Despite advances in antibiotic therapy, sepsis remains a major cause of death in intensive care units. Annually, >750,000 people develop sepsis, and the mortality rate is 30% to 40% in the United States alone.1,2 Severe sepsis also involves organ dysfunction. Indeed, acute kidney injury (AKI) is common during severe sepsis and increases the mortality rate to approximately 75%.3 Although antibiotics may treat the source of sepsis, no specific therapy is available for the associated organ injury.
A growing number of studies report massive cytokine release and oxidative stress as important events in the development of organ dysfunction during sepsis.4,5 As the major source of intracellular reactive oxygen species, mitochondria are particularly vulnerable to reactive oxygen species–mediated damage.6,7 In vitro data obtained from a sepsis model suggest that generation of mitochondrial oxidants—as well as deterioration in mitochondrial function and progression of renal tubular epithelial cell (RTEC) injury—occurs in a time-dependent manner.8
Polydatin (PD), a monocrystalline and polyphenolic drug that can be isolated from a traditional Chinese herb (Polygonum cuspidatum), demonstrates antiinflammatory effects and reduces oxidative stress.9–12 PD also protects neurons and arteriolar smooth muscle cells against mitochondrial injury during severe shock.13,14 The Sino Food and Drug Administration (SFDA) has approved PD medications for use in phase II clinical trials.
The aim of the present study was to determine whether PD decreases sepsis-induced mitochondrial injury in RTECs.
All experimental procedures were performed in accordance with the US National Institutes of Health Guide for the Care and Use of Laboratory Animals after approval was received from the Ethics Committee of Southern Medical University, Guangzhou, China.
Drugs and Reagents
PD was obtained from Neptunus Co. (Shenzhen, China). The CellTiter-Glo kit was purchased from Promega (Madison, WI). JC-1, calcein AM, and MitoTracker dyes were obtained from Molecular Probes (Invitrogen, Carlsbad, CA). Acridine orange (AO) base and all other chemicals were purchased from Sigma (St. Louis, MO).
Cecal Ligation and Puncture Model of Sepsis
Two hundred thirty Sprague-Dawley rats (180–220 g) were anesthetized with isoflurane. Sepsis was introduced using the cecal ligation and puncture (CLP) technique.15 Specifically, midline laparotomy was performed using minimal dissection, and the cecum was ligated just below the ileocecal valve using 4-0 silk ligatures to maintain intestinal continuity. The cecum was perforated at 2 locations 1-cm apart using an 18-gauge needle and gently compressed until the feces were extruded. The bowel was then returned to the abdomen, and the incision was 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 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 hours after CLP.
All rats were catheterized via the tail vein using 24-gauge IV catheters (BD, Sandy, UT). PD was dissolved in the vehicle, 45% ethanol and 20% propylene glycol in a sodium carbonate/sodium bicarbonate solution (Neptunus Co.). PD, vehicle, or NS were administered via the tail vein at 6, 12, and 18 hours after CLP.15
Experimental Groups and Animal Survival
Randomization was performed using computer-generated random numbers. Rats were randomly divided into 6 groups (30 animals/group except sham group): (1) control (sham) group, all rats underwent the midline laparotomy, but the cecum was neither ligated nor punctured; (2) CLP + NS group, all rats underwent CLP and received NS; (3) CLP + vehicle group, all rats underwent CLP and received the vehicle; (4) CLP + PD (15 mg/kg) group, all rats underwent CLP and received 15 mg/kg PD; (5) CLP + PD (30 mg/kg) group; and (6) CLP + PD (45 mg/kg) group. In addition, 8 animals underwent sham surgery. All animals were observed after CLP and treatment, and survival was recorded.
Unlike rats in the other 2 groups, rats given 30 mg/kg PD survived long enough to receive 3 doses. To assess the histologic effects of PD on renal tissue, 30 mg/kg PD was administered in the next experiment.
Rats were randomly divided into 4 groups: control (sham) group, CLP + NS group, CLP + vehicle group, and CLP + PD (30 mg/kg) group. The rats in each group were killed at 24 hours after undergoing CLP, and blood and kidney specimens were collected for subsequent analyses. Forty rats (10 animals/group) were used in the histology analysis, and the other 32 rats (8 animals/group) had cytokine and cellular function analyzed.
Hematoxylin and eosin–stained sections were scored in a blind, semiquantitative manner by 2 pathologists. For each animal, at least 10 high-power fields (×400) were examined. Tubules that demonstrated cellular necrosis, loss of the brush border, cast formation, vacuolization, and tubule dilation were scored as follows: 0, none; 1, <11%; 2, 11% to 25%; 3, 26% to 45%; 4, 46% to 75%; and 5, >75%.
Serum Creatinine and Blood Urea Nitrogen Concentration
Serum creatinine and blood urea nitrogen were assayed using AU680 automatic biochemical analyzer (Beckman Coulter, Brea, CA).
Serum tumor necrosis factor (TNF)-α and interleukin-6 (IL-6) were measured using commercially available enzyme rat immunoassay kits (R&D Systems Europe, Oxford, UK).
Cell Isolation Procedures
Because the tubular epithelium is a major target of sepsis-induced AKI,16–18 mitochondrial function in rat kidney was evaluated in RTECs. We followed the previously described method with some minor modifications.19–21 RTECs were isolated from the kidney cortex at 24 hours after CLP. Briefly, the cortex was cut into fragments, and cells were dissociated by incubation with 1 mg/mL collagenase for 30 minutes at 37°C. The red blood cell lysis method was used to remove all red blood cells. RTECs were separated using Percoll gradient density centrifugation (GE Healthcare Life Sciences, Piscataway, NJ). Immunostaining with cytokeratin 18 and Hoechst (Thermo Fisher Scientific, Inc., Waltham, MA) indicated that the RTEC purity was sufficient (Figure, Supplemental Digital Content, http://links.lww.com/AA/B221).22
To examine morphologic changes in the RTECs, sections of renal cortical tissues were prepared and examined using a transmission electron microscope (Philips CM 10; Philips, Eindhoven, The Netherlands).
Mitochondrial Membrane Potential
Mitochondrial membrane potential (ΔΨm) was measured using JC-1 fluorescent probes. JC-1 is a monomer present in the cytosol (which stains green), and it also accumulates as aggregates in normally polarized mitochondria (which stains red). However, in damaged and depolarized mitochondria, JC-1 exists in its monomeric form and stains the cytosol green. A shift in fluorescence emission from red to green indicates mitochondrial depolarization. Isolated RTECs were exposed to 5 μmol/L JC-1 for 15 minutes at 37°C. A minimum of 10,000 cells per sample were analyzed using flow cytometry (BD FACSVerse, San Jose, CA). Data were analyzed using BD FACSuite software. The percentage of cells with abnormally low ΔΨm (i.e., green fluorescence) was also determined.
ATP levels in the RTECs were assayed using a luciferase-based assay (CellTiter-Glo, Madison, WI). This assay measures ATP via the energy-dependent luciferase/luciferin reaction and thereby provides information on cell viability. The test was performed according to the manufacturer’s instructions. Briefly, after counting the cells in a hemocytometer using the trypan blue exclusion method and adding 100 μL of the CellTiter-Glo reagent per 100 μL of cell suspension (approximately 10,000 isolated cells per well), the cells were allowed to incubate at room temperature for 10 minutes to stabilize the luminescent signal. Luminescence was measured using an automatic microplate reader (SpectraMax M5; Molecular Devices, Sunnyvale, CA).
Determination of Lipid Peroxides
Lipid peroxides (LPOs) were measured using a commercially available kit (Cayman Chemical Co., Ann Arbor, MI). Absorbance was measured at 500 nm using an automatic microplate reader (SpectraMax M5; Molecular Devices) according to the manufacturer’s instructions.
Measurement of Lysosomal Stability
Isolated cells were assessed for lysosomal stability using the AO-uptake method. AO is a lysosomotropic base and a metachromatic fluorescent probe that exhibits red fluorescence when highly concentrated in intact lysosomes. The number of intact lysosomes per cell can be evaluated by assaying red fluorescence (i.e., AO-uptake). Isolated RTECs were incubated with 5 μM AO for 15 minutes at 37°C and examined using a Leica DM IRB/E inverted microscope coupled to a Leica TCS SP confocal system (Leica Microsystems, Wetzlar, Germany). Images were collected using TCS NT software (Leica TCS SP2 AOBS). AO fluorescence was quantitatively measured using flow cytometry (BD FACSVerse). Data were analyzed using BD FACSuite software.
Opening of the Mitochondrial Permeability Transition Pores
Isolated RTECs were incubated with 1 μM calcein AM and 2 mM CoCl2 at room temperature for 15 minutes in the absence of light and then examined using a confocal microscope (LSM780; Zeiss Microsystems, Jena, Germany). Images were collected using the provided software (Zeiss Microsystems). Cells were quantitatively measured using flow cytometry (BD FACSVerse). Data were analyzed using BD FACSuite software.
The homogeneity of variance test (the Levene test) was used to assess whether groups had equal variances. The Lilliefors test and Shapiro-Wilk test were used to assess whether residuals of the analysis of variance (ANOVA) model followed normal distributions. ANOVA was performed when the Levene test indicated homogeneity of variance (P > 0.1). When ANOVA showed significant differences among groups (P < 0.05), the Bonferroni multiple comparison test was performed. When equal variances were not assumed (based on the Levene test; P < 0.1), robust tests of equality of means using the Brown-Forsythe test were performed followed by the Dunnett T3 post hoc comparisons.
If residuals of the ANOVA model of renal tubular injury scores were not normally distributed, the nonparametric Kruskal-Wallis test was performed (nonparametric data presented as median values). When the Kruskal-Wallis test showed significant differences among groups, the Dunn multiple comparison post hoc test was performed (to test for differences between individual groups). Survival analysis was performed using the Kaplan-Meier method, and multiple comparisons of survival curves were performed using the Mantel-Cox log-rank test.
Data are presented as the mean ± SD, except for nonparametric data (presented as median values). For between-group differences, the mean or median differences and 95% confidence intervals (CIs) of the differences were determined. Values were considered statistically significant at P < 0.05, except for the Levene test, the Lilliefors test, and Shapiro-Wilk test (significance defined at P < 0.1).
PD Improved Survival After CLP
The survival rate of septic rats significantly increased from 26.7% and 20% in the CLP + NS and the CLP + vehicle groups, respectively, to 50% in the CLP + PD (30 mg/kg) group (P = 0.017 and P = 0.002, respectively; Fig. 1). Survival was not significantly improved in rats that received either 15 or 45 mg/kg PD after CLP, relative to the CLP + NS and CLP + vehicle groups (CLP + NS versus CLP + PD 15 mg/kg: P = 0.288; CLP + vehicle versus CLP + PD 15 mg/kg: P = 0.078; CLP + NS versus CLP + PD 45 mg/kg: P = 0.073; CLP + vehicle versus CLP + PD 45 mg/kg: P = 0.012). These findings indicate that PD improves survival of septic rats.
PD Attenuated Histopathologic Kidney Damage After CLP
At 24 hours, morphologic changes in the CLP + NS and CLP + vehicle groups were characterized by mild brush border loss, tubular degeneration, and vacuolization in the early segments of the proximal tubules (Fig. 2, A–C). Morphologic damage and renal tubular injury scores were significantly reduced after PD administration (Fig. 2, D and E).
PD Decreased Blood Urea Nitrogen and Creatinine Levels
Three-dose PD (30 mg/kg) treatment (administered at 6, 12, and 18 hours) post-CLP significantly reduced blood urea nitrogen and creatinine at 24 hours (Fig. 3, A and B). Blood urea nitrogen decreased from 63.88 ± 20.57 and 70.75 ± 15.53 mg/dL in the CLP + NS and CLP + vehicle groups, respectively, to 36.00 ± 13.63 mg/dL in the CLP + PD group (CLP + NS versus CLP + PD: P = 0.041, mean difference = 27.88, 95% CI of the difference = 0.94–54.81; CLP + vehicle versus CLP + PD: P = 0.002, mean difference = 34.75, 95% CI of the difference = 12.62–56.88). Blood creatinine decreased from 0.68 ± 0.17 and 0.71 ± 0.20 mg/dL in the CLP + NS and CLP + vehicle groups, respectively, to 0.43 ± 0.14 mg/dL in the CLP + PD group (CLP + NS versus CLP + PD: P = 0.023, mean difference = 0.24, 95% CI of the difference = 0.02–0.46; CLP + vehicle versus CLP + PD: P = 0.007, mean difference = 0.28, 95% CI of the difference = 0.06–0.50).
PD Reduced IL-6 After CLP
No significant differences were found in TNF-α levels for the CLP + PD group relative to the CLP + NS or CLP + vehicle groups (CLP + NS versus CLP + PD: P = 0.424; CLP + vehicle versus CLP + PD: P = 0.676; Fig. 4A). IL-6 levels decreased from 615.13 ± 167.04 and 626.13 ± 150.87 ng/mL in the CLP + NS and CLP + vehicle groups, respectively, to 349.00 ± 84.31 ng/mL in the CLP + PD group (CLP + NS versus CLP + PD: P = 0.012, mean difference = 266.13, 95% CI of the difference = 55.91–476.34; CLP + vehicle versus CLP + PD: P = 0.005, mean difference = 277.13, 95% CI of the difference = 85.10–469.15; Fig. 4B).
PD Attenuated Mitochondrial Damage After CLP
RTEC morphology was examined by transmission electron microscopy. Control cells demonstrated normal mitochondria with preserved membranes and cristae. In contrast, mitochondria in the CLP + NS and CLP + vehicle groups were spherical or irregularly shaped and swollen with electron-lucent matrices and poorly defined cristae. Mitochondrial alterations after CLP appeared to be partially improved with PD treatment (Fig. 5, A–E). These findings indicate that administration of PD may be effective in attenuating severe mitochondrial damage after CLP.
Decrease in Mitochondrial Membrane Potential After CLP Was Attenuated by PD
The fluorescent probe JC-1 was used to measure ΔΨm in RTECs. Cells were incubated with JC-1, and ΔΨm was evaluated using flow cytometry. The proportion of cells with low ΔΨm mitochondria decreased from 66.50% ± 16.02% and 62.00% ± 14.79% in the CLP + NS and CLP + vehicle groups, respectively, to 37.13% ± 7.85% in the CLP + PD group (CLP + NS versus CLP + PD: P = 0.005, mean difference = 29.38, 95% CI of the difference = 9.27–49.48; CLP + vehicle versus CLP + PD: P = 0.009, mean difference= 24.88, 95% CI of the difference = 6.16–43.59; Fig. 6).
PD Restored Mitochondrial ATP Levels After CLP
To further evaluate PD-related protective mechanisms in tubular epithelial mitochondria, the effects of PD on intracellular ATP levels (a key indicator of mitochondrial function23) after CLP were measured using the luciferase bioluminescence method. Intracellular ATP levels increased from 44.00% ± 13.16% and 39.13% ± 17.26% in the CLP + NS and CLP + vehicle groups, respectively, to 67.50% ± 14.51% in the CLP + PD group (CLP + NS versus CLP + PD: P = 0.013, mean difference = −23.50, 95% CI of the difference = −43.18 to −3.82; CLP + vehicle versus CLP + PD: P = 0.002, mean difference = −28.38, 95% CI of the difference = −48.06 to −8.69; Fig. 7).
PD Reduced Lipid Peroxide Levels in RTECs After CLP
Oxidative stress promotes toxic radical formation, lysosomal instability, mitochondrial dysfunction, and cell death.24–26 To evaluate oxidative stress in the kidneys after CLP, we therefore assayed LPO levels in RTECs. Compared with the CLP + NS and CLP + vehicle groups, LPO levels were significantly lower in the PD-treated group. LPO decreased from 186.50% ± 44.91% and 204.50% ± 48.52% in the CLP + NS and CLP + vehicle groups, respectively, to 123.50% ± 25.80% in the CLP + PD group (CLP + NS versus CLP + PD: P = 0.029, mean difference = 63.00, 95% CI of the difference = 5.63–120.37; CLP + vehicle versus CLP + PD: P = 0.009, mean difference = 81.00, 95% CI of the difference =19.61–142.39; Fig. 8).
PD Preserved Lysosomal Stability After CLP
Lysosomal membrane permeability was assessed by counting cells with reduced or diminished intact lysosomes (referred to as “pale” cells), identified by poor intracellular red fluorescence after AO exposure (normal cells with abundant intact lysosomes exhibit bright red fluorescence; Fig. 9A). In the CLP + NS and CLP + vehicle groups, the proportion of pale cells and level of red fluorescence intensity were significantly lower than those in the CLP + PD group (Fig. 9B). The percentage of pale cells markedly decreased from 46.25% ± 16.76% and 47.63% ± 14.11% in the CLP + NS and CLP + vehicle groups, respectively, to 21.13% ± 7.36% in the CLP + PD group (CLP + NS versus CLP + PD: P = 0.018, mean difference = 25.13, 95% CI of the difference = 4.25–46.00; CLP + vehicle versus CLP + PD: P = 0.004, mean difference = 26.50, 95% CI of the difference = 8.68–44.32; Fig. 9C).
PD Inhibited Opening of the Mitochondrial Permeability Transition Pore
The fluorescent molecule calcein, when esterified to calcein AM, becomes nonfluorescent and membrane permeable (membranes are not permeable to unesterified calcein). Once inside the cell, the molecule is de-esterified and trapped in its free fluorophoric form. Bivalent cobalt ions (Co2+) quench cytosolic and nuclear calcein fluorescence, whereas mitochondrial fluorescence is mainly unaffected because Co2+ ions cannot easily cross the normal inner mitochondrial membrane. However, calcein fluorescence in the mitochondrial matrix is quenched by the Co2+ ions if the pores into the inner membrane are opened. RTECs in the sham group exhibited normal mitochondrial fluorescence, but significantly less mitochondrial fluorescence was detected in the CLP + NS and CLP + vehicle groups. In RTECs, mitochondrial fluorescence increased from 364.13 ± 158.85 and 426.25 ± 171.28 in the CLP + NS and CLP + vehicle groups, respectively, to 662.88 ± 148.64 in the CLP + PD group (CLP + NS versus CLP + PD: P = 0.003, mean difference = −298.75, 95% CI of the difference = −514.45 to −83.05; CLP + vehicle versus CLP + PD: P = 0.025, mean difference = −236.63, 95% CI of the difference = −452.32 to −20.93; Fig. 10, A–C).
CLP results in poor survival and is accompanied by impaired renal function and severe kidney damage, as indicated by histologic changes such as loss of the brush border, cast formation, vacuolization, and tubule dilation. In this study, PD decreased CLP-induced mortality in rats, alleviated pathologic markers of severe kidney injury, and improved renal function. Collectively, these results indicate that PD alleviates CLP-induced septic kidney injury in rats.
In the present study, we investigated the acute effects of PD on CLP-induced AKI using a clinically relevant course of therapy (administered after the onset of septic shock).15 The survival rate of rats treated with 30 mg/kg PD was higher than that in rats treated with 15 or 45 mg/kg PD. The mechanisms responsible for the lower survival rate in CLP rats that received 45 mg/kg PD are unknown but may be related to the cumulative effect of the higher PD exposure (i.e., exposure was actually 45 mg/kg greater than in the 30 mg/kg group, as 3 doses were administered). Consistent with a previous study in mice that reported reduced sepsis-induced mortality after PD pretreatment,9 the present study indicates that PD may preserve organ function in rats with severe sepsis.
Mitochondrial dysfunction and inflammation are correlated with sepsis severity and disease outcomes and, therefore, represent potential targets for supporting therapies.27 However, the relevance of mitochondrial dysfunction in the pathogenesis of multiple organ dysfunction and failure in sepsis patients is controversial, and effects of sepsis on mitochondrial function are organ specific.27 The effects of sepsis on kidney mitochondria in rodents have been assessed in only 2 studies.28,29 Therefore, our aim was to determine whether mitochondrial injury occurs in tubular epithelial cells during sepsis. In the present study, observable changes (at the electron microscope level) in markers of mitochondrial damage were apparent during sepsis in rat RTECs. However, this damage could also be attributed to our measurement methods. Most previous studies used freshly isolated mitochondria, whereas the present study used freshly isolated cells and measured mitochondrial function.
Abnormal mitochondrial membrane permeabilization is a critical determinant of mitochondrial dysfunction and apoptosis.30 In pathologic states, the inner mitochondrial membrane may undergo a sudden increase in permeability, allowing entry of solutes <1.5 kDa. This phenomenon is caused by the opening of a mitochondrial permeability transition pore (mPTP) that comprises voltage-dependent, high-conductance channels located in the inner mitochondrial membrane. The mPTP is closed under normal physiologic conditions but can open in pathologic states such as ischemia-reperfusion injury.31,32 In a recent study, septic shock was shown to induce opening of the mPTP in the ventricular myocardium.33
Opening of the mPTP reduces the transmembrane pH gradient, causing dissipation of ΔΨm and inhibition of ATP production.34 Loss of ΔΨm, mitochondrial swelling with poorly defined cristae, and low intracellular ATP are considered signs of mitochondrial injury. In this study, open mPTPs, collapsed ΔΨm, swollen mitochondria, and low ATP in RTECs developed during late sepsis along with impaired renal function, severe pathologic kidney injury, and decreased animal survival. These changes implied that mitochondrial dysfunction with low intracellular ATP in RTECs might be a cause of high mortality rate in sepsis rats.
The factors responsible for the pathologic findings and death associated with sepsis are mediated, in part, by the excessive release of proinflammatory cytokines. TNF appears relatively early in circulation,35 and its circulating levels are not correlated with the lethality of experimental sepsis.36,37 In contrast, circulating IL-6 is key to assessing sepsis severity.38 In this study, the delayed administration of PD attenuated circulating IL-6 but not TNF-α.
Mitochondrial dysfunction is also associated with elevated IL-6 and lipid peroxidation products in circulation, an effect that has been reported in septic patients.27 Inflammatory responses, oxidative stress, and mitochondrial impairment comprise a self-sustaining and amplifying cycle, which contribute to organ failure in sepsis patients.27,39,40 Antioxidants can inhibit inflammatory responses, reduce oxidative stress, and improve mitochondrial function in experimental models of sepsis.5 Polyphenols, such as PD, can potently inhibit inflammatory responses and attenuate oxidative stress.41–43 PD may protect neurons and arteriolar smooth muscle cells against mitochondrial injury by attenuating oxidative stress during severe shock.13,14 Our observations demonstrate that, in a rat model, PD administration after the onset of sepsis reduced IL-6 and oxidative stress, improved mitochondrial function, and prolonged survival. Further work is needed to determine whether PD may have the same effect in humans and whether such effects are clinically significant.
Name: Youguang Gao, MD.
Contribution: This author helped design the study, conduct the study, collect and analyze the data, prepare the manuscript, and approve the final manuscript. This author is the archival author.
Attestation: Youguang Gao attests to the integrity of the original data and the reported analysis.
Name: Zhenhua Zeng, MD.
Contribution: This author helped conduct the study, collect and analyze the data, and approve the final manuscript.
Attestation: Zhenhua Zeng attests to the integrity of the original data and reported analysis.
Name: Tao Li, MD.
Contribution: This author helped collect and analyze the data and approve the final manuscript.
Attestation: Tao Li attests to the integrity of the original data and reported analysis.
Name: Siqi Xu, MM.
Contribution: This author helped conduct the study, collect the data, and approve the final manuscript.
Attestation: Siqi Xu attests to the integrity of the original data and reported analysis.
Name: Xingmin Wang, MD.
Contribution: This author helped design the study, prepare the manuscript, and approve the final manuscript.
Attestation: Xingmin Wang attests to the integrity of the original data and reported analysis.
Name: Zhongqing Chen, MD.
Contribution: This author helped design the study, prepare the manuscript, and approve the final manuscript.
Attestation: Zhongqing Chen attests to the integrity of the original data and reported analysis.
Name: Caizhu Lin, MD.
Contribution: This author helped design the study, prepare the manuscript, and approve the final manuscript.
Attestation: Caizhu Lin attests to the integrity of the original data and reported analysis.
This manuscript was handled by: Avery Tung, MD.
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