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. 2016 Aug 3;68(10):1310–1319. doi: 10.1111/jphp.12608

S-Nitrosoglutathione ameliorates acute renal dysfunction in a rat model of lipopolysaccharide-induced sepsis

Devadoss J Samuvel 1, Anandakumar Shunmugavel 2, Avtar K Singh 1, Inderjit Singh 2, Mushfiquddin Khan 2,✉
PMCID: PMC5028274  NIHMSID: NIHMS801866  PMID: 27484743

Abstract

Objective

Sepsis induces an inflammatory response that results in acute renal failure (ARF). The current study is to evaluate the role of S-Nitrosoglutathione (GSNO) in renoprotection from lipopolysaccharide (LPS)-induced sepsis.

Methods

Rats were divided to three groups. First group received LPS (5 mg/kg body weight), second group was treated with LPS + GSNO (50 μg/kg body weight), and third group was administered with vehicle (saline). They were sacrificed on day 1 and 3 post-LPS injection. Serum levels of nitric oxide (NO), creatinine and blood urea nitrogen (BUN) were analysed. Tissue morphology, T lymphocyte infiltrations, and the expression of inflammatory (TNF-α, iNOS) and anti-inflammatory (IL-10) mediators as well as glutathione (GSH) levels were determined.

Key finding

Lipopolysaccharide significantly decreased body weight and increased cellular T lymphocyte infiltration, caspase-3 and iNOS and decreased PPAR-γ in renal tissue. NO, creatinine and BUN were significantly elevated after LPS challenge, and they significantly decreased after GSNO treatment. TNF-α level was found significantly increased in LPS-treated serum and kidney. GSNO treatment of LPS-challenged rats decreased caspase-3, iNOS, TNF-α, T lymphocyte infiltration and remarkably increased levels of IL-10, PPAR-γ and GSH.

Conclusion

GSNO can be used as a renoprotective agent for the treatment of sepsis-induced acute kidney injury.

Keywords: acute kidney injury, lipopolysaccharide, renoprotection, sepsis, S-Nitrosoglutathione

Introduction

Sepsis is a devastating condition with considerable mortality,[1] and it affects 50–100/100 000 people in developed countries.[2] It is a systemic inflammatory response syndrome, leading to multi-organ dysfunction. Children are more prone to sepsis than adults. In the USA alone, nearly 100 000 children visit emergency departments with severe sepsis.[3] Acute kidney injury (AKI) is the most common organ dysfunction after sepsis,[4,5] contributing to the development of multiple organ dysfunction syndrome.[6,7] AKI has been associated with decreased survival rates from sepsis and septic shock.[8] The presence of antigen species or a bacterial metabolite like LPS has pleiotropic effects on the immune system, activating macrophages, lymphocytes and natural killer cells and altering the homoeostasis of immune function.[9] Recent studies show that this imbalance results in the development and progression of nephropathy.[10]

Nitric oxide (NO) regulates several important aspects of renal function. Decreased NO, with its concomitant increase in reactive oxygen species (ROS), is an early marker of renal failure.[11] Nitric oxide synthases (NOSs) induce exorbitant NO production under inflammatory conditions.[12] NO synthesized thorough inducible NOS (iNOS) under pathological conditions is toxic. However, this toxicity is mitigated by the S-nitrosothiols (SNO) and the antioxidant glutathione (GSH). Further, SNO ensures the sustained availability of NO at a controlled rate and for a longer period of time.[13] S-Nitrosoglutathione (GSNO) is a small S-nitrosylated GSH that serves as a relatively long-lived adduct and reservoir of NO. Interestingly, a time-dependent decrease in the level of S-nitrosylated proteins has been reported during sepsis.[14] Hence, targeting the altered NO/nitrosylation homoeostasis offers a plausible and novel approach to treat endotoxemia.

GSNO covalently modifies the specific sulphhydryl groups of its target proteins and initiates an alternative NO signalling pathway.[15] S-nitrosylation of cysteine residues of these target proteins and downstream signalling pathway modulation are analogous to the phosphorylation signalling pathway.[16] Available reports support the hypothesis that S-nitrosylation regulates the activity of several structural and functional proteins.[17–19] In addition, S-nitrosylation can affect the phosphorylation status of proteins, altering those downstream signalling pathways[20,21] and demonstrating an important link between these two evolutionarily conserved signalling pathways.

Recently, an S-nitrosothiol-mediated therapeutic approach has been proven successful in treating several human disorders and animal models of human diseases. For example, in cystic fibrosis, exogenous GSNO has been successfully used to restore the function of cystic fibrosis transmembrane conductance regulator (CFTR).[22] In addition, GSNO inhibits glomerular sclerosis through inhibiting mesangial cell proliferation.[23,24] Several reports from our laboratories and others have shown that GSNO has antioxidant, anti-inflammatory and neuroprotective properties in various animal models, including experimental autoimmune disease, TBI, stroke, SCI and Alzheimer's disease.[25–32] Recently, we showed that the anti-inflammatory property of GSNO is renoprotective in rats with SCI.[33] In this study, we hypothesized that exogenous administration of GSNO to LPS-induced endotoxemic rats would ameliorate renopathological outcomes and that GSNO-mediated renoprotection would occur through increased expression of anti-inflammatory cytokine IL-10, increased levels of antioxidant GSH and decreased expression of the pro-inflammatory mediators TNF-α and iNOS. GSNO is a natural component of the human body and plants.[34,35] It invokes its anti-inflammatory effects mainly via the mechanism of S-nitrosylation of targeted enzymes, including NF-κB and STAT3.[29,36,37] This study shows, for the time, that GSNO protects against AKI in rats possibly by inhibiting inflammatory and promoting anti-inflammatory pathways. Exogenous administration of GSNO to animals or humans has not shown any toxicity or adverse effect,[38–40] supporting GSNO as a promising candidate to treat sepsis-induced AKI.

Materials and Methods

Experimental animals and drug treatment

Female Sprague Dawley rats (225–250 g) were housed under a 12 h light/dark cycle and fed with a regular rodent diet and water ad libitum. Animal procedures for this study were approved by the Institutional Animal Care and Use Committee of the Medical University of South Carolina. One week after acclimation, morphometry was recorded, and the rats were grouped into a control with two experimental groups. One group received LPS (5 mg/kg body weight) and the other LPS (5 mg/kg body weight) plus GSNO (50 μg/kg body weight). The dose of LPS (5 mg/kg body weight) was selected based on moderate kidney tissue damage, as previously reported,[41] and the dose GSNO (50 μg/kg body weight) was based on our study in a rat model of spinal cord injury.[32] LPS (E. coli, 0111:B4) was purchased from Sigma (Sigma-Aldrich, St. Louis, USA), and GSNO was purchased from World Precision Instruments Inc. (Sarasota, USA). LPS, dissolved in saline, was injected intraperitoneally (ip). GSNO dissolved in sterile saline was gavage fed to rats 1 h after LPS challenge. Another group of control rats received saline. Body weight was monitored on a daily basis before and after the experiments began. Half the rats were sacrificed on day 1 (24 h), and the other half, on day 3 (72 h) post-LPS injection.

Serum isolation, NO and ELISA

On the day of sacrifice, rats were placed in the induction chamber of a Surgivet isoflurane unit. Induction was carried out with 5% isoflurane for 3–5 min, followed by cervical dislocation to ensure death. Blood was collected by direct cardiac puncture, and serum was collected and immediately used for NO measurement and ELISA. Serum levels of TNF-α and IL-10 were determined as described earlier from our laboratory.[42] The NO level was measured as the amount of nitrite/nitrate using the Griess reagent method as described previously.[43]

Real-time PCR and transcript analysis

Total RNA was extracted from kidney tissue using TRIzol reagent (Invitrogen, Carlsbad, USA), and first-strand cDNA was synthesized using the iScript™ cDNA synthesis kit (Bio-Rad, Hercules, USA). Relative levels of the transcript of iNOS gene were determined by real-time PCR (iCycler IQ; Bio-Rad) using a SYBR green PCR mix (Bio-Rad). A rat-specific primer for iNOS was purchased from Qiagen (Gaithersburg, USA). Thermal cycling conditions used in this study were as follows: activation of iTaq DNA polymerase at 95 °C for 10 min, followed by 40 cycles of amplification at 95 °C for 30 s and 60 °C for 1 min. The performance of each real-time PCR run was determined by examining the melting curve. Target-specific mRNA expression was normalized to the endogenous Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) housekeeping gene.

Histology

On days 1 and 3 post-LPS challenge, the rats were anesthetized with an excess dose of Nembutal (100 mg/kg). Transcardial perfusion was carried out with PBS, followed by 10% formalin. Kidneys of control, LPS and LPS + GSNO rats were extracted and preserved in 10% formalin for further fixation. Sections for morphological studies were processed and stained with haematoxylin and eosin (H&E), as described previously.[44]

Immunofluorescence

Paraffin embedded kidney sections (5-μm-thick) were deparaffinized, and rehydrated following the routine procedure.[45] Sections were blocked in TNT buffer with 0.5% blocking reagent (TNB, supplied with TSA-Direct kit; NEN Life Sciences, Boston, USA) for 30 min and incubated overnight with anti-PPAR-γ (1 : 200; Santa Cruz Biotechnology, Inc, Dallas, USA), anti-caspase-3 antibodies (1 : 100; Abcam, Cambridge, USA) and anti-CD3 (as a T lymphocyte marker) antibodies (1 : 100; BD Biosciences, CA, USA) at 4 °C. After washing with PBS, the sections were stained with Alexafluor 488 fluorophore-conjugated secondary antibody (Molecular Probes, Invitrogen, USA). The tissue fluorescence pattern was observed and recorded under an epifluorescence microscope equipped with a DP60 digital camera system (Silver Spring, MD, USA), then analysed with image pro Plus 5.1 software. The number of infiltrated T lymphocytes (CD3+ cells) in kidney tissue was counted as described previously.[46]

Western blot

Kidney tissue was lysed with a lysis buffer (50 mm Tris–HCl (pH 7.5), 250 mm NaCl, 5 mm EDTA, 50 mm NaF and 0.5%NP-40) containing a protease inhibitor cocktail (Boehringer Mannheim, Germany). The lysate was centrifuged at 14 000g for 20 min at 4 °C. Total protein content of the supernatant was estimated by BCA assay (Pierce, Rockford, USA). Proteins were separated by 4–20% SDS-PAGE, transferred to nitrocellulose membranes (Amersham Life Sciences, Arlington Heights, USA) and blocked with 5% nonfat milk in Tween-20 Tris-buffered saline for 1 h at room temperature. The membranes were incubated overnight at 4 °C with primary antibodies at 1 : 1000 dilutions in blocking buffer (TTBS with 2% nonfat milk). The following primary antibodies were used in this study: β-actin (Cell Signaling, Danvers, USA), TNF-α (Abcam), iNOS and IL-10 (Santa Cruz, Dallas, USA). After washing, the membranes were incubated with 1 : 10 000 diluted horseradish peroxidase-conjugated secondary antibody (Jackson Immunoresearch Lab, West Grove, USA) for 1 h at room temperature, again washed, reacted with ECL reagent (Amersham Life Science, Pittsburgh, USA) and subjected to autoradiography. They were scanned and analysed with NIH Image J (Bethesda, MD, USA) software.

Glutathione

Glutathione levels were measured in the kidney tissue using a glutathione assay kit (EMD Millipore, USA), as previously described from our laboratory.[47] The levels of GSH were quantitated by comparison with the standards and then normalized with protein levels.

Statistical evaluation

Statistical analysis was performed by ANOVA followed by Tukey post hoc tests using Graph pad prism 5.0 software (La Jolla, CA, USA). Data are presented as mean ± SD. P < 0.05 was considered statistically significant.

Results

GSNO improved body weight after LPS challenge

Body weight was decreased on day 3 post-LPS challenge. GSNO administration inhibited the LPS-induced body weight loss in rats. The significant difference in weight loss between the LPS and LPS + GSNO groups shows the beneficial effect of GSNO in regaining body weight at a faster rate (Figure 1a). In addition, the ratio of body weight to kidney weight was also calculated. There was no significant difference in the ratio between the two groups (Figure 1a).

Figure 1.

Figure 1

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Effect of GSNO on body and kidney weight as well as renoprotection in a rat model of LPS-induced sepsis. Rats challenged with LPS (5 mg/kg body weight) were administered with GSNO (50 μg/kg body weight) daily for 3 days. (a i) GSNO improved the body weight of LPS- challenge rats on day 3. (a ii) Ratio of body weight to kidney wet weight (n = 6 animals in each group); (b) H&E staining of kidney tissue; (c and e) LPS-induced caspase-3 activation; (d and f) LPS-induced decreased expression of PPARγ. Fluorescence was analysed with image pro Plus 5.1 software. Each photomicrograph represents n = 3 in each group. Data are expressed as mean + SD. *P < 0.05; ***P < 0.001.

GSNO reduced renal injury and dysfunction in LPS-treated rats

Control kidney showed intact glomerular apparatus with well-defined renal tubules. LPS-induced tissue degeneration was observed in the glomerular and renal cortical tubules. Increased infiltration of immune cells was also seen in LPS-challenged kidney. GSNO treatment significantly ameliorated the renal glomerular and tubular degeneration induced by LPS and also decreased immune cell infiltration (Figure 1b). To identify the cellular death pathway involved in glomerular and tubular degeneration, we stained the paraffin embedded kidney sections against active (cleaved) caspase-3 antibodies. LPS administration induced significantly elevated expression of caspase-3 protein on days 1 and 3 after LPS challenge (Figure 1c and 1e). GSNO significantly attenuated the LPS-induced expression of caspase-3. Immunofluorescence studies also showed that GSNO treatment significantly increased the protein expression of renoprotective transcription factor PPAR-γ (Figure 1d and 1f).

To analyse GSNO-mediated renal function recovery of rats exposed to LPS, we measured the serum levels of blood urea nitrogen (BUN) and creatinine (Figure 2). A significant increase in serum creatinine was detected in the LPS group on both days tested while GSNO significantly decreased the serum creatinine level on both days. We also found an increase in BUN with the creatinine level (Figure 2a and 2b). As with creatinine, LPS challenge significantly increased the BUN level on post-LPS days 1 and 3. The LPS-induced increase in BUN was significantly attenuated by the administration of GSNO.

Figure 2.

Figure 2

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Effect of GSNO on creatinine and BUN levels in a rat model of LPS-induced sepsis. The levels of creatinine (a) and BUN (b) were measured in serum. Data (measured as mg/dl) are expressed as mean + SD. ***P < 0.001 (n = 6 in each group).

GSNO decreased the LPS-induced pro-inflammatory cytokine expression

We determined the effect of GSNO on LPS-induced NOS activity measured in terms of total serum nitrite/nitrate using Griess reagent. Serum total nitrite concentration in rats was 235.2 ± 15.66 and 293.3 ± 40.33 μmol/ml on days 1 and 3, respectively, post-LPS challenge. GSNO treatment significantly decreased the total nitrite level, with no significant differences between the levels of the control and LPS + GSNO rats on either day tested (Figure 3a).

Figure 3.

Figure 3

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Effect of GSNO on NO (nitrite/nitrate) levels and the expression of iNOS in a rat model of LPS-induced sepsis. GSNO reduced serum levels of NO (a); kidney tissue levels of iNOS mRNA (b); kidney tissue levels of iNOS protein (c); densitometry of iNOS Western blot using NIH image J software (d). Data are expressed as mean + SD. *P < 0.05; **P < 0.01; ***P < 0.001 vs. control (n = 3 in each group).

The increase in LPS-induced serum nitrite level paralleled the increased transcription of iNOS mRNA (Figure 3b). The RT-PCR data show an increase in the transcription of iNOS over the control on days 1 and 3 post-LPS challenge. GSNO treatment significantly decreased the transcription rate of iNOS mRNA. The data show that the GSNO-mediated decrease in systemic nitrite level is due to the inhibition of iNOS at the transcription level (Figure 3a and 3b). The decreased transcription of iNOS is also reflected at the translation level as determined by Western blot analysis (Figure 3c and 3d). The data show that GSNO treatment significantly decreased iNOS expression at the transcription level and total nitrite/nitrate in the serum.

Available studies strongly suggest that the organ dysfunction seen during sepsis is mediated through the release of inflammatory cytokines.[48] In this study, we determined the effect of GSNO on LPS-induced increases in serum cytokine levels. Western blot analysis shows that the expression of the inflammatory marker TNF-α was increased in the kidney tissue on days 1 and 3 post-LPS challenge (Figure 4a). There was also a significant increase in the serum TNF-α level in LPS-challenged rats (Figure 4c). GSNO treatment significantly decreased the serum TNF-α in LPS rats. ELISA and Western blot analyses confirmed the anti-inflammatory properties of GSNO in rats challenged with LPS.

Figure 4.

Figure 4

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Effect of GSNO on kidney tissue protein expression and serum protein levels of TNF-α and IL-10 in a rat model of LPS-induced sepsis. GSNO reduced kidney tissue expression of TNF-α (a; Western blot and densitometry) and serum levels of TNF-α (c; ELISA). In contrast to the effects on TNF-α, GSNO treatment increased kidney tissue expression of IL-10 (b; Western blot and densitometry) and serum levels of IL-10 (d, ELISA). Data are expressed as mean + SD. **P < 0.01; ***P < 0.001 (n = 3 in each group).

Figure 4d shows the expression level of cytokine IL-10 in the serum of rats. LPS treatment did not result in increased expression of IL-10 on either day tested. GSNO treatment significantly increased the expression of serum IL-10 in LPS rats on day 1 (207.34 ± 12.24 pg/ml). There was no significant difference in the expression level of IL-10 between the groups on day 3 post-LPS challenge (Figure 4d). The expression of IL-10 in tissue was determined using Western blot analysis (Figure 4b). Increased serum expression of IL-10 was also reflected in the tissue level. The data show that GSNO treatment increased the expression of the anti-inflammatory cytokine IL-10.

GSNO reduced the LPS-induced infiltration of T lymphocytes

To further confirm the immune filtrations as indicated in Figure 1b, we evaluated the infiltration of T lymphocytes using CD3+ as a T-cell marker.[49] The LPS-challenged kidney had significantly increased infiltration of T lymphocytes, whereas GSNO treatment decreased the number of infiltrated T lymphocytes (Figure 5a and 5b).

Figure 5.

Figure 5

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Effect of GSNO on the infiltration of kidney T lymphocytes in a rat model of LPS-induced sepsis. GSNO treatment of LPS-administered rats reduced the infiltration of T lymphocytes. (a) Photomicrographs of T lymphocyte stainings; (b) Counting of infiltrated T lymphocytes. Cell counting data are expressed as mean ± SD. ***P < 0.001 (n = 3 in each group).

GSNO alleviated LPS-induced loss of renal GSH

Lipopolysaccharide challenge decreased the glutathione levels in kidney tissue significantly when compared with corresponding control animals at days 1 and 3. GSNO treatment markedly increased the levels of glutathione in the LPS-challenged rats (Figure S1).

Discussion

The present study shows that the exogenous administration of GSNO significantly ameliorated LPS-induced experimental AKI in rats and suggests a novel therapeutic potential of GSNO in protecting sepsis-induced renal dysfunction. Several animal models, including the caecal ligation and puncture models,[50] are available to examine the pathophysiology of sepsis. In the present study, we used the LPS-induced sepsis rat model to study the renoprotective effects of GSNO. In evaluating these effects, we found that the administration of GSNO significantly increased the body weight of LPS-challenged rats. LPS-induced alterations in body weight regulation are correlated with endocrine, neuropeptide and cytokine regulation systems.[51] Histological examination of the kidney of LPS-challenged rats showed increased glomerular degeneration, exudate (oedema) (Figure 1b) and cellular infiltration (Figure 5). GSNO treatment significantly decreased the glomerular degeneration. Fluid filled oedema was also decreased. Further, cellular infiltration of T lymphocytes in the kidney tissue was decreased after GSNO treatment, indicating the anti-inflammatory and immunomodulatory properties of GSNO.

IL-10 is a pleiotropic molecule with immuno-regulatory and anti-inflammatory properties. It downregulates the expression of Th1 cytokines and NF-kB activity, and it upregulates B-cell survival, proliferation and antibody production. The immunosuppressive property of IL-10 is clinically significant.[52] In the present study, GSNO-mediated increase of IL-10 expression supports the idea that GSNO may have influenced differentiation of T cells towards a Th2 phenotype. This observation corroborates a report of Corinti et al.[53] about immunomodulatory regulation by NO. Further, the stimulation of Toll-like receptor 4 by LPS induces the TLR signalling pathway, resulting in the release of pro-inflammatory cytokines and a potent immune response.[54] Interleukin-1 receptor-associated kinase M (IRAK-M) is a negative regulator of TLR signalling. TLR signalling involves the disassociation of the Myeloid Differentiation 88 (MyD88) and IRAKs complex. The disassociated IRAKs combine with TRAF (TNF receptor-associated factor) and induce the overexpression of cytokines.[55] One key negative regulator of cytokines is SOCS-1 (Suppressor of cytokine signalling), whose transcription and translation, along with that of IRAK-M, is induced by GSNO.[56] Hence, the decrease in TNF-α cytokine level in endotoxemic rats treated with GSNO may be due to the induction of IRAK-M and SOCS-1, important negative regulators of cytokine production.

Having established that GSNO administration significantly decreased LPS-induced inflammation and renal dysfunction, we assessed GSNO's effects on PPAR-γ, a molecule that plays several important anti-inflammatory and antifibrotic roles.[57,58] In the present study, we observed a significant decrease in renal PPAR-γ level after LPS challenge. GSNO treatment ameliorated this decrease in PPAR-γ expression. Hence, the renoprotective effect of GSNO may also be attributed to a GSNO-induced increase in PPAR-γ protein expression. However, the molecular mechanism by which GSNO increases the expression of this transcription factor needs further investigation.

Lipopolysaccharide-induced expression of inflammatory cytokines stimulates robust NO production. Of the three isoforms of NOS, neuronal NOS (nNOS) and endothelial NOS (eNOS) produce low levels of NO required for normal physiological functioning. On the other hand, iNOS, induced by LPS and inflammatory cytokines, produce cytotoxic and pathologic high levels of NO. Studies from our laboratories and others have shown that GSNO inhibits iNOS expression, thereby limiting cytotoxicity.[59] NO is a highly reactive molecule involved in numerous physiological and pathological processes.[60] NO is short-lived but exists in other physiological forms, including GSNO.[61] GSNO in equilibrium with other SNOs functions as part of a bioavailable intracellular NO pool involved in NO-mediated signalling pathways. For example, S-nitrosylation of NADPH oxidase (NOX), a major source of superoxide implicated in oxidative stress, significantly decreased the enzyme activity of the protein,[62] resulting in a decreased level of ROS and superoxide production, thereby relieving oxidative stress.[15] However, on LPS challenge, the endogenous level of S-nitrosylated proteins decreases with a concomitant increase in the level of peroxynitrite.[14] The functional impact of S-nitrosylation on proteins of physiological significance is considered equivalent to other post-translational modifications like phosphorylation. Both the modifications are reversible and specific, allowing cells to manoeuvre the protein's function in accordance with environmental cues.[63] GSNO is also reported to restore the homoeostasis of GSH, a potent thiol-based antioxidant,[64,65] in several neurodegenerative diseases. LPS-induced GSH depletion has also been observed.[66,67] Elsewhere, we reported that GSNO promoted neurorepair processes in traumatic brain injury (TBI) by increasing GSH production.[28] As in TBI, GSNO here increased GSH levels in the LPS-challenged kidney, indicating that GSNO protects against AKI, at least in part, via an antioxidant mechanism.

The pharmacological implications of S-nitrosylating agents such as GSNO have recently been given prime importance. Enhancing GSNO levels by inhibiting the activity of GSNO reductase has been shown to improve the endothelial function.[61] Interestingly, we also found that an administration of GSNO decreases peroxynitrite levels under animal models of neuropathological conditions like TBI and stroke.[27,28] Therefore, it is intriguing that exogenous administration of GSNO can also ameliorate LPS-induced renal pathology through the S-nitrosylation of critical proteins of interest. The specific protein targets and status of S-nitrosylation under LPS-induced renal dysfunction need to be investigated.

Conclusions

The study demonstrates the beneficial effects of GSNO in protecting rats against LPS-induced AKI. GSNO-mediated amelioration is associated with its anti-inflammatory and antioxidant effects. These results indicate that GSNO may be used alone or as an adjunct therapy for the treatment of sepsis-induced AKI.

Declarations

Conflict of interest

The Author(s) declare that they have no competing interest.

Funding

This work was supported in part by grants from The Heather Perkins Trew Foundation and grants from the NIH (NS72511) and (CO6 RR018823 and CO6 RR0015455 from the Extramural Research Facilities Program of the National Center for Research Resources).

Supplementary Material

jphp12608-sup-0001-FigS1

Figure S1. Effect of GSNO on levels of reduced glutathione (GSH) in a rat model of LPS-induced sepsis.

Click here for additional data file. (348.4KB, png)

Acknowledgements

We thank Joyce Bryan for procurement of animals and chemicals, and secretarial assistance. We also acknowledge Dr. Tom Smith from the MUSC Writing Center for his valuable editing of the manuscript.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

jphp12608-sup-0001-FigS1

Figure S1. Effect of GSNO on levels of reduced glutathione (GSH) in a rat model of LPS-induced sepsis.

Click here for additional data file. (348.4KB, png)

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