https://orcid.org/0000-0001-7580-7069
Retraction
After this article [1] was published, concerns were raised about some of the panels in Figs 5–8. Specifically:
- In Fig 5A, the two H&E stain Sham panels are duplicated in the cortex Sham panels in Fig 4A in [2].
- In Fig 5B, the two Masson stain Sham panels are duplicated in the cortex Sham panels in Fig 4B in [2].
- In Fig 5C, the two H&E stain Sham panels are duplicated in the medulla Sham panels in Fig 4A in [2].
- In Fig 5D, the two Masson stain Sham panels are duplicated in the medulla Sham panels in Fig 4B in [2].
- The β-actin 42kDa panel in Fig 6A appears similar to the β-actin 42kDa panel in Fig 8A.
- In Fig 6C, the two Sham panels are duplicated in the Sham panels in Fig 7C when rotated 180°, the cortex Sham panels in Fig 5B in [2], and the cortex Sham panels in Fig 6B in [2] when rotated 180°.
- In Fig 6D, the two Sham panels are duplicated in the medulla Sham panels in Fig 5B in [2].
- In Fig 6D, the two CKD panels are duplicated in the CKD panels in Fig 7D.
- In Fig 7D, the two Sham panels are duplicated in the medulla Sham panels in Fig 6B in [2].
- In Fig 8E, the two Sham panels appear similar to the Sham panels in Fig 7B in [2].
- In Fig 8F, the two Sham panels appear similar to the Sham panels in Fig 7C in [2].
During editorial follow-up on these issues, the corresponding author stated that the eight Sham panels in Figs 5A-D, the four Sham panels in Figs 6C-D, the two CKD panels in Fig 6D, and the four Sham panels in Figs 7C-D in [1] were included in error.
In light of the extent of the above issues with Figs 5–8 that question the reliability of these data, the PLOS ONE Editors retract this article.
CTC agreed with the retraction. YHC, CAY, CCY, and SPH either did not respond directly or could not be reached.
Owing to the concerns about similarities with previously published content [2], published in 2021 by S. Karger AG [2], which is not offered under a CC BY license, the eight Sham panels in Figs 5A-D, the four Sham panels in Figs 6C-D, the four Sham panels in Figs 7C-D, and the four Sham panels in Figs 8E-F are excluded from this article’s [1] license. At the time of retraction, the article [1] was republished to note these exclusions in the legends of Figs 5–8 and the article’s copyright statement.
28 Nov 2023: The PLOS ONE Editors (2023) Retraction: Sodium thiosulfate through preserving mitochondrial dynamics ameliorates oxidative stress induced renal apoptosis and ferroptosis in 5/6 nephrectomized rats with chronic kidney diseases. PLOS ONE 18(11): e0295162. https://doi.org/10.1371/journal.pone.0295162 View retraction
Figures
Abstract
Chronic kidney disease (CKD) progression may be evoked through dysregulated mitochondrial dynamics enhanced oxidative stress and inflammation contributing to high cardiovascular morbidity and mortality. Previous study has demonstrated sodium thiosulfate (STS, Na2S2O3) could effectively attenuate renal oxidative injury in the animal model of renovascular hypertension. We explored whether the potentially therapeutic effect of STS is available on the attenuating CKD injury in thirty-six male Wistar rats with 5/6 nephrectomy. We determined the STS effect on reactive oxygen species (ROS) amount in vitro and in vivo by an ultrasensitive chemiluminescence-amplification method, ED-1 mediated inflammation, Masson’s trichrome stained fibrosis, mitochondrial dynamics (fission and fusion) and two types of programmed cell death, apoptosis and ferroptosis by western blot and immunohistochemistry. Our in vitro data showed STS displayed the strongest scavenging ROS activity at the dosage of 0.1 g. We applied STS at 0.1 g/kg intraperitoneally 5 times/week for 4 weeks to these CKD rats. CKD significantly enhanced the degree in arterial blood pressure, urinary protein, BUN, creatinine, blood and kidney ROS amount, leukocytes infiltration, renal 4-HNE expression, fibrosis, dynamin-related protein 1 (Drp1) mediated mitochondrial fission, Bax/c-caspase 9/c-caspase 3/poly (ADP-ribose) polymerase (PARP) mediated apoptosis, iron overload/ferroptosis and the decreased xCT/GPX4 expression and OPA-1 mediated mitochondrial fusion. STS treatment significantly ameliorated oxidative stress, leukocyte infiltration, fibrosis, apoptosis and ferroptosis and improved mitochondrial dynamics and renal dysfunction in CKD rats. Our results suggest that STS as drug repurposing strategy could attenuate CKD injury through the action of anti-mitochondrial fission, anti-inflammation, anti-fibrosis, anti-apoptotic, and anti-ferroptotic mechanisms.
Citation: Cheng Y-H, Yao C-A, Yang C-C, Hsu S-P, Chien C-T (2023) Sodium thiosulfate through preserving mitochondrial dynamics ameliorates oxidative stress induced renal apoptosis and ferroptosis in 5/6 nephrectomized rats with chronic kidney diseases. PLoS ONE 18(2): e0277652. https://doi.org/10.1371/journal.pone.0277652
Editor: Hiroyasu Nakano, Toho University Graduate School of Medicine, JAPAN
Received: July 20, 2022; Accepted: November 1, 2022; Published: February 16, 2023
Copyright: © 2023 Cheng et al. Except for the eight Sham panels in Fig 5A–5D, the four Sham panels in Fig 6C–6D, the four Sham panels in Fig 7C–7D, and the four Sham panels in Fig 8E–8F, this is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All relevant data are within the paper and its Supporting Information files.
Funding: This work was partly supported from MOST-102-2320-B-003-001-MY3 (Dr. Chiang-Ting Chien).
Competing interests: The authors have declared that no competing interests exist.
Introduction
Chronic kidney disease (CKD) is a chronic progressive renal parenchyma damage caused by a variety of risk factors like hypertension, diabetes or obesity. CKD is characterized by the progressive loss of discharging metabolic waste, dysregulation of the water-salt and acid-base balance related renal function, chronic inflammation, oxidative stress, vascular remodeling, as well as glomerular and tubulointerstitial scarring [1]. The number of potential CKD patients, however, is increasing worldwide [2] and with the exception of renal replacement therapy and kidney transplantation, the alternative therapies, including hemodialysis and peritoneal dialysis, are unable to improve the pathological damage of kidney tissues [3–5]. During the progression of CKD, exaggerated production of reactive oxygen species (ROS) from the damaged mitochondria in renal tubular cells may evoke abnormal signal transduction or cellular dysfunction and initiates the cascade of apoptosis contributing to further severe injury in the kidney. Several types of programmed cell death can be distinguished based on its morphologic and biochemical criteria and can also be induced by the increasing release of ROS [6, 7]. For example, excess ROS induce apoptosis by causing DNA damage and enhancement of Bax/Bcl-2 ratio, expression of caspase 3, and caspase-mediated cleavage of poly-(ADP-ribose)-polymerase [7, 8]. Ferroptosis, another type of programmed cell death, is also evoked by the excess ROS mediated lipid peroxidation and downregulation in GPX4/xCT expression and is found in acute kidney injury and CKD [9]. Targeting ferroptosis with its inducers/initiators and inhibitors can modulate the progression of kidney diseases in animal models. On the other hand, mitochondria are dynamic organelles constantly undergoing fission and fusion events. Mitochondrial fusion is majorly regulated by optic atrophy 1 (OPA-1). The main regulator of mitochondrial fission is dynamin-related protein 1 (Drp-1) [10]. Increased Drp-1 mediated mitochondrial fission or decreased OPA-1 mediated mitochondrial fusion may further impair mitochondrial dynamics and function. Therefore, we aimed to screen potential drugs to inhibit CKD involved mitochondrial fission, apoptosis and ferroptosis in the present study.
To confer the ease and immediate use of potential drugs in preventing the CKD progression, the application of well developed drugs, or new potential uses for currently available drugs is crucial for treating or inhibiting the CKD progress. Sodium thiosulfate (STS), a major metabolite of H2S with strong antioxidant and vasodilatory activity, has been proven safe for the treatment of calciphylaxis, renal hypertension, cyanide poisoning, and cisplatin toxicity, cerebral vasospasm, angiotensin II-induced hypertension, renovascular hypertension and improving renal function in hyperoxaluric rats [11–18]. Because these cumulated evidence and our recent data implicated the beneficial effects of STS, we therefore suggest that the use of STS may provide therapeutic potential in improving and delaying the progress of CKD injury.
The sources of ROS generated in CKD may be from the damaged mitochondria in several types of resident cells such as proximal tubules, distal tubules and infiltrated leukocytes. However, most of the antioxidants tested to date confer adverse side effects that preclude their utility in human clinical trials [3, 19]. In the present study, we aimed to explore the therapeutic effect of drug repurposing strategy of STS on CKD induced oxidative stress, mitochondrial dynamics, inflammation, apoptosis, ferroptosis and renal dysfunction in the rats.
Methods and materials
Animals
Thirty six male Wistar rats (200–250 g) were obtained from BioLASCO Taiwan Co. Ltd. (I-Lan, Taiwan) and housed at the Experimental Animal Center, National Taiwan Normal University, at a constant temperature and with a consistent light cycle (light from 07:00 to 18:00 o’clock). The rats were fed with standard chow of Laboratory Rodent Diet 5001 containing 0.4% sodium and tap water ad libitum. All surgical and experimental procedures were approved by Institutional Animal Care and Use Committee of National Taiwan Normal University with the Approval number 107026 (on the date of 2018 September 26) and were in accordance with the guidelines of the National Science Council of Republic of China (1997) and the ARRIVE guidelines 2.0. All animal experiments were performed under anesthesia, all efforts were made to minimize suffering and finally sacrificed with overdose of anesthesia. These messages were described in detail in following experiments.
Induction of CKD and grouping
Male Wistar rats underwent 5/6 nephrectomy to induce CKD or sham operation were described previously [19–21]. Briefly, rats were anesthetized with avertin (250mg/kg, ip). Under avertin anesthesia, the left kidney was exposed, and two-thirds of the left kidney was infarcted by selective ligation of left renal artery, followed by complete removal of the right kidney. The sham rats underwent the same procedure except for renal artery ligation or nephrectomy. All rats were recovered from anesthesia and placed in an individual cage for 4 weeks to induce CKD. Following surgery, the rats were randomized to divide into three groups of rats: sham-control rats (Sham, n = 6), CKD rats with intraperitoneal saline (CKD, n = 6) and CKD rats with STS treatment at the dosage of 0.1 g/kg intraperitoneally three times/week for 4 weeks (CKD STS, n = 6). The rats were given standard feed and povidone iodine to prevent wound infection. For analgesia to minimize suffering, 20 mg/kg dose paracetamol was added to daily drinking waters of the rats in all groups.
Renal arterial blood flow determination
The determination of renal arterial blood flow by placing the renal artery on a blood flow probe was performed ad described previously [6]. The determined renal blood flow was connected to a transonic flow meter (T420, Transonic System Inc. Ithaca, NY, USA) and recorded on a Power Lab system (ML870 PowerLab 8/30, ADInstruments Pty Ltd, New South Wales, Australia). After CKD model induction and renal arterial blood flow measurement, all the rats were sutured and recovered. On the day of the experiments, all rats were anesthetized with urethane (1.2 g/kg subcutaneously) and were sacrificed with overdose of urethane from the tail vein.
In vitro and in vivo recording for STS effect on scavenging ROS activity
In the first part of study, the scavenging H2O2 and HOCl level of the different concentration of STS was measured by luminol-amplification chemiluminescence (CL) detection method in our laboratory as described before. In the second part of experiment, three groups of rats (n = 6 in each group) were used for measurement of ROS from the kidney by 2-Methyl-6-(4-methoxyphenyl)-3,7-dihydroimidazo-[1,2-a]-pyrazin-3-one-hydrochloride (MCLA)-amplification method or whole-blood samples by luminol-amplification method as described in our previous report [7]. The assay was performed in duplicate for each sample and was expressed as CL counts/10 s for CL.
Malondialdehyde (MDA) assay
We measured the lipid peroxidation product malondialdehyde (MDA) in these three groups of kidneys. The MDA level in renal tissue was measured using the Lipid Peroxidation (MDA) Assay Kit (ab118970, Abcam, Cambridge, USA) according to the manufacturer’s instructions. In brief, 10 mg of renal tissue was mixed with 300 μl MDA Lysis Buffer and 3 μl butylated hydroxytoluene, and centrifuged at 13,000 × for 10 minutes. Then, 600 μl of thiobarbituric acid solution reagent was added with 200 μl of standard solution or supernatant, and the mixture were incubated in a dry bath at 95°C for 1 hour. After the sample was cooled down to room temperature on ice, 200 μl mixture was added to the 96-well plate for detecting the absorbance at OD 532 nm by an ELISA Reader.
Histologic studies
The renal tissues were fixed in 4% paraformaldehyde and embedded in paraffin. The 4-μm tissue section was stained with hematoxylin & eosin (Sigma) for evaluating the degree of morphologic changes and leukocyte infiltration.
Western blotting
We used western blotting technique to determine the level of lipid peroxidation, apoptosis, ferroptosis and mitochondrial fission and fusion in the kidney samples. In brief, the kidneys were homogenized in liquid nitrogen and were lysed in RIPA buffer (Bio Basic, Amherst, NY, USA) supplemented with protease inhibitor (Roche, Basel, Switzerland) for 10 min at 4°C. The degree of expression of apoptosis-related proteins including Bcl-2, Bax, cleaved caspase 3 (c-caspase 3) and PARP, oxidative stress 4-HNE and ferroptosis-related proteins like GPX4 were assayed in the homogenized kidney tissues. These antibodies raised against 4-HNE (bs-6313R, 1:200; Bioss, Mass, USA), Bax (bs-0032R, 1:100; Bioss, Mass, USA), Bcl-2 (1:500; Transduction, Bluegrass-Lexington, KY, USA), c-caspase 9 (1:1000, Chemicon International, Temecula, CA, USA), c-caspase 3 (1:1000, Chemicon International, Temecula, CA, USA), poly(ADP-ribose) polymerase (PARP, #9532, 1:1000; Cell Signaling Technology, Mass, USA), GPX4 (1:500, Abcam, UK), dynamin-related protein 1 (Drp-1, 1:1000) (Cell Signaling Technology, Beverly, MA, USA), and optic atrophy 1 (OPA-1, 1:2000) (BD Biosciences, San Jose, CA, USA) and β-actin (1:1000; Sigma, St. Louis, MO, USA) were adapted in the present study.
Measurement of oxidative stress, iron accumulation, apoptosis, ferroptosis, and mitochondrial fission in CKD kidneys
To investigate the possible effect of STS on CKD kidneys, several oxidative stress parameters determined by immunohistochemistry, including 4-Hydroxynonenal (4-HNE, ab46545, Abcam, UK), proapoptotic Bax (ab32503, Abcam, UK), anti-apoptotic Bcl-2 (3498, Cell Signaling, USA), and PARP (9542, Cell Signaling, USA), Drp-1 (Cell Signaling, USA), cytochrome c (Santa Cruz Biotechology, Inc., Santa Cruz, CA) and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL assay) were measured the degree of lipid peroxidation, apoptosis, iron accumulation by Prussian blue stain and mitochondrial fission in CKD kidneys.
Biochemical parameters in blood and urine
We examined the STS effect on urinary protein, blood urea nitrogen and creatinine in CKD rats. Urinary protein concentration was measured by a BCA protein assay kit (Bio-Rad, Hercules, CA, USA). The blood sample was used to determine blood urea nitrogen (BUN) and creatinine.
Statistical analysis
All values were expressed as mean ± standard error mean. Data were subjected to one-way analysis of variance (ANOVA), followed by Duncan’s multiple-range test for assessment of the difference among groups. Differences within groups were evaluated by paired t test. Differences were regarded as significant if P < 0.05 was attained. Statistical analyses were performed using SPSS 18.0 statistics software (IBM Corp., Armonk, NY).
Results
STS inhibited H2O2 and HOCl activity in a dose-dependent manner
The two major ROS, hydrogen peroxide and hypochlorite (HOCl), played an important role in the inflammation primarily generated from activated neutrophils through the enhanced myeloperoxidase activity [3]. The chemical STS structure was indicated in Fig 1A. Our data demonstrated that STS at a dose-dependent manner (0.001–0.5 g) reduced H2O2 (Fig 1B) and HOCl (Fig 1C) activity. We also found that STS demonstrated a maximal scavenging H2O2 and HOCl effect at the dosage of 0.1 g.
The structure (A) and effect of STS on H2O2 (B) and HOCl (C) scavenging activity. Data shows that STS displays a dose-dependent manner in scavenging H2O2 and HOCl activity and a maximal effect is at the dosage of 0.1 g. * P < 0.05 vs. saline control (0). Data are expressed as mean ± SEM (n = 3).
CKD model displayed a decrease renal microcirculation
As shown in Fig 2, we examined the left kidney microcirculation and renal arterial blood flow in response to renal arterial partial ligation in the CKD rats. Fig 2A demonstrated the ligation of left renal arterial branches close to the renal hilum. The partially ligated kidney microcirculation (Fig 2B) and renal arterial blood flow (Fig 2C) were significantly decreased to a half value of the microcirculation or renal blood flow after partial ligation. After 4 weeks of CKD model, the level of left renal arterial blood flow determined by an ultrasound flowmeter was significantly decreased to 2.5 ml/min in the CKD rats as compared to 6.4 ml/min in Sham control rats (Fig 2D).
Evaluation of the CKD (5/6 nephrectomized) model (A) on left renal microcirculation (B), left renal arterial blood flow (C) for developing CKD. Partial ligation of left renal artery led to a near 2/3 reduction of left renal microcirculation (B) and renal arterial blood flow (C, D) in CKD group vs. Sham group. Data are expressed as mean ± SEM (n = 6). * P < 0.05 vs. Sham group.
STS attenuated CKD enhanced hypertension and urinary protein
It has been reported that signals arising from the kidney which activate the renin-angiotensin system and afferent renal nerves increase sympathetic activity importantly contributing to the pathogenesis of hypertension secondary to renal artery stenosis and end-stage renal disease in CKD [20]. Our data consistently found an increased 32 mmHg of arterial blood pressure in CKD rats (151 ± 5 mmHg) as compared to Sham control rats (129 ± 4 mmHg) (Fig 3A). STS treatment in the CKD rats effectively reduced 7.9% (139 ± 7 mmHg in CKD STS) of arterial blood pressure as compared to CKD rats (151 ± 5 mmHg). We also determined the STS effect on CKD induced urinary protein level in Fig 3B. The urinary protein concentration was significantly increased in CKD rats (856 ±168 mg/dL) as compared to the Sham controls (125 ±10 mg/dL) indicating the glomerular injury in CKD. We also found that STS efficiently depressed CKD enhanced urinary protein concentration in CKD STS rats (620 ± 160 mg/dL) vs. CKD group (856 ±168 mg/dL).
The STS effect on CKD induced renal hypertension (A), urinary protein (B), blood ROS (C) and kidney ROS amount (D). Our data show that the elevated arterial blood pressure, urinary protein, blood and kidney ROS amount are found in the CKD rats, whereas the increased level of urinary protein, blood and kidney ROS amount is reduced in STS treated CKD rats (CKD STS). All the data are expressed as mean ± SEM (n = 6). * P < 0.05 vs. Sham. # P < 0.05 vs. CKD.
STS reduced blood and kidney ROS amount in CKD rats
As demonstrated in Fig 3C, the blood ROS amount was higher in the CKD rats (3714 ± 142 counts/10 sec) than that of Sham control animals (2809 ± 132 counts/10 sec). STS treatment significantly depressed blood ROS amount (1741 ± 133 counts/10 sec) vs. CKD rats (3714 ± 142 counts/10 sec). Fig 3D also consistently displayed that kidney ROS level was significantly increased in the CKD group vs. Sham group, whereas the increased kidney ROS level was significantly inhibited in CKD STS rats vs. CKD rats.
STS improved CKD induced renal dysfunction
CKD significantly elevated the value of BUN (Fig 4A) and creatinine (Fig 4B) vs. Sham control rats implicating the tubular injury in CKD. However, STS treatment significantly reduced BUN and creatinine levels in CKD STS rats compared to CKD rats.
The increased value of blood urea nitrogen (BUN, A) and creatinine (B) was denoted in the CKD rats. STS significantly depressed the increased level of BUN and creatinine in CKD STS rats as compared to CKD rats. Data are expressed as mean ± SEM (n = 6). * P < 0.05 vs. Sham. # P < 0.05 vs. CKD.
STS reduced leukocyte infiltration and inflammation in the CKD kidneys
Leukocyte infiltration including neurophils or monocytes/macrophages into the kidney may contribute to kidney inflammation by the release of ROS and inflammatory cytokines in several types of renal diseases [3, 5–7]. Our data demonstrated that in Sham control rats, few leukocyte located in the glomerular and tubular area (Fig 5A and 5C). CKD significantly increased the degree of leukocytes infiltration in the glomerular and tubular areas vs. Sham control rats implicating the increased oxidative stress and inflammation in the CKD kidneys. STS treatment efficiently reduced the degree of infiltrated leukocytes in glomerular and tubular area as compared to CKD kidneys (Fig 5E) implicating a reduction in glomerular and tubular inflammation in CKD.
Histologic graphs from H&E stain (A, C) and Masson stain (B, D) were displayed in sham, CKD and STS treated CKD (CKD STS) rats. The increased leukocytes infiltration (yellow arrows) is indicated in the tubular area of the CKD kidney (A, C). Increased masson stain by blue color is indicated in the glomerular (B) and tubular areas (D) of the CKD kidneys. The mean data of number of leukocyte infiltration and % of blue Masson stain for each section is indicated in E and F, respectively. Data are expressed as mean ± SEM (n = 6). * P < 0.05 vs. Sham. # P < 0.05 vs. CKD. The eight Sham panels in Figures 5A-D are excluded from this article’s CC-BY license. See the accompanying retraction notice for more information.
STS reduced renal fibrosis in the CKD kidneys
We determined the STS effect on renal fibrosis in the CKD kidneys by using Masson stain for measuring the blue collagen deposits in these rats. Fig 5B and 5D indicated that the blue Masson stain markedly identified in the glomerular area and tubular area of the CKD kidneys as compared to Sham control kidneys. STS treatment significantly decreased the blue Masson stain in the glomerular and tubular areas as compared to CKD kidneys (Fig 5F).
Effect of STS on renal 4-HNE expression in CKD kidneys by Western blot and immunohistochemistry
We first explored the STS effect on renal 4-HNE, GPX4 and xCT expression by western blot and renal MDA assay in CKD kidneys. Our results indicated that CKD significantly increased renal 4-HNE expression, a lipid peroxidation biomarker, associated with the increased MDA content as compared to that in Sham control kidneys (Fig 6A and 6B). Our data also implicated that the expression of xCT and GPX4 of ferroptotic biomarkers was significantly decreased in CKD group vs. Sham control group (Fig 6A and 6B) implicating the ferroptosis formation in CKD kidneys. STS treatment significantly decreased renal 4-HNE expression and renal MDA content and restored xCT and GPX4 expression in CKD STS group vs. CKD group. The immunohistochemistry consistently demonstrated an increase of brown 4-HNE expression in the glomerular and tubular areas of the CKD kidneys as compared to Sham control kidneys (Fig 6C and 6D). STS also efficiently reduced brown 4-HNE expression in the CKD STS kidneys vs. CKD kidneys. The Prussian blue stain for indicating iron-overload ferroptosis was primarily identified in CKD as compared to Sham control kidneys. STS treatment markedly depressed the Prussian stain as indicated with red arrows in the CKD STS kidneys vs. CKD kidneys (Fig 6E).
Western blot of 4-HNE, GPX4 and xCT expression and MDA assay in CKD induced kidney injury (A, B). Immunohistochemistry of brown 4-HNE expression (red arrows) is indicated in the glomerular and tubular areas of the CKD and CKD STS kidneys (C, D). Prussian blue stain for iron-overloaded ferroptosis is indicated in E (red arrows). Data are expressed as mean ± SEM (n = 3). * P < 0.05 vs. Sham. # P < 0.05 vs. CKD. The four Sham panels in Figures 6C-D are excluded from this article’s CC-BY license. See the accompanying retraction notice for more information.
STS reduced renal Bax expression, Bax/Bcl-2 ratio and PARP expression in CKD kidneys
We examined the effect of STS on CKD evoked apoptosis formation in the kidneys by western blot and immunohistochemistry. Our finding indicated that the renal Bax expression was significantly (P < 0.05) increased in the tubular area of CKD kidneys as compared to Sham control kidneys (Fig 7A and 7B). STS treatment significantly reduced renal tubular Bax expression in CKD STS vs. CKD group. The renal Bcl-2 expression was significantly decreased in CKD vs. Sham group. STS treatment significantly recovered renal Bcl-2 expression in CKD STS group vs. CKD group. We further found that the ratio of Bax/Bcl-2 was significantly increased in CKD group vs. Sham group. STS administration significantly reduced the increased Bax/Bcl-2 ratio in CKD STS group vs. CKD group. The renal Bax immunohistochemistry consistently demonstrated that CKD significantly enhanced Bax expression in the renal tubular areas. STS treatment significantly reduced renal tubular Bax expression in the kidneys of CKD STS group as compared to CKD group (Fig 7C and 7D). The renal Bcl-2 expression by immunohistochemistry was markedly decreased in CKD group vs. Sham group. STS partly recovered the decreased renal Bcl-2 expression in CKD STS group when compared to CKD group (Fig 7E and 7F).
Western blot of Bax and Bcl-2 expression in the kidneys with CKD (A, B). Immunohistochemistry of Bax is primarily enhanced in the tubular area of the CKD and partly in CKD STS kidneys (C, D). Less expression of Bcl-2 in glomerular or tubular areas is denoted (E, F). Data are expressed as mean ± SEM (n = 3). * P < 0.05 vs. Sham. # P < 0.05 vs. CKD. The four Sham panels in Figures 7C-D are excluded from this article’s CC-BY license. See the accompanying retraction notice for more information.
We further determined the effect of STS on CKD enhanced c-caspase 9, c-caspase 3 and PARP expression by western blot. Our results displayed that the renal c-caspase 9, c-caspase 3 and PARP expression was significantly (P < 0.05) increased in CKD group as compared to Sham group (Fig 8A–8D). STS also effectively depressed c-caspase 9, c-caspase 3 and PARP expression in CKD STS kidneys as compared to CKD kidneys. We further examined the renal TUNEL immunohistochemistry in the glomerular area (Fig 8E) and tubular area (Fig 8F). Our data observed a significant increase of TUNEL positive stain cells in the glomerular and tubular areas of CKD as compared to Sham control group. A significant decrease of TUNEL apoptosis positive stain cells was found in both glomerular and tubular areas of CKD STS group as compared to CKD group (Fig 8G and 8H).
Western blot of renal c-caspase 9 (A, B) and c-caspase 3 (A, C) and PARP (A, D) expression in CKD induced kidney injury and TUNEL immunohistochemistry of renal glomeruli (E, F) and tubules (G, H) for TUNEL positive cells indicated with red arrows counting in CKD induced kidney injury. Data are expressed as mean ± SEM (n = 3). * P < 0.05 vs. Sham. # P < 0.05 vs. CKD. The four Sham panels in Figures 8E-F are excluded from this article’s CC-BY license. See the accompanying retraction notice for more information.
STS modulated mitochondrial dynamics in CKD rats
Mitochondrial dynamics was regulated by the balance of mitochondrial fission and fusion. Drp-1 and OPA-1 are the key regulators in regulating mitochondrial fission and fusion, respectively [10]. Our results observed that Drp-1 was upregulated and OPA-1 was downregulated in CKD rats vs. Sham control rats (Fig 9A). STS treatment could significantly reverse the expression pattern of OPA-1 (Fig 9B) and Drp-1 (Fig 9C). Immunohistochemistric data evidenced that brown Drp-1 color indicated by blue arrows was highly expressed in CKD kidneys, whereas STS treatment decreased the high expression of Drp-1 in the CKD STS kidneys (Fig 9D). It has been reported that the leakage of cytochrome c from mitochondria to cytosol is required for triggering the apoptotic pathway. We further performed the cytosolic cytochrome c stain for denoting the mitochondrial injury in the CKD tissue. Our data demonstrated that the cytosolic cytochrome c released from the damaged mitochondria and an increased brown color of cytosolic cytochrome c was found in the CKD kidney (Fig 9E). STS treatment markedly reduced the brown cytosolic cytochrome c expression.
Western blot of renal OPA-1 and Drp-1 expression in CKD kidneys (A). The statistical data of densitometry is indicated in B and C, respectively. Immunohistochemistry of mitochondrial fission Drp-1 indicated by blue arrows is indicated in D. Mitochondrial injury indicated by the appearance of cytosolic cytochrome C by blue arrows is demonstrated in E. Data are expressed as mean ± SEM (n = 3). * P < 0.05 vs. Sham. # P < 0.05 vs. CKD.
Discussion
We have well-established the CKD model with two-thirds of the left kidney infarcted by selective ligation of left renal artery, followed by complete removal of the right kidney in the rats with a high survival rate (> 90%). This CKD model displayed an increased oxidative stress (enhanced H2O2 and HOCl amount in blood and kidney ROS), systemic arterial blood pressure associated with renal dysfunction including the elevation of urinary protein, BUN and creatinine in the rats. These data implicated that CKD impaired both glomeruli and tubules in the rats. Upon treatment of STS, our data demonstrated that STS efficiently attenuated oxidative stress and hypertension and conferred significantly improvements in renal function and partial attenuation of glomerulosclerosis and tubulointerstitial lesions. To further explore the underlying mechanisms of the beneficial effects of the STS on renal histology in CKD animals, we have examined its influence on several oxidative stress parameters including mitochondrial fission mediated mitochondrial injury, inflammation, ROS amount, apoptosis, ferroptosis and fibrosis that trigger CKD progression. Our data evidenced that STS could partially ameliorate CKD-induced oxidative, apoptotic, ferroptosis, inflammatory, and fibrotic pathways and subsequently improve the CKD induced renal dysfunction.
We evidenced that STS can efficiently and dose-dependently scavenge H2O2 and HOCl in vitro and can reduce blood and kidney ROS in vivo in the present study. Our recent data [13] indicated that the antioxidant, anti-apoptotic and anti-fibrotic effects of STS confer more locally renal protection in reducing proteinuria than in reducing systematically arterial blood pressure levels in the renovascular hypertension model. Chou et al. [13] found that a less leukocyte infiltration, NADPH oxidase gp91 and 4-HNE expression and fibrosis were identified in the renal cortex and medulla with renovascular hypertension induced injury. We suggest that STS through the antioxidant activity could inhibit angiotensin II/angiotensin type 1 receptor/NADPH oxidase gp91(phox) signaling mediated ROS production, vasoconstriction, inflammation and fibrosis in renovascular hypertension model [13]. Our present study further indicated the increase in blood and kidney ROS, renal leukocyte infiltration and 4-HNE expression in the CKD kidneys implicating CKD enhanced oxidative stress in the impaired kidneys. STS treatment decreased all the oxidative stress parameters including ROS amount, 4-HNE expression, apoptosis, ferroptosis, and fibrosis in the CKD kidneys possibly through the antioxidant action.
STS has been adopted clinically in treating calciphylaxis [12, 22], cyanide poisoning [14], cisplatin toxicity [15, 23], cerebral vasospasm [16], hypertension and renaovascular hypertension [13, 17], however, some adverse effects of STS have been recognized in some patients. It is well-known that intravenous STS was the most common and acceptable route for administration to patients. However, some patients with intravenous STS are more prone to sepsis with 20% occurring rate [24]. In addition, intravenous STS could result in the alterations in osmotic pressure, followed by the release of H+ ions, resulting in severe metabolic acidosis in the patient with calcific uremic arteriolopathy [25]. Previous reports have demonstrated that intraperitoneal administration of STS could induce chemical peritonitis [24, 26]. On the other hand, some reports indicated that STS preconiditioning or post-conditioning provided protective capability against rat heart ischemia/reperfusion injury via the reduction of apoptosis and oxidative stress [27, 28]. Shirozu et al. [29] delineated that intraperitoneal administration of STS attenuated D-Galactosamine and lipopolysaccharide-induced liver injury through activation of Akt- and Nrf2-dependent signaling and inhibition of D-Galactosamine and lipopolysaccharide-induced JNK phosphorylation in the mice. According to our survey, the possible role and mechanism of STS on inhibiting CKD induced renal dysfunction has not been clearly explored. In the present study, we found that STS can reduce CKD-enhanced leukocyte infiltration, blood and kidney ROS levels, renal 4-HNE expression, glomerulerosclerosis, tubulointerstitial fibrosis, Bax/caspase 3/PARP/apoptosis production, and GPX4/xCT mediated ferroptosis associated with the elevated urinary protein concentration, BUN and creatinine levels in the CKD kidneys, implicating STS reducing oxidative stress-induced inflammation, fibrosis, apoptosis and ferroptosis mechanisms in CKD.
On the other hand, the kidneys in CKD displayed severe glomerulosclerosis and interstitial fibrosis in our animal model. The fibrosis appeared in the glomerular and tubular areas of cortex and medulla can be effectively inhibited by intraperitoneal STS treatment. Recently, Nguyen et al. [30] reported that oral treatment with STS for two weeks ameliorated hypertension and improved systolic function, left ventricular hypertrophy, cardiac fibrosis and oxidative stress, without causing metabolic acidosis as is sometimes observed following parenteral administration of this drug. Its protection could be due to the vasodilator and antioxidant potential. In the antioxidant STS treated animals with paraquat induced lung fibrosis, there was a lower severity of elastofibrosis with thinner fascicles and destruction of the collagenous and elastic fibers [31]. Nephrogenic fibrosing dermopathy/nephrogenic systemic fibrosis is an emerging scleromyxedema-like cutaneous disorder of unknown cause that is seen in patients with renal failure and this symptom can be markedly improved after a trial of intravenous sodium thiosulfate [32]. Oxidative stress was raised by the disruption of the redox system leading to an overproduction of ROS. The sources of excess ROS may be produced from NADPH oxidases, mitochondria, nitric oxide synthases, and xanthine oxidases. Accumulating evidences have identified that oxidative stress plays an essential role in tubulointerstitial fibrosis by myofibroblast activation as well as in glomerulosclerosis by mesangial sclerosis, podocyte abnormality, and parietal epithelial cell injury [33]. However, there is lack of underlying mechanisms involving STS on reducing fibrosis-related findings. This requires further experiment to determine.
Renal tubular cells, particularly proximal tubular cells, are rich in mitochondria with high oxygen consumption to maintain a prolonged metabolically active state, which makes the renal tubular cells more vulnerable to hypoxic injury during pathology, leading to mitochondrial dysfunction, production of ROS and secretion of profibrotic cytokines [34]. Mitochondrial dynamics is regulated by Drp-1 mediated mitochondrial fission and OPA-1 mediated mitochondrial fusion and the dysregulated mitochondrial dynamics resulted in several types of kidney diseases [10, 34]. These messages informed that a normal mitochondrial function can be regulated by the balance control of mitochondrial dynamics. Our data informed that an upregulation in Drp-1, a downregulation in OPA-1 and an increased release of cytosolic cytochrome c were noted in the CKD kidney, whereas STS treatment efficiently recovered the Drp-1, OPA and cytosolic cytochrome c expression toward normal status, implicating the role of STS in preservation of mitochondrial dynamics and integrity.
The occurrence of hypertension in several types of CKD is usually found possibly by the impaired endocrine and nervous systems. For example, the contribution of the vasoconstrictor systems (renin-angiotensin-II and sympathetic nervous system) was increased following hypertension induction in CKD models. The role of NO-dependent vasodilation was gradually decreased in 5/6 nephrectomized rats [34]. We suggest that STS may decrease hypertension possibly through the inhibition of renin-angiotensin-II axis and sympathetic nerve activity or preservation of NO mediated vasodilatory function in these CKD rats. However, it requires further experiments to confirm.
It is mentioned that CKD is characterized with the progression of inflammation and oxidative injury in the kidneys leading to severe renal dysfunction. We suggest that STS could confer anti-inflammation through the action of H2S. A previous report stated that STS is a recognized drug devoid of major side effects, which could attenuate murine acute lung injury and cerebral ischemia/reperfusion injury through the action of H2S [35]. The present study indicated that the increased leukocyte infiltration by CKD can be attenuated with STS treatment. We suggest that STS treatment can reduce the severity of inflammation in several kinds of diseases possibly by the antioxidant mechanisms.
Conclusion
In summary, the present study suggest that STS treatment via the intraperitoneal route can mildly counteract CKD-induced hypertension and efficiently improved renal dysfunction possibly through the action of antioxidant, anti-inflammatory, anti-fibrotic, anti-apoptotic and anti-ferroptotic mechanisms. Furthermore, STS treatment could preserve mitochondrial function by depressed mitochondrial fission and restored mitochondrial fusion in the kidneys.
Supporting information
S1 Checklist. The ARRIVE guidelines 2.0: Author checklist.
https://doi.org/10.1371/journal.pone.0277652.s001
(PDF)
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