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Nuclear factor erythroid 2-related factor 2 (Nrf2) is a critical regulator of the cellular-defense response in protection against oxidative injury. Several studies have demonstrated that propofol ameliorates ischemia/reperfusion injury in a number of organs. However, whether propofol exerts renal protection against liver transplantation via Nrf2 activation remains to be elucidated. The aim of the present study was to investigate the effects of orthotopic liver autotransplantation (OLAT) on renal Nrf2 expression and to determine whether propofol protects against kidney injury induced by OLAT via Nrf2 activation. A total of 24 male Sprague Dawley rats were randomly divided into four groups: sham surgery + normal saline (sham group); OLAT + normal saline (OLAT group); OLAT + propofol 50 mg/kg (L-Prop group) and OLAT + propofol 100 mg/kg (H-Prop group). Normal saline and propofol were administered for 3 consecutive days through an intraperitoneal injection prior to surgery. Kidney pathology, blood urea nitrogen (BUN), creatinine (Cr), superoxide anion (O2•−), hydroxyl radical (·OH), maleic dialdehyde (MDA) and expression levels of Nrf2, Kelch-like ECH-associated protein 1 (Keap1), heme oxygenase-1 (HO-1) and NADP quinine oxidoreductase 1 (NQO1) were assessed 8 h after OLAT. It was demonstrated that OLAT induced remote kidney damage. Pretreatment with propofol significantly ameliorated renal pathology and abrogated the increase of the Cr and BUN concentrations, O2•− and ·OH activities, and MDA levels induced by OLAT. In the H-Prop group, Keap1 expression in the cytoplasm was decreased and Nrf2 expression in the nucleus was upregulated, accompanied by an increase of HO-1 and NQO1 expression. The present results suggest that propofol pretreatment exerted renal protection against OLAT, with the upregulation of nuclear Nrf2 expression as a potential mechanism.
The incidence of acute kidney injury (AKI) following liver transplantation has been reported to range widely-between 8% and 78% (
Previously, it has been demonstrated that transcription nuclear factor erythroid 2-related factor 2 (Nrf2), characterized as ‘an oxidative stress-sensing guarding regulator’, combines with the activating response element and regulates a multitude of cytoprotective genes acting in synergy to remove reactive oxygen species (ROS) through sequential enzymatic reactions, including heme oxygenase-1 (HO-1) and nicotinamide adenine dinucleotide phosphate quinine oxidoreductase1 (NQO1) (
Propofol, (
A total of 24 male Sprague Dawley (SD) rats (220–280 g) were provided by the Medical Experimental Animal Center of Guangdong Province (Foshan, China). Ethical approval for the present study was provided by the Institutional Animal Care and Use Committee of Sun Yat-Sen University (Guangzhou, China). The animal studies were performed in accordance with the Guide for the Care and Use of Laboratory Animals, issued by the National Institutes of Health (Bethesda, MD, USA).
SD rats were randomly assigned to four groups (n=6 in each group). The sham group (sham), which was subjected to abdominal incision, vascular dissection and wound closure without hepatic vascular exclusion and perfusion, and the orthotopic liver autotransplantation group (OLAT) were administered isotonic sodium chloride solution (Sigma-Aldrich, St. Louis, MO, USA) through intraperitoneal (i.p.) injection on each of three consecutive days prior to the experiment. The OLAT + low dose propofol-treated group (L-Prop) received propofol at 50 mg/kg i.p. for 3 consecutive days prior to OLAT and the OLAT + high dose propofol-treated group (H-Prop) received propofol (Corden Pharma S.P.A, Caponago, Italy) at 100 mg/kg i.p. for 3 consecutive days prior to OLAT. All rats were sacrificed 8 h after the sham or OLAT surgeries and kidney tissue was collected for analysis.
Following administration of an ether inhalation anesthetic (Sanpin Chemical Technology, Co., Ltd, Shenzen, China), surgery was performed on the OLAT model, as originally described by Jin
The harvested kidneys were fixed in a 10% formaldehyde solution (Sigma-Aldrich) and embedded in paraffin (Leica Microsystems, Wetzlar, Germany) for histopathological analysis. The kidney paraffin sections (5 μm) were stained with hematoxylin and eosin (BeiJingDingGuoChangShengBiotech. Co., Ltd, Beijing, China). The severity of kidney injury was evaluated, in a blinded manner, using a semi-quantitative scale evaluating morphological characteristics of the tubules as suggested by Paller
Blood urea nitrogen (BUN) and Creatinine (Cr), which were used as renal functional indices, were detected in blood samples with an automatic biochemistry analyzer (Hitachi 7600-020/7170A; Hitachi, Tokyo, Japan).
The collected kidney tissue was frozen at −80°C. The frozen kidney tissue was homogenized on ice in 10 volumes of frozen saline using a homogenizer (Polytron; Kinematica, Lucerne, Switzerland). The homogenates were centrifuged for 10 min at 5,000 × g and the supernatant was allocated into 6–8 separate tubes and preserved at −80°C until use in biochemical assays. The protein content was determined using a bicinchoninic acid (BCA) protein assay kit (KeyGen Biotech Co., Ltd, Nanjing, China) according to the manufacturer’s instructions.
O2•− and ·OH, the main oxygen free radicals, were quantified using assay kits (KeyGen Biotech Co., Ltd.) according to the manufacturer’s instructions. The quantity of O2•− and ·OH in the kidney tissue was expressed in units per milligram of protein (U/mg).
The content of MDA was determined as an index of lipid peroxidation, as described by Draper and Hadley (
Total protein was extracted from frozen kidney tissue using total protein extraction kits (KeyGen Biotech Co., Ltd.) for NQO1 measurement. Cytosolic and nuclear protein extracts were prepared using nuclear and cytoplasmic protein extraction kits (KeyGen Biotech Co., Ltd.) according to the manufacturer’s instructions. The cytoplasmic protein was collected for Keap1, NQO1 and HO-1 measurements, and the nuclear protein was extracted for Nrf2 detection. Protein concentrations were determined using a BCA protein assay reagent kit (KeyGen Biotech Co., Ltd).
Primary antibodies were added to the samples to detect Nrf2 (1:250 dilution, Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), Keap1 (1:1,000 dilution, Millipore, Billerica, MA, USA), HO-1 (1:250 dilution, Santa Cruz Biotechnology, Inc.), NQO1 (1:250 dilution, Santa Cruz Biotechnology, Inc.), β-actin (1:2,000 dilution, Santa Cruz Biotechnology, Inc.) and Lamin B (1:2,000 dilution, Millipore). The relative density of bands was quantified by computerized scanning of the images using AlphaView software version 2.2.14407 (ProteinSimple, Santa Clara, CA, USA) and normalized by β-actin immunoreactivity to correct for any loading and transfer differences between samples.
For immunohistochemical staining, the kidney paraffin sections (5 μm) were stained using the immunohistochemistry technique for Nrf2 as described by Tanaka
Quantitative data, expressed as the mean ± standard deviation, were statistically analyzed using a one-way analysis of variance for inter-group comparisons using SPSS 17.0 (IBM, Armonk, NY, USA). P<0.05 was considered to indicate a statistically significant difference.
As shown in
Plasma Cr and BUN are classical indicators, which reflect renal function. OLAT, involving 20 min of ischemia followed by 8 h of reperfusion resulted in significant increases in the serum concentration of Cr and BUN (
O2•− and ·OH are the main oxygen free radicals. As depicted in
MDA is the product of lipid peroxidation damage. The MDA content in the kidney was significantly higher in the OLAT group than in the sham group (P<0.05, versus the sham group). However, this increase was significantly reduced by pretreatment with propofol (P=0.04, L-Prop group, versus the OLAT group; P=0.009, H-Prop group, versus the OLAT group;
Nrf2 is an unstable protein with a short half-life (<20 min) and is dependent on the status of the Keap1/Nrf2 system. Keap1, which sequesters Nrf2 in the cytoplasm and prevents it from translocation to the nucleus, is crucial in the regulation of the Nrf2 pathway (
Consistent with the western blot analysis, the expression of Nrf2 was minimal, as demonstrated by the lack of clear visualization of its immunoreactivity in the sham group. Compared with the sham group, the proportion of Nrf2-positive cells with brown staining in the nuclei was significantly higher in the OLAT, L-Prop and H-Prop groups. Furthermore, compared with the OLAT group, positive expression of Nrf2 was markedly increased in the H-Prop group (
HO-1 and NQO1 are two typical downstream phase II antioxidant enzymes mediated by Nrf2 (
OLAT is a well-established liver transplantation model, including a superior vena cava, inferior vena cava and hepatic portal vein block, hepatic I/R and cold liver preserving fluid perfusion. The present study provided evidence that liver transplantation induces remote kidney damage, which contributes to the progression of oxidative damage. Pretreatment with propofol, a widely used anesthetic, significantly ameliorated renal dysfunction and pathology injury induced by OLAT. Furthermore, propofol inhibited the expression of Keap1 in the cytoplasm, upregulated the expression of Nrf2 in the nucleus and increased HO1 and NQO1 expression. Propofol also reduced OLAT-induced increases in O2•− and ·OH activity as well as the MDA content. These results suggest that propofol’s renal protective effects against OLAT may be partly due to activation of the Keap1/Nrf2 pathway.
It has been established that remote kidney injury is frequently induced by I/R injury during liver transplantation (
Nrf2 is a critical regulator of the cellular defense response to protect against oxidative injury (
Several studies have demonstrated that propofol ameliorates I/R injury in several organs, including the heart, liver, kidney and brain, through improvement of antioxidant enzyme activity (
In conclusion, propofol pretreatment exerted a renal protective effect against OLAT. The potential mechanism for this effect is through upregulation of nuclear Nrf2 expression.
Financial support and sponsorship: The present study was in part supported by the National Natural Science Foundation of China (grant nos. 81170449 and 81372090).
Histopathological analysis of a kidney under microscopy (magnification, ×200). L-Prop, the OLAT + low-dose propafol-treated group; H-Prop, the OLAT + high-dose propafol-treated group; OLAT, orthotopic liver autotransplantation.
Changes in serum (A) BUN and (B) Cr levels. Results are expressed as the mean ± standard deviation (n=6). *P<0.05 compared with the Sham group; #P<0.05 compared with the OLAT group. L-Prop, the OLAT + low-dose propafol-treated group; H-Prop, the OLAT + high-dose propafol-treated group; OLAT, orthotopic liver autotransplantation; BUN, blood urea nitrogen; Cr, creatinine.
Changes in (A) superoxide anion and (B) ·OH activity and (C) the MDA content in kidney tissue. Results are expressed as the mean ± standard deviation (n=6). *P<0.05 compared with the Sham group; #P<0.05 compared with the OLAT group. L-Prop, the OLAT + low-dose propafol-treated group; H-Prop, the OLAT + high-dose propafol-treated group; OLAT, orthotopic liver autotransplantation; ·OH, hydroxyl radical; MDA, maleic dialdehyde.
Western blot analysis of expression of Nrf2 in the nucleus and Keap1 in the cytoplasm of the kidney tissue. Results are expressed as the mean ± standard deviation (n=6). *P<0.05 compared with the Sham group; #P<0.05 compared with the OLAT group. L-Prop, the OLAT + low-dose propafol-treated group; H-Prop, the OLAT + high-dose propafol-treated group; OLAT, orthotopic liver autotransplantation; Nrf2, nuclear factor erythroid 2-related factor 2; Keap1, Kelch-like ECH-associated protein 1.
Immunohistochemical analysis of expression of nuclear factor erythroid 2-related factor 2 in kidney (magnification ×400). L-Prop, the OLAT + low-dose propafol-treated group; H-Prop, the OLAT + high-dose propafol-treated group; OLAT, orthotopic liver autotransplantation.
Western blot analysis of the expression of (A) HO-1 and (B) NQO1 in renal tissue. Results are expressed as the mean ± standard deviation (n=6). *P<0.05 compared with the Sham group; #P<0.05 compared with the OLAT group; L-Prop, the OLAT + low-dose propafol-treated group; H-Prop, the OLAT + high-dose propafol-treated group; OLAT, orthotopic liver autotransplantation; HO-1, heme oxygenase-1; NQO1, NADP quinine oxidoreductase 1.