and renal tissues, which could
407
possibly be attributed to its anti-inflammatory effects that ameliorated the peculiar fibrotic
408
changes induced by BDL.
409
Collectively, our study highlights the importance of liver inflammation and oxidative
410
stress in the development of inflammatory cell infiltration, oxidative/nitrative injury and
411
microcirculation collapse in the kidneys of mice subjected to BDL, ultimately leading to fibrotic
412
remodeling and renal dysfunction/failure. CB2-R activation exerts anti-inflammatory effects and
413
reduces the extent of oxidative/nitrative tissue injury both in liver and kidneys and improves
414
microcirculation in the kidneys. Thus, targeting the CB2-R in HRS may emerge as a promising
415
new therapeutic avenue.
416
417
Acknowledgements
418
The research was financed by the NIH/NIAAA Intramural Research Program (to PP). ET
419
received financial support from the Rosztoczy Foundation and the NTP-NFTÖ-17 project by the
420
Human Capacities Grant Management Office and the Hungarian Ministry of Human Capacities.
421
The authors are indebted for Prof. Dr. Raphael Mechoulam (Institute for Drug Research, Medical
422
Faculty, Hebrew University, Jerusalem, Israel) for providing HU-910 for their studies.
423
424
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425 426 427 428 429 430 431 432 433 434 435 436 437 438 439 440 441 442 443 444 445 446 447 448 449 450 451 452 453 454 455 456 457 458 459 460 461 462 463 464 465 466 467 468 469
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586
Table 1. Gene symbol
Accession number
Forward primer
Reverse primer
Custom designed primers CD11b CD68 Col1a1 Col3a1 F4/80 p22phox gp91phox MCP1 MIP1α MIP2 NOX4
NM_0010 82960.1 NM_0012 91058.1 NM_0077 42.3 NM_0099 30.2 NM_0101 30 NM_0013 01284.1 NM_0078 07.5 NM_0113 33.3 NM_0113 37.2 NM_0091 40.2 NM_0012 85835.1
GGATCCGGAAAGTAGTGAGAGA AC
CCGAATTTTTCTCCATCTGTGAT
GGGGCTCTTGGGAACTACAC
GTACCGTCACAACCTCCCTG
TGGCCTTGGAGGAAACTTTG
CTTGGAAACCTTGTGGACCAG
GAGGAATGGGTGGCTATCCG
TCGTCCAGGTCTTCCTGACT
CTTTGGCTATGGGCTTCCAGT C ATGGAGCGATGTGGACAGAA G
GCAAGGAGGACAGAGTTTATCG TG
GACCATTGCAAGTGAACACCC
AAATGAAGTGGACTCCACGCG
TGCCCTTGCTGTTCTTCTCTG
CAACGATGAATTGGCGTGG
TGCCCTTGCTGTTCTTCTCTG
CAACGATGAATTGGCGTGG
AGTGAACTGCGCTGTCAATGC
AGGCAAACTTTTTGACCGCC
ACCAAATGTTGGGCGATTGTG
TCCTGCTAGGGACCTTCTGT
TAGATCACACTGGCAATGGCC
p40phox
NM_0086 77.2
TTCAAAGACCTGCTAGCGCT
TCCTTCTGTGTGACATGCAGC
p47phox
NM_0012 86037.1
TTCCATCCCCAAATGCAAAG
TCAGATGCCCTAAAACCGGAG
GCCTTCACCAAAAGCATCAAC
ACCTCACAGGCAAACAGCTTG
GCCAGTTCATGTGCGATGAA
GGCGAAGATTCCTGGACACTT
GTAACCCGTTGAACCCCATT
CCATCCAATCGGTAGTAGCG
TGTGACTGCTGGAAAGGACG
ACTGCGCAATCCCAATCACT
GTGGGAGTCCAAGGTTCAGG
TAGTAAGCGTGCTCCCACAC
TCTACAACCAACACAACCCGG
GAGCGCACAATCATGTTGGAC
p67phox P-selectin Rn18s SOD1 SOD2 TGFβ
NM_0108 77.5 NM_0091 51.3
NR_0032 78.3 NM_0114 34.1 NM_0136 71.3 NM_0115 77.1
TNFα Vcam-1
NM_0136 93.3 NM_0116 93.3
TCTCATTCCTGCTTGTGGCAG
TCCACTTGGTGGTTTGCTACG
CTGGGAAGCTGGAACGAAGT
GCCAAACACTTGACCGTGAC
Commercially available primers Gene symbol Catalase Cnr2 iNOS
587
Accession number NM_0098 04 NM_0099 24 NM_0109 27
Catalog number
Vendor
QT01058106
Qiagen (Germantown, MD, USA)
QT00159558
Qiagen (Germantown, MD, USA)
QT00100275
Qiagen (Germantown, MD, USA)
588
Figure legends
589 590
Figure 1. BDL-induced liver fibrosis, inflammation and oxidative damage are attenuated
591
by HU-910
592
Representative images of (A) Sirius red, F4/80 and 4-HNE-stained liver sections (magnification
593
100x) of each study group, with (B) fibrosis scores of N=6-8 sections in each group. mRNA
594
expression of markers of (C) fibrotic remodeling, (D) inflammatory cell markers, (E)
595
inflammatory cytokines and chemokines and (F) oxidative stress markers evaluated by
596
quantitative real time PCR in N=4-7 samples in each study group. Data are shown as mean with
597
the standard error of mean, with alternating colors reflecting each group (Sham – white,
598
Sham+HU-910 – grey, BDL – black, BDL+HU-910 – red). Statistical analysis was performed
599
with the One way analysis of variance followed by the Tukey’s post hoc test, statistical
600
significance is indicated by asterisks (*p<0.05 vs. Sham, #p<0.05 vs. BDL).
601 602
Figure 2. HU-910 mitigates the BDL-associated renal dysfunction and kidney fibrosis
603
(A) Serum levels of kidney injury markers are displayed including creatinine, blood urea
604
nitrogen (BUN), kidney injury molecule-1 (KIM-1), osteopontin (OPN) and neutrophil
605
gelatinase-associated lipocalin (NGAL) in each study group. (B) Representative images of the
606
hematoxylin & eosin, periodic acid-Schiff (PAS) (magnification 100x) and Masson’s trichrome
607
staining (magnification 200x) together with (C) the pathological scoring of the PAS-stained
608
sections are shown in the renal tissue of BDL- and Sham-operated animals with or without HU-
609
910 therapy. (D) Fibrosis scoring together with the (E) mRNA expression level of various
610
markers of fibrotic remodeling are displayed, showing results of the quantitative real time PCR
611
analysis. Data are shown as mean with the standard error of mean of N=4-8 in each study group,
612
with alternating colors reflecting each group (Sham – white, Sham+HU-910 – grey, BDL –
613
black, BDL+HU-910 – red). Statistical analysis was performed with the One way analysis of
614
variance followed by the Tukey’s post hoc test, statistical significance is indicated by asterisks
615
(*p<0.05 vs. Sham, #p<0.05 vs. BDL).
616 617
Figure 3. Renal inflammation is ameliorated by the application of HU-910
618
(A) Representative images the F4/80 positive inflammatory cell infiltration and of the renal
619
ICAM-1 immunohistochemistry (magnification 200x) are displayed in the renal tissue of BDL
620
and Sham animals with or without HU-910 therapy, with arrows in the enlarged panels
621
indicating the F4/80 positive inflammatory cells. (B) Scoring of the F4/80 staining intensity. (C-
622
D) mRNA expression of various inflammatory markers are presented, showing results of the
623
quantitative real time PCR analysis. (E) Scoring of the ICAM-1 staining intensity. Data are
624
expressed as mean with the standard error of mean of N=4-8 samples in each study group, with
625
alternating colors reflecting each group (Sham – white, Sham+HU-910 – grey, BDL – black,
626
BDL+HU-910 – red). Statistical analysis was performed with the One way analysis of variance
627
followed by the Tukey’s post hoc test, statistical significance is indicated by asterisks (*p<0.05
628
vs. Sham, #p<0.05 vs. BDL).
629 630
Figure 4. Oxidative damage is a key feature of renal injury in BDL
631
(A) Representative images of the 4-hydroxy-2-nonenal (4-HNE), malondialdehyde (MDA) and
632
3-nitrotyrosine (3-NT) staining (magnification 200x) are displayed in the renal tissue of BDL-
633
and Sham-operated animals with or without HU-910 therapy. (B-D) mRNA expression of
634
various antioxidants and indicators of oxidative/nitrative damage are displayed, showing results
635
of the quantitative real time PCR analysis. Data are expressed as mean with the standard error of
636
mean of N=4-8 samples in each study group, with alternating colors reflecting each group (Sham
637
– white, Sham+HU-910 – grey, BDL – black, BDL+HU-910 – red). Statistical analysis was
638
performed with the One way analysis of variance followed by the Tukey’s post hoc test,
639
statistical significance is indicated by asterisks (*p<0.05 vs. Sham, #p<0.05 vs. BDL).
640 641
Figure 5. Renal microvascular flow is recovered by CB2-R activation in BDL-nephropathy
642
(A) Representative images of the renal microvascular flow are displayed in each study group,
643
supplemented by (B) the quantification of the flow intensity. (C) mRNA expression of vascular
644
injury markers are displayed, showing results of the quantitative real time PCR analysis. (D)
645
Hepatic and renal Cnr2 expression is shown, evaluated by droplet digital PCR analysis. Data are
646
expressed as mean with the standard error of mean of N=4-8 samples in each study group, with
647
alternating colors reflecting each group (Sham – white, Sham+HU-910 – grey, BDL – black,
648
BDL+HU-910 – red). Statistical analysis was performed with the One way analysis of variance
649
followed by the Tukey’s post hoc test, statistical significance is indicated by asterisks (*p<0.05
650
vs. Sham, #p<0.05 vs. BDL).
651
Figure 1. A
F4/80
4-HNE
BDL+HU910
BDL
Sham+HU910
Sham
Sirius red
-
+
-
-
-
+
Sham
# +
+
BDL
-
+
Sham
+
BDL
HU910
-
-
+
Sham
CD11b
Col3a1
BDL
-
* -
+
Sham
-
200 150 100 50 0
*
#
40
#
20 0
+
HU910
BDL
+
HU910
BDL
-
+
Sham
-
*
5
-
+
Sham
-
# +
BDL
10 8 6 4 2 0
*
HU910
-
+
Sham
-
p=0.06
+
BDL
*
150 100
#
50 0
+
HU910
BDL
Oxidative stress markers
10
HU910
40 30 20 10 0
HU910
BDL
0
HU910
*
60
-
+
Sham
-
# +
BDL
Inflammatory cytokines and chemokines
15
#
0
+
#
*
*
6 4 2 0
HU910
-
+
Sham
-
-
+
Sham
-
+
BDL
# +
BDL
p22phox
HU910
Sham
5
Inflammatory cell markers 10 8 6 4 2 0
MCP1
*
#
-
+
10
p67phox
* Sham
25 20 15 10 5 0
-
*
15
MIP2
BDL
HU910
MIP1α
Sham
+
*
40 30 20 10 0
gp91phox
p40phox
mRNA expression (fold change)
F
HU910
-
+
mRNA expression (fold change)
E
-
p47phox
0
Col1a1
5
HU910
8 6 4 2 0
mRNA expression (fold change)
* *#
10
TNFα
Fibrosis score
15
D
Markers of fibrotic remodelling
100 µm
F4/80
C
B
100 µm
mRNA expression (fold change)
200 µm
*
6 4 2 0
HU910
-
+
Sham
-
+
BDL
HU910
B
-
+ -
Sham
+
-
HU910
BDL
-
+
Sham
+
0
HU910
BDL
- +
- +
Sham
HU910
BDL
-
PAS
*#
-
+
Sham
1000 800 600 400 200 0
* *#
+
HU910
BDL
-
-
+
Sham
+
BDL
Masson`s trichrome
BDL+HU910
BDL
Sham+HU910
Sham
Hematoxylin-eosin
*#
NGAL
1000
*
2000 1500 1000 500 0
(ng/mL)
2000
OPN
*#
KIM-1
*
*
3000
(ng/mL)
0
#
Serum markers of kidney injury
200 150 100 50 0
(pg/mL)
0.2
BUN
0.4
(mg/dL)
*
0.6
(mg/dL)
Creatinine
Figure 2. A
0
HU910
-
+
Sham
-
+
BDL
HU910
-
+
Sham
-
+
BDL
4 2 0
HU910
-
+
Sham
-
+
BDL
Markers of fibrotic remodelling 5 4 3 2 1 0
*#
HU910
-
+
Sham
-
+
BDL
2.0 1.5 1.0 0.5 0
*#
Tgf β
#
* *#
6
100 µm
Col3a1
5
*
E
Col1a1
* *#
10
5 4 3 2 1 0
200 µm
mRNA expression (fold change)
15
fold change
D
Fibrosis area
C
Pathological score
200 µm
HU910
-
+
Sham
-
+
BDL
Figure 3. A
ICAM-1
BDL+HU910
BDL
Sham+HU910
Sham
F4/80
100 µm
5 0
+
#
-
+
BDL
HU910
-
+
Sham
*
#
-
+
10 8 6 4 2 0
HU910
BDL
5 0
HU910
-
+
Sham
-
# +
BDL
* *#
HU910
-
+
Sham
-
+
BDL
2.0 1.5 1.0 0.5 0
*
TNFα
10
50 40 30 20 10 0
MIP2
*
15
HU910
-
-
*# -
+
Sham
+
BDL
E
Inflammatory chemokines and cytokines MCP1
mRNA expression (fold change)
D
-
Sham
*
4 3 2 1 0
+
Sham
-
#
+
BDL
*
15
ICAM-1 Score
10
CD11b
F4/80 Score
15
Inflammatory cell markers CD68
C
mRNA expression (fold change)
B
HU910
100 µm
25 µm
10 5 0
HU910
-
+
Sham
-
*# +
BDL
Figure 4. A
MDA
3-NT
BDL+HU910
BDL
Sham+HU910
Sham
4-HNE
100 µm
+
BDL
*
6 4 2 0
HU910
-
+
Sham
-
# +
BDL
HU910
-
+
Sham
D
-
HU910
BDL
-
+
Sham
-
+
HU910
BDL
*#
2.5 2.0 1.5 1.0 0.5 0
-
-
+
Sham
+
BDL
*
12
NOX4
* *#
gp91phox
p67phox
+
4 3 2 1 0
8 4 0
HU910
# -
+
Sham
-
+
BDL
Antioxidant enzymes 1.5
1.5
1.0
1.0
0.5 0
HU910
-
+
Sham
** -
#
+
BDL
1.5
0.5 0
HU910
**
#
-
+
Sham
-
+
BDL
1.0
SOD2
-
#
SOD1
+
*
100 µm
Oxidative stress markers
Catalase
-
Sham
5 4 3 2 1 0
mRNA expression (fold change)
HU910
* *#
p47phox
p40phox
4 3 2 1 0
iNOS
C
mRNA expression (fold change)
B
mRNA expression (fold change)
100 µm
0.5 0
HU910
-
+
Sham
# * * -
+
BDL
Microcirculation Sham+HU910
Sham
BDL+HU910
BDL
HU910
* -
+
Sham
-
+
BDL
D
5 0
HU910
-
+
Sham
-
+
BDL
10 8 6 4 2 0
*
P-selectin
*
#
*#
10
HU910
-
+
Sham
-
*# +
BDL
Cnr2 expression by ddPCR
*
15 10
Liver
2500 2000 1500 1000 500 0
INCREASE
Vascular injury markers 15
Vcam-1
Kidney microcirculation
Copy number (fold change)
Flux
B
C
mRNA expression (fold change)
Laser Speckle intensity (flux)
DECREASE
5 0
HU910
-
+
Sham
-
*# +
BDL
15
Kidney
Figure 5. A
10
* *#
5 0
HU910
-
+
Sham
-
+
BDL
S0891-5849(19)31745-9
DOI:
https://doi.org/10.1016/j.freeradbiomed.2019.11.027
Reference:
FRB 14498
To appear in:
Free Radical Biology and Medicine
Received Date: 15 October 2019 Accepted Date: 21 November 2019
Please cite this article as: E. Trojnar, K. Erdelyi, C. Matyas, S. Zhao, J. Paloczi, P. Mukhopadhyay, Z.V. Varga, G. Hasko, P. Pacher, Cannabinoid-2 receptor activation ameliorates hepatorenal syndrome, Free Radical Biology and Medicine (2019), doi: https://doi.org/10.1016/j.freeradbiomed.2019.11.027. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Inc.
Cannabinoid-2 receptor activation
Inflammation
Inflammation
Bile duct ligation
Oxidative stress
Oxidative stress
Fibrosis
Fibrosis
Microcirculation
ROS
Liver failure
Paracrine mediators (cytokines, chemokines, oxidants)
Activation
Endothelium
ROS
Hepatorenal syndrome
1
Cannabinoid-2 receptor activation ameliorates hepatorenal syndrome
2 3
Eszter Trojnar*1, Katalin Erdelyi*1, Csaba Matyas1, Suxian Zhao1, Janos Paloczi1, Partha
4
Mukhopadhyay1, Zoltan V. Varga1, Gyorgy Hasko2, and Pal Pacher1
5 6
*These authors contributed equally to this work
7 8
Affiliations:
9
1
Laboratory of Cardiovascular Physiology and Tissue Injury, National Institute on Alcohol
10
Abuse and Alcoholism (NIAAA), 5625 Fishers Lane, 20852 Rockville, MD, USA
11
2
Department of Anesthesiology, Columbia University, New York, NY 10032, USA
12 13
Corresponding author:
14
Pal Pacher, MD, PhD, FAHA, FACC
15
Laboratory of Cardiovascular Physiology and Tissue Injury
16
5625 Fishers Lane, Room 2N-17; Bethesda, MD 20892-9413
17
Phone: (301)443-4830
18
Email: [email protected]
19 20
Email addresses of each author:
21
[email protected]
22
[email protected]
23
[email protected]
24
[email protected]
25
[email protected]
26
[email protected]
27
[email protected]
28
[email protected]
29
[email protected]
30
Conflict of interest disclosure: The authors have no conflict of interest to disclose.
31
Keywords: Hepatorenal syndrome, Cannabinoid-2 receptor, HU-910, bile-duct ligation,
32
endocannabinoid system
33
34 35
Highlights
36
• Bile duct ligation (BDL) causes hepatorenal syndrome (HRS)
37
• Oxidative damage/inflammation drives liver and kidney injury following BDL
38
• Cannabinoid-2 receptor (CB2-R) activation attenuates hepatic damage in BDL
39
• CB2-R activation mitigates the renal inflammation and oxidative damage in BDL
40
• CB2-R activation attenuates renal microcirculatory dysfunction in BDL
41
42
Abstract
43
Study rationale: Hepatorenal syndrome (HRS) is a life-threatening complication of end-stage
44
liver disease characterized by the rapid decline of kidney function. Herein, we explored the
45
therapeutic potential of targeting the cannabinoid 2 receptor (CB2-R) utilizing a commonly used
46
mouse model of liver fibrosis and hepatorenal syndrome (HRS), induced by bile duct ligation
47
(BDL).
48
Methods: Gene expression analysis, histological evaluation, determination of serum levels of
49
renal injury-biomarkers were used to characterize the BDL-induced organ injury; laser speckle
50
analysis to measure microcirculation in the kidneys.
51
Key results: We found that liver injury triggered marked inflammation and oxidative stress also
52
in the kidneys of BDL-operated mice. We detected pronounced histopathological alterations with
53
tubular injury paralleled with increased inflammation, oxidative/nitrative stress and fibrotic
54
remodeling both in hepatic and renal tissues as well as endothelial activation and markedly
55
impaired renal microcirculation. This was accompanied by increased CB2-R expression in both
56
liver and the kidney tissues of diseased animals. A selective CB2-R agonist, HU-910, markedly
57
decreased numerous markers of inflammation, oxidative stress and fibrosis both in the liver and
58
in the kidneys. HU-910 also attenuated markers of kidney injury and improved the impaired
59
renal microcirculation in BDL-operated mice.
60
Conclusions: Our results suggest that oxidative stress, inflammation and microvascular
61
dysfunction are key events in the pathogenesis of BDL-associated renal failure. Furthermore, we
62
demonstrate that targeting the CB2-R by selective agonists may represent a promising new
63
avenue to treat HRS by attenuating tissue and vascular inflammation, oxidative stress, fibrosis
64
and consequent microcirculatory dysfunction in the kidneys.
65
66
Abbreviations
67
4-HNE – 4-hydroxy-2-nonenal
68
BDL – bile duct ligation
69
BUN – blood urea nitrogen
70
CB1-R – cannabinoid-1 receptor
71
CB2-R – cannabinoid-2 receptor
72
DAB – 3,3'-diaminobenzidine
73
ddPCR – droplet digital PCR
74
ECS – endocannabinoid system
75
FFPE – formalin-fixed and paraffin-embedded
76
HRP – horseradish peroxidase
77
HRS – hepatorenal syndrome
78
ICAM-1 – intercellular-adhesion molecule 1
79
KIM-1 – kidney injury molecule-1
80
MDA – malondialdehyde
81
NADPH oxidase– nicotinamide adenine dinucleotide phosphate oxidase
82
NGAL – neutrophil gelatinase-associated lipocalin
83
NO – nitrogen monoxide
84
OPN – osteopontin
85
3-NT – 3-nitrotyrosine
86
iNOS – inducible nitric-oxide synthase
87
88
Introduction
89
End-stage liver disease has been associated with increasing mortality rate during the past
90
two decades representing a major epidemiological burden in developed countries [1].
91
Independent of the etiology of liver cirrhosis, its association with kidney function decline, and
92
subsequent development of extrahepatic complications is a major determinant of the clinical
93
outcome [2]. Up to 50% of hospitalized patients with liver cirrhosis may develop renal
94
dysfunction [1, 2], 20% of which fulfills the diagnostic criteria of HRS [1]. HRS is associated
95
with a poor prognosis and high mortality [3], with more than half of the patients with
96
concomitant cirrhosis and renal failure dying within one month of diagnosis [4]. Despite the
97
significant advancement in the understanding of the pathophysiology of HRS, still no specific
98
pharmacological approaches are available to treat this devastating condition. The exact molecular
99
steps leading to kidney function decline during chronic liver diseases/cirrhosis are poorly
100
understood, necessitating further investigations to identify novel therapeutic approaches for the
101
management of this condition.
102
Bile duct ligation (BDL) is an established animal model of cholestatic liver damage and
103
fibrosis [5]. Following BDL, due to exhaustive bile flow blockade, increased levels of
104
intrahepatic and circulatory bile acids provoke an extensive inflammatory response and oxidative
105
stress with subsequent production of free radicals that ultimately lead to cytotoxicity and tissue
106
fibrosis in the hepatic tissue [6]. Time-dependent features of progressive renal dysfunction
107
mimicking HRS in humans associated with BDL in mice have been recently described [7],
108
promoting this model as a valuable in vivo tool to study the HRS pathogenesis and management.
109
The cannabinoid-2 receptor (CB2-R), primarily expressed in inflammatory cells and
110
activated endothelium, is a G-protein coupled receptor. The activation of this receptor by
111
endocannabinoids or synthetic ligands has been reported to exert anti-inflammatory effects in
112
models of atherosclerosis, stroke, myocardial, hepatic, and renal injury [8-13], [14-16].
113
In this study, we explored the potential role of inflammation and the disruption of renal
114
redox homeostasis in the evolvement of BDL-associated HRS. We also demonstrate by using a
115
selective CB2-R agonist HU-910 [17, 18] that targeting CB2-R attenuates not only liver injury,
116
but also kidney inflammation, oxidative/nitrative stress, and microcirculatory collapse in BDL,
117
suggesting a potential clinical utility of selective CB2-R agonists in HRS in humans.
118
119
Methods
120
Study design and animals
121
All animal experiments reported in this manuscript complied with the National Institutes of
122
Health “Guide for the Care and Use of Laboratory Animals” (NIH publication 86-23 revised
123
1985) and were approved by the Institutional Animal Care and Use Committee of the National
124
Institute on Alcohol Abuse and Alcoholism (Bethesda, MD). Animals were kept in the institute`s
125
designated animal facility under constant temperature (22±2°C) and humidity, with 12-hour
126
alternating light and dark cycles. For all experiments listed, 12-16 week-old C57BL/6J male
127
mice were used (The Jackson Laboratory, Bar Harbor, ME, USA) and bile duct ligation was
128
performed as described earlier [7, 19], with Sham-operated mice used as controls. Following
129
surgery, the animals were randomly assigned to one of four groups: Sham (vehicle-treated),
130
Sham (HU-910-treated), BDL (vehicle-treated) and BDL (HU-910-treated). Starting on post-
131
operative day 1, all animals received a daily intraperitoneal injection of treatment or vehicle
132
only, consisting of 10mg/kg of the CB2-R agonist HU-910 (Institute for Drug Research, The
133
Hebrew University of Jerusalem, Israel), dissolved in dimethyl sulfoxide, Tween-80 and distilled
134
water in a ratio of 1:1:8. Animals were sacrificed on post-operative day 14, and respective tissues
135
were harvested following exsanguination and perfusion with sterile, cold phosphate buffered
136
saline.
137 138
Microvascular flow determination
139
On the post-operative day 14, kidney microcirculation was assessed by the application of the
140
laser speckle contrast method. Animals were anesthetized with 1-2% isoflurane, while their body
141
temperature was maintained at 37ºC. After opening the abdominal cavity, the left kidney was
142
exposed and scanned with MoorFLPI-2 blood flow imager (Moor Instruments, Wilmington, DE,
143
USA). Microcirculation was quantified and expressed as flux arbitrary units with the analysis
144
software provided by Moor Instruments.
145 146
Histological evaluation of organ impairment
147
For pathological evaluation of structural alterations, 5 µm thick liver and kidney sections were
148
cut from formalin-fixed and paraffin-embedded (FFPE) tissues, and stained for hematoxylin &
149
eosin, periodic acid-Schiff, Masson’s trichrome (Thermo Fisher Scientific, Waltham, MA, USA)
150
and Sirius red (Electron Microscopy Sciences, PA, USA) using conventional staining methods
151
following the manufacturer’s instructions.
152
Evaluation of fibrosis was performed on Sirius red-stained liver and Masson`s trichrome-stained
153
kidney tissue sections. Quantification of the Sirius-red stained area and staining intensity was
154
performed by a blinded investigator using the following scoring system: staining intensity: 1: no
155
staining, 2: weak staining, 3: moderate staining, 4: strong staining; area positivity: 1: 0-25%, 2:
156
25-50%, 3: 50-75%, 4: 75-100%. Area*intensity score values were used to compare intergroup
157
differences. Intensity of the Masson`s-trichrome staining was evaluated by Image J.
158
Histopathological alterations in the kidneys were assessed on periodic acid-Schiff-stained tissue
159
sections. A blinded investigator evaluated five randomly selected areas under 100x magnification
160
and scored the extent of tubular dilatation, brush border loss, intraluminal cast formation, the
161
presence of interstitial edema and inflammatory cell infiltration on a scale of zero to five (0: 0-
162
10%, 1: 11-25%, 2: 25-50%, 3: 50-75%, 4: 75-100%). The thus obtained values were added up
163
and the average summary scores of each study group are presented in the respective figure panel.
164 165
Immunohistochemistry
166
Evaluation of oxidative and nitrative organ damage and endothelial activation was performed on
167
FFPE tissue sections using the Mouse on Mouse (M.O.M.®) ImmPRESS® HRP (Peroxidase)
168
Polymer Kit or the ImmPRESS® Excel Amplified HRP Polymer Staining Kit (Anti-Rabbit IgG)
169
(Vector Laboratories, Burlingame, CA, USA), respectively. Following deparaffination and
170
antigen retrieval with 1x citrate buffer (95ºC for 15 minutes), endogenous peroxidase activity
171
was blocked by BloxAll reagent (Vector Laboratories), while the aspecific binding sites were
172
blocked with 2.5% normal-horse serum (Vector Laboratories). The below primary antibodies
173
were applied overnight at 4ºC: anti-4-hydroxy-2-nonenal (4-HNE) monoclonal antibody (1:200,
174
Japan Institute for the control of Aging # MHN-100P, Japan), anti-malondialdehyde (MDA)
175
monoclonal antibody (1:100, Japan Institute for the control of Aging #MMD-030n, Japan), anti-
176
3-nitrotyrosine (3-NT) polyclonal antibody (1:500, Cayman Chemicals #10189540, Ann Arbor,
177
MI, USA) and anti ICAM-1 monoclonal antibody (1:4000, SinoBiological #50440-R020,
178
Wayne, PA, USA). Horseradish peroxidase (HRP)-conjugated secondary antibodies were used
179
conforming to the antibody host and the manufacturer's instructions. Signal development was
180
carried out with 3,3'-diaminobenzidine (DAB), followed by hematoxylin counter-staining.
181
For the assessment of inflammatory cell infiltration staining of F4/80 positive macrophages on
182
FFPE tissue sections was performed using the ImmPRESS® HRP Anti-Rat IgG (Peroxidase)
183
Polymer Detection Kit, made in Goat (Vector Laboratories). Following dehydration of the tissue
184
sections, antigen retrieval was performed via Proteinase K-based digestion in Tris-EDTA buffer
185
(pH: 8). The primary antibody, F4/80 (1:50, BioRad #MCA497, Hercules, CA, USA) was
186
applied overnight at 4oC and signal development was performed with an HRP-conjugated
187
secondary antibody and DAB.
188
Quantification of the stained area and staining intensity was performed by two blinded
189
investigators using the following scoring system: staining intensity: 1: no staining, 2: weak
190
staining, 3: moderate staining, 4: strong staining; area positivity: 1: 0-25%, 2: 25-50%, 3: 50-
191
75%, 4: 75-100%. Area*intensity score values were used to compare intergroup differences.
192 193
Serum chemistry analysis
194
Laboratory markers of kidney function were assessed from sera of the animals collected on post-
195
operative day 14. Creatinine and blood urea nitrogen (BUN) levels were determined via the
196
Idexx VetTest 8008 (Idexx Laboratories, Westbrook, ME) system, while Kidney Injury
197
Molecule-1 (KIM-1), Neutrophil Gelatinase-Associated Lipocalin (NGAL) and Osteopontin
198
(OPN) levels were determined using commercially available ELISA assays (R&D Systems,
199
Minneapolis, MN, USA).
200 201
Gene expression analysis by quantitative real-time PCR and droplet digital PCR
202
RNA isolation for gene expression analysis was performed from hepatic and renal tissues using
203
the Direct-zol RNA Miniprep Kit (Zymo Research, Irvine, CA, USA) according to the
204
manufacturer’s instructions, following the homogenization of 15-20 mg tissue pieces in TRIzol
205
reagent (Thermo Fisher Scientific, Waltham, MA, USA) and separation of the RNA-rich
206
aqueous layer with chloroform. From each sample, the isolated RNA was reverse transcribed
207
using the High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific). cDNA
208
samples were then used for subsequent quantitative real-time PCR experiments with SyberSelect
209
PCR Master Mix (Thermo Fisher Scientific) on an ABI 7900HT Realtime PCR Instrument
210
(Applied Biosystems, Foster City, CA, USA) with each reaction performed in duplicates.
211
Relative quantification of target gene expression was calculated with the comparative CT
212
method. The average value of the housekeeping gene encoding the 18S ribosomal RNA (Rn18s)
213
was used as reference. Expression levels of the following target genes were analyzed with primer
214
pairs listed in Table 1.: CD11b, CD68, CB2-R (Cnr2), collagen, type I, alpha 1 (Col1a1),
215
collagen, type III, alpha 1 (Col3a1), F4/80, monocyte chemoattractant protein 1 (MCP1),
216
macrophage inflammatory protein 1α (MIP1α), chemokine (C-X-C motif) ligand 2 (MIP2),
217
NADPH oxidase 4 (NOX4), p22phox, gp91phox, p40phox, p47phox, p67phox, P-selectin,
218
superoxide dismutase 1-2 (SOD1, SOD2), transforming growth factor beta (Tgfβ), tumor necrosis
219
factor alpha (TNFα), vascular adhesion molecule 1 (Vcam-1), Catalase, inducible nitric-oxide
220
synthase (iNOS).
221
Expression of the CB2-R in the hepatic and renal tissues was further assessed by droplet digital
222
PCR (ddPCR) analysis using the QX200 ddPCR System (Bio-Rad, Hercules, CA, USA). For the
223
absolute quantification of the Cnr2 copy numbers EvaGreen-based chemistry (Bio-Rad) was
224
applied and equal amounts of cDNA, isolated from livers and kidneys of BDL and Sham
225
operated animals were loaded into separate assay wells. Following droplet generation and
226
thermal cycling, the Cnr2 copy number was determined using the QX200 Droplet Reader and the
227
QuantaSoft Software (Bio-Rad), with the same threshold applied in the corresponding study
228
groups for the determination of positive droplet count.
229 230
Statistical analysis
231
For statistical comparison of datasets, the Graph Pad Prism software version 6.00 was used
232
(GraphPad Software, La Jolla, CA, www.graphpad.com). In all figures, group mean values with
233
the standard error of mean are presented with the respective statistical test indicated in each
234
figure legend and significance level set at p<0.05.
235
236
Results
237
The BDL-induced hepatic inflammatory response, oxidative damage and liver fibrosis in mice is
238
attenuated by a CB2-R agonist
239
14 days post-operation peculiar structural alterations evolved in the livers of BDL-operated mice.
240
Prominent dilatation of the intrahepatic bile ducts was observed and signs of parenchymal
241
fibrosis was apparent on the Sirius red-stained tissue sections (Figure 1A-B). Massive
242
inflammatory cell infiltration of the portal areas was detected and confirmed by
243
immunohistochemistry using a specific cell surface marker of macrophages (F4/80) (Figure 1A,
244
middle panels). Inflammation and liver injury were accompanied by signs of oxidative damage in
245
BDL, as evidenced by the increased staining intensity of the lipid peroxidation marker 4-HNE in
246
the hepatic tissues (Figure 1A, right panels).
247
Histological features of fibrosis were paralleled by significantly elevated expression levels of
248
multiple markers of fibrotic remodeling such as Col1a1 and Col3a1 mRNA in BDL compared to
249
Sham (Figure 1C). Inflammatory cell infiltration was accompanied by a substantially increased
250
expression level of various proinflammatory cytokines and chemokines as well as a notable
251
increase in the expression level of inflammatory cell markers in the hepatic tissue of BDL-
252
operated animals in comparison to Sham-operated ones (Figure 1D-E). In agreement with
253
increased lipid peroxidation, we detected a markedly elevated expression level of multiple
254
isoforms and subunits of the phagocytic reactive oxygen species generating NADPH oxidases
255
(p40phox, p47phox, gp91phox, p67phox, p22phox), in BDL versus Sham mice (Figure 1F).
256
Chronic treatment with HU-910 attenuated the BDL-induced hepatic damage. CB2-R activation
257
alleviated the observed fibrotic changes (Figure 1A-C), exerted an anti-inflammatory effect via
258
both mitigation of the inflammatory cell infiltration (Figure 1A) and significant reduction of the
259
expression levels of inflammatory cell markers, cytokines and chemokines (Figure 1D-E). In line
260
with the attenuation of the inflammatory response, treatment with HU-910 in BDL also
261
effectively decreased the extent of oxidative injury, as shown by the reduced hepatic 4-HNE
262
staining intensity (Figure 1A), and moderation of the BDL-induced increment in gene expression
263
of NADPH oxidase isoforms (Figure 1F). The treatment by itself had no effect on the above-
264
mentioned variables in Sham-operated animals.
265
The BDL-induced extensive kidney injury is attenuated by a CB2-R agonist
266
As an extrahepatic complication of liver fibrosis, a progressive kidney function decline following
267
BDL was observed in mice (Figure 2). Classical laboratory signs of renal dysfunction (BUN and
268
creatinine levels) in BDL- compared to Sham-operated mice indicated reduction of renal
269
filtration capacity (Figure 2A). In addition to that, significantly increased KIM-1, OPN and
270
NGAL serum levels were measured, suggesting the presence of both proximal and distal tubular
271
damage in the kidneys (Figure 2A). Consistently with prior observations, histological signs of
272
marked renal tubular injury were evident by 14 days post-operation [7] (Figure 2B-C). In
273
vehicle-treated BDL mice we detected extensive tubular dilatation with signs of epithelial cell
274
necrosis, leukocyte infiltration and nuclear enlargement or loss of nuclear staining, respectively
275
(Figure 2B). In some areas flattening of the epithelial cells and the loss of cellular architecture
276
indicated atrophy of the tubuli. BDL was also associated with visible signs of interstitial fibrosis
277
in the kidneys of mice. These included a notable expansion of the collagenous area on Masson’s
278
trichrome-stained tissue sections (Figure 2B, D), corroborated by the increased expression level
279
of various indicators of fibrotic remodeling (Col1a1, Col3a1, TGFβ mRNA) in the renal tissue
280
(Figure 2E).
281
Similar to the BDL-induced hepatic injury, histological signs of kidney damage were
282
significantly attenuated by HU-910 treatment. HU-910 treatment in BDL attenuated tubular
283
dilatation with better preservation of the cellular architecture in large parts of the cortical area
284
(Figure 2B). Laboratory signs of kidney dysfunction were also ameliorated in the treatment
285
group, suggesting a partial rescue of the reduced renal filtration capacity and tubular injury
286
(Figure 2A). Furthermore, HU-910 also improved both the histological and genetic signs of renal
287
fibrosis (Figure 2B, D), while the drug by itself exerted no effect in Sham-operated animals.
288 289
The BDL-induced renal inflammation and oxidative/nitrative stress is attenuated by a CB2-R
290
agonist
291
To explore the molecular background of the observed organ impairment, we evaluated the extent
292
of inflammation and oxidative damage in the kidneys. We found a notable inflammatory cell
293
infiltration of the cortical area in BDL, shown by the increased F4/80 staining intensity
294
compared to the lack of F4/80 positive cells in the control groups (Figure 3A, left and middle
295
panels, Figure 3B). We also detected an increased staining intensity of the intercellular adhesion
296
molecule ICAM-1 in BDL compared to Sham-operated as well as HU-910-treated BDL animals
297
(Figure 3A, right panels, Figure 3E). The histological signs of inflammatory cell infiltration were
298
corroborated by increases in the expression levels of inflammatory cell markers (CD68 and
299
CD11b) (Figure 3C), as well as inflammatory cytokines and chemokines (MCP1, MIP2, TNFα)
300
(Figure 3D) in the kidneys of BDL-mice compared to Sham-operated ones.
301 302
The extensive renal inflammatory response was accompanied by signs of markedly enhanced
303
oxidative damage in the kidneys (Figure 4). An increased intensity of the lipid peroxidation
304
markers 4-HNE and MDA, as well as an enhanced renal 3-NT staining intensity were detected in
305
BDL (Figure 4A). The histological signs of oxidative and nitrative damage were further
306
supported by the increased renal expression of the mRNAs of p40phox, p47phox, p67phox,
307
gp91phox and NOX4 (Figure 4B) and an elevated iNOS expression level (Figure 4C), in parallel
308
with a significant reduction in the renal expression level of mRNA of cellular antioxidant
309
enzymes (Catalase, SOD1, SOD2) (Figure 4D).
310 311
HU-910 treatment in BDL not only attenuated the inflammatory cell infiltration (Figure 3), but it
312
also significantly abrogated the renal oxidative/nitrative stress, as evidenced by the reduced
313
staining intensity of the 4-HNE, MDA and 3-NT stainings, and the significant reduction of iNOS
314
and NADPH oxidase isoforms mRNA expression with partial recovery of the expression levels
315
of cellular antioxidant enzymes (Figure 4).
316 317
Nephropathy in BDL is associated with endothelial activation/inflammatory response and
318
decline of the renal microvascular flow, which is attenuated by CB2-R activation
319
In addition to the prominent inflammatory response and oxidative damage observed in BDL, the
320
progressive renal injury also involved notable endothelial activation and the reduction of the
321
microvascular flow in the kidneys, as confirmed by the reduced laser speckle intensity on the
322
surface of the renal tissue (Figure 5A-B). The reduction of the microvascular flow was
323
associated with a significant increment in the expression level of the endothelial
324
activation/inflammation markers Vcam-1 and P-selectin (Figure 5C). All of these pathological
325
alterations were partially restored in BDL-animals by HU-910 treatment.
326 327
Enhanced CB2-R expression in BDL is attenuated by CB2-R agonist
328
With the application of droplet digital PCR analysis we determined the absolute copy number of
329
the Cnr2 gene in both the hepatic and renal tissues of Sham and BDL-operated animals and
330
found that the CB2-R expression was significantly elevated in BDL, in both the liver and the
331
kidneys, whereas its levels remained very low in the control group (Figure 5D). We also
332
analyzed in detail the gene expression level of the Cnr2 gene in the kidneys and liver of BDL
333
and Sham operated mice following the application of the CB2-R agonist, HU-910. Consistently
334
with the attenuation of inflammatory cell infiltration and vascular inflammation (inflammatory
335
cells and activated endothelium express CB2-R) we found that chronic treatment with HU-910
336
significantly reduced the Cnr2 expression levels both in the liver and kidneys of BDL-operated
337
mice.
338
339
Discussion
340 341
In this study we confirm that BDL leads to progressive renal failure on the basis of liver
342
inflammation, oxidative stress and fibrosis, resembling characteristic features of HRS. We
343
conclude that tissue inflammation and oxidative damage are major driving forces of kidney
344
injury in BDL, leading to endothelial activation/inflammatory response and collapse of the renal
345
microcirculation. We also show increased expression of CB2-R in the liver and kidneys of
346
animals exposed to BDL and that a selective CB2-R agonist, HU-910, by attenuating tissue and
347
vascular inflammation, oxidative/nitrative stress and fibrosis significantly improves the HRS in
348
the mouse model of BDL.
349
Under physiological conditions CB1- and CB2-R expression are low in the hepatic tissue
350
[20-22]. However, dysregulation of the endocannabinoid system (involving CB1/2 receptors,
351
endocannabinoids and their metabolizing and synthetic enzymes) have been shown to play an
352
important role in the pathogenesis of liver cirrhosis and its complications [11, 22]. Activation of
353
the CB1-R on parenchymal and inflammatory cells and the vascular endothelium was reported to
354
stimulate hepatic inflammation and fibrogenesis [11, 20, 23], as well as to promote vasodilation
355
and cardiac dysfunction thus contributing to the decompensation of liver cirrhosis and
356
development of cirrhotic cardiomyopathy [24].
357
In normal liver CB2-R is primarily expressed in Kupffer cells (resident macrophages of
358
the liver) [25, 26]. Increment of hepatic CB2-R expression under pathological conditions is
359
mostly attributed to influx/activation of inflammatory cells and activated sinusoidal endothelial
360
cells [25, 27, 28]. In contrast to the CB1-R mediated effects, signaling through CB2-R has been
361
shown to exert anti-inflammatory and anti-fibrogenic effects in numerous pathological
362
conditions and in vivo as well as in vitro disease models of hepatic injury [18, 23, 28-31].
363
Activation of the CB2-R has also been shown to be beneficial in cisplatin- and diabetes-induced
364
nephropathy models [14, 16, 23, 32, 33].
365
We detected a notable elevation of the CB2-R expression not only in the liver, but also in
366
the kidneys of BDL-operated animals. Therefore, we hypothesized that its activation may exert
367
an anti-inflammatory effect in BDL-induced HRS by limiting infiltration of CB2-R positive
368
immune cells and by attenuating the vascular inflammatory response. To test this hypothesis we
369
used HU-910, one of the most selective CB2-R agonists with proven specificity and negligible
370
off-target activity in vivo [17], as a chronic therapy in the BDL-induced HRS model. We
371
observed that chronic treatment with HU-910 significantly attenuated inflammatory cell
372
infiltration and endothelial activation in both the liver and kidneys of mice subjected to BDL.
373
Inflammatory cell infiltration was accompanied by direct signs of oxidative/nitrative
374
damage in our mouse model of HRS. Overproduction of superoxide radicals in both the liver and
375
kidneys has been previously described in BDL [34, 35], likewise increased nitric oxide
376
production, in part attributed to the infiltrating inflammatory cells in the kidneys of rats subjected
377
to BDL [36]. Our observations are in agreement with these reports, since we detected a
378
significantly increased iNOS expression in the kidneys which could have contributed to the
379
enhanced peroxynitrite generation from the diffusion limited reaction of superoxide anion and
380
iNOS-derived nitric oxide, consequent protein nitration [37] observed as increment in the renal
381
3-NT staining intensity, as well as to increased lipid peroxidation. Elevation of multiple markers
382
of oxidative stress such as MDA and 4-HNE have also been described in patients with end-stage
383
renal disease [38], [39], with decreased levels following hemodialysis [40], [41]. Moreover,
384
MDA and 4-HNE were shown to correlate with the levels of proinflammatory cytokines [42] and
385
were reported to have direct proinflammatory effects in vitro [43] [44-46].
386
In addition to evident signs of oxidative damage and inflammation, we also detected
387
profound microvascular dysfunction in the kidneys, conforming clinical observations of vascular
388
alterations in renal biopsies of cirrhotic patients [47] or patients with obstructive hepatic damage
389
[48]. This microvascular dysfunction can be the consequence of decreased nitrogen monoxide
390
(NO) bioavailability as a result of increased peroxynitrite generation from superoxide and NO,
391
and also a consequence of increased endothelial activation and vascular inflammation.
392
Altogether,
the
observed
renal
inflammation,
oxidative/nitrative
damage
and
393
microvascular dysfunction might have led to tubular damage and kidney dysfunction [49, 50],
394
which suggest the decline of renal filtration capacity and the presence of both proximal and distal
395
tubular injury in BDL-induced HRS. Since treatment with HU-910 significantly decreased the
396
serum levels of all laboratory indicators of renal impairment, our results indicate that CB2-R
397
activation indeed attenuated the observed renal damage and possibly contributed to the
398
improvement of fluid balance.
399
Development of hepatic fibrosis is a hallmark of the BDL mouse model, which closely
400
resembles the features of end-stage liver disease in human patients [51]. The sustained
401
proinflammatory response induced by cholestasis in BDL significantly contributes to the fibrotic
402
remodeling of the affected organs. Herein, besides the confirmation of liver fibrosis, we detected
403
histological signs of tubulointerstitial fibrosis and a significantly elevated mRNA expression of
404
multiple markers of fibrotic remodeling in the kidneys, histological alterations that have been
405
described in patients with cholestatic liver diseases, too [52, 53]. Treatment with HU-910
406
significantly decreased the signs of fibrosis both in the hepatic and renal tissues, which could
407
possibly be attributed to its anti-inflammatory effects that ameliorated the peculiar fibrotic
408
changes induced by BDL.
409
Collectively, our study highlights the importance of liver inflammation and oxidative
410
stress in the development of inflammatory cell infiltration, oxidative/nitrative injury and
411
microcirculation collapse in the kidneys of mice subjected to BDL, ultimately leading to fibrotic
412
remodeling and renal dysfunction/failure. CB2-R activation exerts anti-inflammatory effects and
413
reduces the extent of oxidative/nitrative tissue injury both in liver and kidneys and improves
414
microcirculation in the kidneys. Thus, targeting the CB2-R in HRS may emerge as a promising
415
new therapeutic avenue.
416
417
Acknowledgements
418
The research was financed by the NIH/NIAAA Intramural Research Program (to PP). ET
419
received financial support from the Rosztoczy Foundation and the NTP-NFTÖ-17 project by the
420
Human Capacities Grant Management Office and the Hungarian Ministry of Human Capacities.
421
The authors are indebted for Prof. Dr. Raphael Mechoulam (Institute for Drug Research, Medical
422
Faculty, Hebrew University, Jerusalem, Israel) for providing HU-910 for their studies.
423
424
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425 426 427 428 429 430 431 432 433 434 435 436 437 438 439 440 441 442 443 444 445 446 447 448 449 450 451 452 453 454 455 456 457 458 459 460 461 462 463 464 465 466 467 468 469
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586
Table 1. Gene symbol
Accession number
Forward primer
Reverse primer
Custom designed primers CD11b CD68 Col1a1 Col3a1 F4/80 p22phox gp91phox MCP1 MIP1α MIP2 NOX4
NM_0010 82960.1 NM_0012 91058.1 NM_0077 42.3 NM_0099 30.2 NM_0101 30 NM_0013 01284.1 NM_0078 07.5 NM_0113 33.3 NM_0113 37.2 NM_0091 40.2 NM_0012 85835.1
GGATCCGGAAAGTAGTGAGAGA AC
CCGAATTTTTCTCCATCTGTGAT
GGGGCTCTTGGGAACTACAC
GTACCGTCACAACCTCCCTG
TGGCCTTGGAGGAAACTTTG
CTTGGAAACCTTGTGGACCAG
GAGGAATGGGTGGCTATCCG
TCGTCCAGGTCTTCCTGACT
CTTTGGCTATGGGCTTCCAGT C ATGGAGCGATGTGGACAGAA G
GCAAGGAGGACAGAGTTTATCG TG
GACCATTGCAAGTGAACACCC
AAATGAAGTGGACTCCACGCG
TGCCCTTGCTGTTCTTCTCTG
CAACGATGAATTGGCGTGG
TGCCCTTGCTGTTCTTCTCTG
CAACGATGAATTGGCGTGG
AGTGAACTGCGCTGTCAATGC
AGGCAAACTTTTTGACCGCC
ACCAAATGTTGGGCGATTGTG
TCCTGCTAGGGACCTTCTGT
TAGATCACACTGGCAATGGCC
p40phox
NM_0086 77.2
TTCAAAGACCTGCTAGCGCT
TCCTTCTGTGTGACATGCAGC
p47phox
NM_0012 86037.1
TTCCATCCCCAAATGCAAAG
TCAGATGCCCTAAAACCGGAG
GCCTTCACCAAAAGCATCAAC
ACCTCACAGGCAAACAGCTTG
GCCAGTTCATGTGCGATGAA
GGCGAAGATTCCTGGACACTT
GTAACCCGTTGAACCCCATT
CCATCCAATCGGTAGTAGCG
TGTGACTGCTGGAAAGGACG
ACTGCGCAATCCCAATCACT
GTGGGAGTCCAAGGTTCAGG
TAGTAAGCGTGCTCCCACAC
TCTACAACCAACACAACCCGG
GAGCGCACAATCATGTTGGAC
p67phox P-selectin Rn18s SOD1 SOD2 TGFβ
NM_0108 77.5 NM_0091 51.3
NR_0032 78.3 NM_0114 34.1 NM_0136 71.3 NM_0115 77.1
TNFα Vcam-1
NM_0136 93.3 NM_0116 93.3
TCTCATTCCTGCTTGTGGCAG
TCCACTTGGTGGTTTGCTACG
CTGGGAAGCTGGAACGAAGT
GCCAAACACTTGACCGTGAC
Commercially available primers Gene symbol Catalase Cnr2 iNOS
587
Accession number NM_0098 04 NM_0099 24 NM_0109 27
Catalog number
Vendor
QT01058106
Qiagen (Germantown, MD, USA)
QT00159558
Qiagen (Germantown, MD, USA)
QT00100275
Qiagen (Germantown, MD, USA)
588
Figure legends
589 590
Figure 1. BDL-induced liver fibrosis, inflammation and oxidative damage are attenuated
591
by HU-910
592
Representative images of (A) Sirius red, F4/80 and 4-HNE-stained liver sections (magnification
593
100x) of each study group, with (B) fibrosis scores of N=6-8 sections in each group. mRNA
594
expression of markers of (C) fibrotic remodeling, (D) inflammatory cell markers, (E)
595
inflammatory cytokines and chemokines and (F) oxidative stress markers evaluated by
596
quantitative real time PCR in N=4-7 samples in each study group. Data are shown as mean with
597
the standard error of mean, with alternating colors reflecting each group (Sham – white,
598
Sham+HU-910 – grey, BDL – black, BDL+HU-910 – red). Statistical analysis was performed
599
with the One way analysis of variance followed by the Tukey’s post hoc test, statistical
600
significance is indicated by asterisks (*p<0.05 vs. Sham, #p<0.05 vs. BDL).
601 602
Figure 2. HU-910 mitigates the BDL-associated renal dysfunction and kidney fibrosis
603
(A) Serum levels of kidney injury markers are displayed including creatinine, blood urea
604
nitrogen (BUN), kidney injury molecule-1 (KIM-1), osteopontin (OPN) and neutrophil
605
gelatinase-associated lipocalin (NGAL) in each study group. (B) Representative images of the
606
hematoxylin & eosin, periodic acid-Schiff (PAS) (magnification 100x) and Masson’s trichrome
607
staining (magnification 200x) together with (C) the pathological scoring of the PAS-stained
608
sections are shown in the renal tissue of BDL- and Sham-operated animals with or without HU-
609
910 therapy. (D) Fibrosis scoring together with the (E) mRNA expression level of various
610
markers of fibrotic remodeling are displayed, showing results of the quantitative real time PCR
611
analysis. Data are shown as mean with the standard error of mean of N=4-8 in each study group,
612
with alternating colors reflecting each group (Sham – white, Sham+HU-910 – grey, BDL –
613
black, BDL+HU-910 – red). Statistical analysis was performed with the One way analysis of
614
variance followed by the Tukey’s post hoc test, statistical significance is indicated by asterisks
615
(*p<0.05 vs. Sham, #p<0.05 vs. BDL).
616 617
Figure 3. Renal inflammation is ameliorated by the application of HU-910
618
(A) Representative images the F4/80 positive inflammatory cell infiltration and of the renal
619
ICAM-1 immunohistochemistry (magnification 200x) are displayed in the renal tissue of BDL
620
and Sham animals with or without HU-910 therapy, with arrows in the enlarged panels
621
indicating the F4/80 positive inflammatory cells. (B) Scoring of the F4/80 staining intensity. (C-
622
D) mRNA expression of various inflammatory markers are presented, showing results of the
623
quantitative real time PCR analysis. (E) Scoring of the ICAM-1 staining intensity. Data are
624
expressed as mean with the standard error of mean of N=4-8 samples in each study group, with
625
alternating colors reflecting each group (Sham – white, Sham+HU-910 – grey, BDL – black,
626
BDL+HU-910 – red). Statistical analysis was performed with the One way analysis of variance
627
followed by the Tukey’s post hoc test, statistical significance is indicated by asterisks (*p<0.05
628
vs. Sham, #p<0.05 vs. BDL).
629 630
Figure 4. Oxidative damage is a key feature of renal injury in BDL
631
(A) Representative images of the 4-hydroxy-2-nonenal (4-HNE), malondialdehyde (MDA) and
632
3-nitrotyrosine (3-NT) staining (magnification 200x) are displayed in the renal tissue of BDL-
633
and Sham-operated animals with or without HU-910 therapy. (B-D) mRNA expression of
634
various antioxidants and indicators of oxidative/nitrative damage are displayed, showing results
635
of the quantitative real time PCR analysis. Data are expressed as mean with the standard error of
636
mean of N=4-8 samples in each study group, with alternating colors reflecting each group (Sham
637
– white, Sham+HU-910 – grey, BDL – black, BDL+HU-910 – red). Statistical analysis was
638
performed with the One way analysis of variance followed by the Tukey’s post hoc test,
639
statistical significance is indicated by asterisks (*p<0.05 vs. Sham, #p<0.05 vs. BDL).
640 641
Figure 5. Renal microvascular flow is recovered by CB2-R activation in BDL-nephropathy
642
(A) Representative images of the renal microvascular flow are displayed in each study group,
643
supplemented by (B) the quantification of the flow intensity. (C) mRNA expression of vascular
644
injury markers are displayed, showing results of the quantitative real time PCR analysis. (D)
645
Hepatic and renal Cnr2 expression is shown, evaluated by droplet digital PCR analysis. Data are
646
expressed as mean with the standard error of mean of N=4-8 samples in each study group, with
647
alternating colors reflecting each group (Sham – white, Sham+HU-910 – grey, BDL – black,
648
BDL+HU-910 – red). Statistical analysis was performed with the One way analysis of variance
649
followed by the Tukey’s post hoc test, statistical significance is indicated by asterisks (*p<0.05
650
vs. Sham, #p<0.05 vs. BDL).
651
Figure 1. A
F4/80
4-HNE
BDL+HU910
BDL
Sham+HU910
Sham
Sirius red
-
+
-
-
-
+
Sham
# +
+
BDL
-
+
Sham
+
BDL
HU910
-
-
+
Sham
CD11b
Col3a1
BDL
-
* -
+
Sham
-
200 150 100 50 0
*
#
40
#
20 0
+
HU910
BDL
+
HU910
BDL
-
+
Sham
-
*
5
-
+
Sham
-
# +
BDL
10 8 6 4 2 0
*
HU910
-
+
Sham
-
p=0.06
+
BDL
*
150 100
#
50 0
+
HU910
BDL
Oxidative stress markers
10
HU910
40 30 20 10 0
HU910
BDL
0
HU910
*
60
-
+
Sham
-
# +
BDL
Inflammatory cytokines and chemokines
15
#
0
+
#
*
*
6 4 2 0
HU910
-
+
Sham
-
-
+
Sham
-
+
BDL
# +
BDL
p22phox
HU910
Sham
5
Inflammatory cell markers 10 8 6 4 2 0
MCP1
*
#
-
+
10
p67phox
* Sham
25 20 15 10 5 0
-
*
15
MIP2
BDL
HU910
MIP1α
Sham
+
*
40 30 20 10 0
gp91phox
p40phox
mRNA expression (fold change)
F
HU910
-
+
mRNA expression (fold change)
E
-
p47phox
0
Col1a1
5
HU910
8 6 4 2 0
mRNA expression (fold change)
* *#
10
TNFα
Fibrosis score
15
D
Markers of fibrotic remodelling
100 µm
F4/80
C
B
100 µm
mRNA expression (fold change)
200 µm
*
6 4 2 0
HU910
-
+
Sham
-
+
BDL
HU910
B
-
+ -
Sham
+
-
HU910
BDL
-
+
Sham
+
0
HU910
BDL
- +
- +
Sham
HU910
BDL
-
PAS
*#
-
+
Sham
1000 800 600 400 200 0
* *#
+
HU910
BDL
-
-
+
Sham
+
BDL
Masson`s trichrome
BDL+HU910
BDL
Sham+HU910
Sham
Hematoxylin-eosin
*#
NGAL
1000
*
2000 1500 1000 500 0
(ng/mL)
2000
OPN
*#
KIM-1
*
*
3000
(ng/mL)
0
#
Serum markers of kidney injury
200 150 100 50 0
(pg/mL)
0.2
BUN
0.4
(mg/dL)
*
0.6
(mg/dL)
Creatinine
Figure 2. A
0
HU910
-
+
Sham
-
+
BDL
HU910
-
+
Sham
-
+
BDL
4 2 0
HU910
-
+
Sham
-
+
BDL
Markers of fibrotic remodelling 5 4 3 2 1 0
*#
HU910
-
+
Sham
-
+
BDL
2.0 1.5 1.0 0.5 0
*#
Tgf β
#
* *#
6
100 µm
Col3a1
5
*
E
Col1a1
* *#
10
5 4 3 2 1 0
200 µm
mRNA expression (fold change)
15
fold change
D
Fibrosis area
C
Pathological score
200 µm
HU910
-
+
Sham
-
+
BDL
Figure 3. A
ICAM-1
BDL+HU910
BDL
Sham+HU910
Sham
F4/80
100 µm
5 0
+
#
-
+
BDL
HU910
-
+
Sham
*
#
-
+
10 8 6 4 2 0
HU910
BDL
5 0
HU910
-
+
Sham
-
# +
BDL
* *#
HU910
-
+
Sham
-
+
BDL
2.0 1.5 1.0 0.5 0
*
TNFα
10
50 40 30 20 10 0
MIP2
*
15
HU910
-
-
*# -
+
Sham
+
BDL
E
Inflammatory chemokines and cytokines MCP1
mRNA expression (fold change)
D
-
Sham
*
4 3 2 1 0
+
Sham
-
#
+
BDL
*
15
ICAM-1 Score
10
CD11b
F4/80 Score
15
Inflammatory cell markers CD68
C
mRNA expression (fold change)
B
HU910
100 µm
25 µm
10 5 0
HU910
-
+
Sham
-
*# +
BDL
Figure 4. A
MDA
3-NT
BDL+HU910
BDL
Sham+HU910
Sham
4-HNE
100 µm
+
BDL
*
6 4 2 0
HU910
-
+
Sham
-
# +
BDL
HU910
-
+
Sham
D
-
HU910
BDL
-
+
Sham
-
+
HU910
BDL
*#
2.5 2.0 1.5 1.0 0.5 0
-
-
+
Sham
+
BDL
*
12
NOX4
* *#
gp91phox
p67phox
+
4 3 2 1 0
8 4 0
HU910
# -
+
Sham
-
+
BDL
Antioxidant enzymes 1.5
1.5
1.0
1.0
0.5 0
HU910
-
+
Sham
** -
#
+
BDL
1.5
0.5 0
HU910
**
#
-
+
Sham
-
+
BDL
1.0
SOD2
-
#
SOD1
+
*
100 µm
Oxidative stress markers
Catalase
-
Sham
5 4 3 2 1 0
mRNA expression (fold change)
HU910
* *#
p47phox
p40phox
4 3 2 1 0
iNOS
C
mRNA expression (fold change)
B
mRNA expression (fold change)
100 µm
0.5 0
HU910
-
+
Sham
# * * -
+
BDL
Microcirculation Sham+HU910
Sham
BDL+HU910
BDL
HU910
* -
+
Sham
-
+
BDL
D
5 0
HU910
-
+
Sham
-
+
BDL
10 8 6 4 2 0
*
P-selectin
*
#
*#
10
HU910
-
+
Sham
-
*# +
BDL
Cnr2 expression by ddPCR
*
15 10
Liver
2500 2000 1500 1000 500 0
INCREASE
Vascular injury markers 15
Vcam-1
Kidney microcirculation
Copy number (fold change)
Flux
B
C
mRNA expression (fold change)
Laser Speckle intensity (flux)
DECREASE
5 0
HU910
-
+
Sham
-
*# +
BDL
15
Kidney
Figure 5. A
10
* *#
5 0
HU910
-
+
Sham
-
+
BDL