Sepsis often results in damage to multiple organ systems, possibly due to severe mitochondrial dysfunction. Two members of the sirtuin family, SIRT1 and SIRT3, have been implicated in the reversal of mitochondrial damage. The aim of this study was to determine the role of SIRT1/3 in acute kidney injury (AKI) following sepsis in a septic rat model. After drug pretreatment and cecal ligation and puncture (CLP) model reproduction in the rats, we performed survival time evaluation and kidney tissue extraction and renal tubular epithelial cell (RTEC) isolation. We observed reduced SIRT1/3 activity, elevated acetylated SOD2 (ac-SOD2) levels and oxidative stress, and damaged mitochondria in RTECs following sepsis. Treatment with resveratrol (RSV), a chemical SIRT1 activator, effectively restored SIRT1/3 activity, reduced acetylated SOD2 levels, ameliorated oxidative stress and mitochondrial function of RTECs, and prolonged survival time. However, the beneficial effects of RSV were greatly abrogated by Ex527, a selective inhibitor of SIRT1. These results suggest a therapeutic role for SIRT1 in the reversal of AKI in septic rat, which may rely on SIRT3-mediated deacetylation of SOD2. SIRT1/3 activation could therefore be a promising therapeutic strategy to treat sepsis-associated AKI.
Sepsis is a frequently fatal condition characterized by uncontrolled and adverse host reactions to microbial infection [
There is increasing evidence that silent mating type information regulation 2 homolog 1 (sirtuin 1, SIRT1) plays an important role in mitochondrial protection. Through deacetylation of histone and nonhistone substrate, SIRT1 is involved in various metabolic and inflammatory diseases [
SOD2 Activity kit was purchased from Dojindo (Kumamoto, Japan). Antibodies against SOD2 and acetylated superoxide dismutase 2 (ac-SOD2) as well as SIRT1 and SIRT3 Deacetylase Fluorometric Assay kits were obtained from Cyclex (Nagano, Japan). Antibody against SIRT1 was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibody against SIRT3 was purchased from ABClonal (Boston, MA, USA). Antibodies against acetylated lysine and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were purchased from CST (Danvers, MA, USA). Immunoprecipitation kits were obtained from Proteintech (Chicago, IL, USA). Membrane-permeant JC-1 dye and calcein-AM were purchased from Molecular Probes (Eugene, OR, USA). Assay kits for reduced glutathione/oxidized glutathione (GSH/GSSG) and catalase (CAT) were obtained from Beyotime Biotech (Beijing, China). A CellTiter-Glo® Assay kit and terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining kit were purchased from Promega (Madison, WI, USA). Polyvinylidene fluoride (PVDF) membranes were obtained from Millipore (Billerica, MA, USA). 3-(1H-1,2,3-Triazol-4-yl)pyridine (3-TYP), a selective inhibitor of SIRT3, was synthesized and characterized by the School of Pharmaceutical Sciences, Southern Medical University, Guangzhou, China, based on our previous work [
The present study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals (US National Institutes of Health, Bethesda, MD, USA). The study protocol was approved by the Committee on Ethics in Animal Experiments of Southern Medical University.
In total, 64 specific pathogen-free Sprague-Dawley rats (male or female) weighing 180–220 g were used in this study. The rats were housed in plastic cages with a controlled temperature of 25°C, humidity of 50–55%, and a 12 h light/dark cycle. All the animals had free access to food and distilled water. All rats were anesthetized and maintained with isoflurane (RWD Life Science, Shenzhen, China) and were randomly divided into six groups: the control, vehicle, resveratrol (RSV), SRT1720 (a selective activator of SIRT1), RSV+Ex527, and RSV+3-TYP groups. In the control group, rats were anesthetized and underwent surgery without any other treatment. In the vehicle group, the rats were given vehicle (0.3 mL) and subjected to cecal ligation and puncture (CLP) after 30 min. In the RSV group, the rats were given RSV (0.3 mL; 50 mg/kg) and subjected to CLP after 30 min. In the SRT1720 group, the rats were given SRT1720 (0.3 mL; 0.2 mg/kg) [ In the RSV+Ex527 group, rats were given RSV+Ex527 (0.3 mL, 50 mg/kg, and 5 mg/kg, resp.) and subjected to CLP after 30 min. In the RSV+3-TYP group, the rats were given RSV+3-TYP (0.3 mL, 50 mg/kg, and 5 mg/kg, resp.) [
Sepsis was introduced by using the CLP technique. Specifically, midline laparotomy was performed using minimal dissection, and the cecum was ligated just below the ileocecal valve by using 4-0 silk ligatures to maintain intestinal continuity. The cecum was perforated at 2 locations 1 cm apart by using an 18-gauge needle and gently compressed until the feces were extruded. The bowel was then returned to the abdomen and the incision closed. Control rats underwent the same surgical procedures, but the cecum was neither ligated nor punctured. At the end of the operation, all rats were subcutaneously resuscitated with vehicle (normal saline, NS; 20 mL/kg). All rats were deprived of food but had free access to water after the operation. All rats received subcutaneous injections of imipenem/cilastatin (14 mg/kg) in 8 mL NS solution (40 mL/kg) at 6 h after CLP.
After the CLP model was created, 36 rats (6 in each group) were killed for tissue extraction, observation of mitochondrial morphology, and renal tubular epithelial cell (RTEC) isolation; the remaining animals (8 in each group) were used for survival analyses.
A portion of extracted renal tissue from each group was used for isolation of RTECs by our previously described methods [
Both kidney tissue and isolated RTECs were centrifuged at 14000 rpm for 10 minutes after homogenization in Radio-Immunoprecipitation Assay (RIPA) lysis buffer, and the clear supernatants were collected. Total protein concentrations in the supernatants were determined by the bicinchoninic acid (BCA) method. Then, the protein was boiled at 98°C for 5 to 10 minutes and stored at −80°C for later analysis. Equal amounts of protein samples were electrophoresed through a 7.5% SDS-polyacrylamide gel and then transferred onto polyvinylidene difluoride (PVDF) membrane using wet transfer at 100 V for 90 minutes at 4°C. Nonspecific binding sites were blocked by 1% BSA in 0.05% Tween-20 Tris-buffered saline (TBST) and then incubated overnight at 4°C with primary antibodies. After incubation with primary antibodies (against SIRT1, SIRT3, SOD2, cytochrome C, and GAPDH) and secondary antibodies, protein bands were detected using chemiluminescence detection reagents. GAPDH was used as an internal reference. Ac-SOD2 levels on immunoprecipitated SOD2 protein were measured. Band intensity was quantified by scanning densitometry. Each measurement was made at least 3 times.
Activity of SIRT1 deacetylase was detected using SIRT1 (Cyclex, Cat#CY-1151V2) and SIRT3 Deacetylase Fluorometric Assay kits (Cyclex, Cat#CY-1153V2) as described previously [
SOD2 activity was measured with a commercially available kit using water-soluble tetrazolium salt- (WST-) 1 as a substrate [
GSH, the GSSG/GSH ratio, and CAT activity in isolated RTECs were evaluated using kits according to manufacturer instructions and standard methods. Briefly, 200
All rats (6 in each group) were killed and the renal tissue was used for morphological observation and protein extraction. Morphological changes in the mitochondria of cells from renal tissue were observed using transmission electron microscopy. Renal tissues were fixed with 2.5% glutaraldehyde and stained with cacodylate-buffered osmium tetroxide. Sections were prepared and examined under an electron microscope (H-7500; Hitachi, Tokyo, Japan) [
Isolated cells were used for the detection of mitochondrial function (mitochondrial membrane potential (
For detection of apoptotic cells, TUNEL staining was carried out using a Promega apoptosis detection kit. Immunofluorescence for TUNEL staining was performed with fluorescein isothiocyanate (FITC). The glass was mounted with cover slips containing 4′,6-diamidino-2-phenylindole (DAPI) and imaged under a Confocal Microscope (LSM 780; Carl Zeiss, Oberkochen, Germany). Cells stained green fluorescence and DAPI stained blue fluorescence were detected as TUNEL-positive apoptotic cells. Both TUNEL-positive and DAPI-positive cells were counted in 10 random high-power fields (HPF; 300-cells each). Data are expressed as the number of apoptotic cells/HPF (400x magnification).
Some of the animals in each group (32 rats in total, 8 in each group) were assigned to a subgroup for survival analyses. To minimize suffering, pentobarbital sodium (30 mg/kg, i.p.) was given intermittently to conscious animals. Animals had access to food and water
Median survival was analyzed using Kaplan–Meier plots and compared using the log-rank test. Other data were presented as the mean ± standard deviation and were analyzed using SPSS 20.0 (IBM, Armonk, NY, USA). Levene’s test was used to ascertain if groups had equal variance. Moreover, one-way analysis of variance (ANOVA) followed by Tukey’s test was applied. If equal variances were not assumed (based on Levene’s test;
To determine whether SIRT1 is involved in the pathogenesis of sepsis-induced AKI, we studied both the activity and protein expression of SIRT1 in renal tissue (Figure
Expression and activity of SIRT1 protein in renal tissue after cecal ligation and puncture (CLP). (a) Representative western blot of SIRT1 proteins (upper panel) and densitometric analyses (lower panel); (b) SIRT1 activity was determined using a SIRT1 Assay kit and normalized to that of the control group. The time points were set at 4, 8, 16, and 24 h after CLP treatment. Values shown are the mean ± SEM.
We then explored which kind of renal cell contributed to the decreased SIRT1 activity. Both the RTECs and glomerular cells were isolated immediately after the rats were executed. To further test the role of SIRT1 in AKI induced by CLP, the SIRT1 chemical activator RSV and SRT1720 and inhibitor Ex527 were used. SIRT1 protein expression and activity were considerably reduced in RTECs compared to that in glomerular cells (see Supplementary Figure
Furthermore, RSV greatly restored SIRT1 activity and slightly elevated the SIRT1 protein expression. Interestingly, SRT1720 addition increased the SIRT1 activity slightly more than RSV treatment alone. In contrast, the beneficial effect of RSV was blocked after Ex527 was added (Figure
Expression and activity of SIRT1 protein in renal tubular epithelial cells after CLP. (a) Representative western blot of SIRT1 proteins (upper panel) and densitometric analyses (lower panel); (b) SIRT1 activity was determined using a SIRT1 Assay kit and normalized to that of the control group. Values shown are the mean ± SEM.
Based on the importance of the SIRT1/3 axis in the pathogenesis of severe shock [
Expression and activity of SIRT3 protein in renal tubular epithelial cells after CLP. (a) Representative western blot of SIRT3 proteins (upper panel) and densitometric analyses (lower panel); (b) SIRT3 activity was determined using a SIRT3 Assay kit and normalized to that of the control group. Values shown are the mean ± SEM.
Since several reports claim that SOD2 is one of the deacetylated targets of SIRT1/3, we determined protein expression, acetylation level, and activity of SOD2 in RTECs following CLP. As expected, RSV treatment slightly restored the SOD2 protein content and remarkably decreased the acetylated SOD2 level (Ac-lys), leading to elevated SOD2 activity (Figure
Expression of SOD2 protein, acetylation level, and activity in renal tubular epithelial cells after CLP. (a) Representative western blot of SOD2 proteins (upper panel) and densitometric analyses (lower panel); (b) level of acetylated SOD2 was determined using an acetylated SOD2 (Ac-SOD2) antibody normalized to purified SOD2 protein by immunoprecipitation (IP SOD2); (c) SOD2 activity was determined using a SOD2 Assay kit and normalized to that of the control group. Results are expressed as fold change over control. Values shown are the mean ± SEM.
Since SOD2 activity was significantly restored by SIRT1 activation and SOD2 is a key antioxidative enzyme, we tested GSH content, GSH/GSSG ratio, and CAT activity in RTECs. The GSH content, GSH/GSSG ratio, and CAT activity were considerably decreased after CLP in the vehicle group and were partially restored by SIRT1 activation in the RSV treatment group; however, after Ex527 was added, the beneficial effect of RSV was greatly blocked and was almost equivalent to that in the vehicle group (Figure
Indices of oxidative stress in the renal tubular epithelial cell after CLP. (a) GSH content; (b) GSH/GSSG ratio; (c) CAT activity. Results are expressed as fold change over control. Values shown are the mean ± SEM.
Due to the positive effect of SIRT1 activation on SOD2, an antioxidative stress enzyme located in the mitochondria, we also tested the mitochondrial morphology and function in RTECs. An elliptical shape with well-developed cristae and electron-dense matrices was noted in the control group. Severe MD was found after CLP treatment (vehicle group), evidencing by irregularly shaped, swollen, and disrupted mitochondria with poorly defined cristae and electron-lucent matrices. Surprisingly, these changes were restored by treatment with RSV. Moreover, RSV treatment restored the mitochondrial transmembrane potential (JC-1 aggregates/monomer), enhanced ATP content, and inhibited the opening of mPTP. However, these beneficial effects of RSV were considerably blocked by Ex527 addition (Figure
Mitochondrial morphology and function of renal tubular epithelial cells after CLP. (a) Representative transmission electron microscope images of the mitochondria. Healthy mitochondria (control group) have intact mitochondrial membranes and cristae. Mitochondria are swollen, with poorly defined cristae and more mitochondrial vacuolization in the vehicle group, whereas these changes are prevented partially in RSV group. (b) Representative flow cytometry scatter plots of the JC-1 probe. Cells were stained with the JC-1 probe; regions of reduced polarization are indicated by the JC-1 monomers. Regions of high polarization of mitochondrial membranes are indicated by the formation of J-aggregates. (c)
We subsequently tested the effect of SIRT1 activation on apoptosis in RTECs; a mitochondria-related apoptosis index (cytochrome C) and a general apoptosis index (TUNEL) were selected. The mitochondrial cytochrome C (mito-cyt C) was decreased and the cytoplasmic cytochrome C (cyto-cyt C) was increased after CLP treatment. Moreover, the number of TUNEL-positive cells was significantly increased. As expected, RSV administration reduced the cyto-cyt C content and decreased the number of TUNEL-positive cells. However, the beneficial effects of RSV were reversed considerably by Ex527 addition (Figure
Cytochrome C expression and TUNEL staining of apoptotic cells in the kidneys of rats after CLP. (a) Representative western blot of cytochrome C protein in mitochondria (mito-CytC) and cytoplasm (cyto-CytC). (b) Densitometric analyses of cytochrome C protein in mitochondria (mito-CytC) and cytoplasm (cyto-CytC). (c) TUNEL staining. Original magnification ×400.
Finally, we tested the effect of SIRT1 activation on renal function and survival time. Both the creatinine and urea nitrogen were elevated in septic rat, accompanied by shortened survival time. SIRT1 activation by RSV considerably reduced the level of creatinine and blood urea nitrogen. Importantly, RSV administration greatly prolonged the survival of sepsis animal. However, the beneficial effect of RSV on renal function and survival time were partially abrogated by Ex527 (Supplementary Figure
Our results showed that SIRT1/3 activity was reduced in the RTECs of septic rat and was accompanied by higher acetylated SOD2 levels, swollen mitochondria, and increased cell apoptosis. Moreover, SIRT1 activation by RSV could partially restore SIRT3 activity and attenuate MD, leading to improved mitochondrial function and reduced apoptosis. Furthermore, all the beneficial effects of SIRT1 activation were considerably reduced by a selective inhibitor. To the best of our knowledge, this is a first report describing a role for SIRT1/3 in the pathogenesis of sepsis-associated AKI.
SIRTs, first discovered in yeast as NAD+-dependent epigenetic and metabolic regulators, have comparable activities in human physiology and disease [
In terms of mitochondrial function, SIRT3 is another known deacetylating enzyme. A growing body of evidence has confirmed that SIRT3 defends against oxidative stress in multiple diseases including ischemia and neurodegenerative disease. Genetic inference or genetic knockout of SIRT3 accelerates diet-induced obesity, type 2 diabetes, and nonalcoholic fatty liver disease [
Some reports have confirmed that the dysfunction of the renal peritubular microenvironment leads to sepsis-induced AKI [
Our study has some potential limitations. Firstly, sepsis is a dynamic disease. It changes from the hyperinflammatory to hypoinflammatory phase. In this study, we only assessed the early (hyperinflammatory) phase of sepsis; further studies regarding therapeutic options during the hypoinflammatory phase are needed. However, this study provides a prevention strategy for sepsis AKI treatment, which partially elucidates the SIRT1-SIRT3 axis in the pathogenesis of sepsis. Secondly, we applied a chemical activator and a selective inhibitor against SIRT1; more intensive methods such as genetic inference or genetic knockout may provide stronger evidence. Thirdly, we only explored the effects of SIRT1 in RTECs; reduction of SIRT1 activity also occurs in other types of kidney cells and will certainly be a worthwhile direction for future studies.
This study outlines an important role for SIRT1 in the reversal of AKI following sepsis. In addition, our findings suggest that RSV treatment exerts a profound protective effect against renal injury caused by sepsis. This protection appears to be largely due to the ability to activate SIRT1 and SIRT3, augment SOD2-mediated antioxidative ability, and mitochondrial protection.
Acute kidney injury
Renal tubular epithelial cell
Cecal ligation and puncture
Resveratrol
Acetylated superoxide dismutase 2
Superoxide dismutase
Sirtuin
Mitochondrial dysfunction
Reduced form of glutathione
Oxidized form of glutathione
Catalase
Terminal deoxynucleotidyl transferase dUTP nick-end labeling
Polyvinylidene fluoride
Water-soluble tetrazolium salt
Glyceraldehyde 3-phosphate dehydrogenase
Mitochondrial membrane potential
Adenosine triphosphate
Mitochondrial permeability transition pore
High-power field.
The authors declare that there is no conflict of interests regarding the publication of this paper.
Ke-seng Zhao and Zhenhua Zeng prepared the concept and designed the research; Siqi Xu, Youguang Gao, Qin Zhang, Siwei Wei, and Xingui Dai performed the experiments; Zhenhua Zeng, Zhongqing Chen, and Ke-seng Zhao analyzed data; Xiqi Xu and Youguang Gao prepared the figures; Siqi Xu, Qin Zhang, Zhenhua Zeng, and Ke-seng Zhao interpreted the results of experiments; Siqi Xu and Youguang Gao drafted the paper; Zhenhua Zeng and Ke-seng Zhao edited and revised the paper. All the authors read and approved the final version of the paper. Siqi Xu and Youguang Gao contributed equally to this paper.
This work was supported by the Natural Science Foundation of Fujian Province, China [Grant no. 2016J01451] and the President Foundation of Nanfang Hospital, Southern Medical University, Guangzhou, China [Grant nos. 2015C005 and 2015Z001].