Free Radical Biology and Medicine 112 (2017) 423–432
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Original article
Hydrogen sulfide-producing cystathionine γ-lyase is critical in the progression of kidney fibrosis
MARK
Sang Jun Hana, Mi Ra Noha, Jung-Min Jungb, Isao Ishiic, Jeongsoo Yoob, Jee In Kimd, ⁎ Kwon Moo Parka, a Department of Anatomy, Cardiovascular Research Institute and BK21 Plus, Kyungpook National University School of Medicine, 680 Gukchaebosang-ro, Junggu, Daegu 41944, Republic of Korea b Department of Molecular Medicine, BK21 Plus, Kyungpook National University School of Medicine, 680 Gukchaebosang-ro, Junggu, Daegu 41944, Republic of Korea c Laboratory of Health Chemistry, Showa Pharmaceutical University, Machida, Tokyo 194-8543, Japan d Department of Molecular Medicine and MRC, College of Medicine, Keimyung University, 1095 Dalgubeol-daero 250-gil, Dalseogu, Daegu 42601, Republic of Korea
A R T I C L E I N F O
A B S T R A C T
Keywords: Chronic kidney disease Cystathionine γ-lyase Reactive oxygen species Kidney fibrosis Hydrogen sulfide
Cystathionine γ-lyase (CSE), the last key enzyme of the transsulfuration pathway, is involved in the production of hydrogen sulfide (H2S) and glutathione (GSH), which regulate redox balance and act as important antioxidant molecules. Impairment of the H2S- and GSH-mediated antioxidant system is associated with the progression of chronic kidney disease (CKD), characterized by kidney fibrosis and dysfunction. Here, we evaluated the role of CSE in the progression of kidney fibrosis after unilateral ureteral obstruction (UUO) using mice deficient in the Cse gene. UUO of wild-type mice reduced the expression of H2S-producing enzymes, CSE, cystathionine βsynthase, and 3-mercaptopyruvate sulfurtransferase in the obstructed kidneys, resulting in decreased H2S and GSH levels. Cse gene deletion lowered H2S and GSH levels in the kidneys. Deleting the Cse gene exacerbated the decrease in H2S and GSH levels and increase in superoxide formation and oxidative damage to proteins, lipids, and DNA in the kidneys after UUO, which were accompanied by greater kidney fibrosis, deposition of extracellular matrixes, expression of α-smooth muscle actin, tubular damage, and infiltration of inflammatory cells. Furthermore, Cse gene deletion exacerbated mitochondrial fragmentation and apoptosis of renal tubule cells after UUO. The data provided herein constitute in vivo evidence that Cse deficiency impairs renal the H2S- and GSH-producing activity and exacerbates UUO-induced kidney fibrosis. These data propose a novel therapeutic approach against CKD by regulating CSE and the transsulfuration pathway.
1. Introduction Pyridoxal-5′-phosphate (P5P)-dependent cystathionine γ-lyase (CSE), cystathionine β-synthase (CBS), and P5P-independent 3-mercaptopyruvate sulfurtransferase (3-MST) produce hydrogen sulfide (H2S) [1]. CSE and CBS are involved in the synthesis of glutathione (GSH) by suppling endogenous cysteine, a component of GSH. These two molecules, H2S and GSH, produced by those enzymes, play important roles in the progression and development of various fibrogenic diseases, which are associated with oxidative tissue damage [1]. H2S controls cellular redox status by regulating NF–E2-related factor 2 (Nrf2)/antioxidant responsive element (ARE) signaling and directly
scavenging free radicals [2,3]. GSH is the most abundant antioxidant molecule and plays a critical role in maintaining cellular redox balance [4,5]. Emerging evidence has demonstrated that H2S and GSH are associated with the progression of fibrosis in various organs [6,7]. However, the roles of these enzymes in kidney fibrosis remain to be defined. Kidney fibrosis is a major cause in the development and progression of chronic kidney disease (CKD), which evokes severe clinical problems [8]. Kidney fibrosis is characterized by increased numbers of myofibroblasts, infiltration and accumulation of inflammatory cells, and excessive accumulation of extracellular matrix components such as collagen and fibronectin [9,10]. Recently, it was reported that
List of abbreviations: CSE, Cystathionine γ-lyase; H2S, Hydrogen sulfide; GSH, Glutathione; CKD, Chronic kidney disease; UUO, Unilateral ureteral obstruction; P5P, Pyridoxal-5′phosphate; CBS, Cystathionine β-synthase; 3-MST, 3-mercaptopyruvate sulfurtransferase; Nrf2, NF–E2-related factor 2; ARE, Antioxidant responsive element; ESRD, End-stage renal disease; α-SMA, α-smooth muscle actin; Gpx, Glutathione peroxide; 8-OHdG, 8-hydroxy-2'-deoxyguanosine; Fis1, Fission 1 protein; PAG, Propargylglycine; NAC, N-acetylcysteine; TGFβ1, Transforming growth factor β1; 4-HNE, 4-hydroxynonenal; MnSOD, Manganese superoxide dismutase; CuZnSOD, Copper-zinc superoxide dismutase; TNF-α, Tumor necrosis factor α; Opa1, Optic atrophy 1; XIAP, X-linked inhibitor-of-apoptosis protein ⁎ Corresponding author. E-mail address: [email protected] (K.M. Park). http://dx.doi.org/10.1016/j.freeradbiomed.2017.08.017 Received 8 June 2017; Received in revised form 29 July 2017; Accepted 21 August 2017 Available online 24 August 2017 0891-5849/ © 2017 Elsevier Inc. All rights reserved.
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signals and myofibroblast activation after UUO.
impairments in H2S production and GSH-associated antioxidant system play a critical role in the development and progression of kidney fibrosis; plasma H2S and CSE mRNA levels in end-stage renal disease (ESRD) patients are lower than levels observed in healthy people. Furthermore, levels of plasma homocysteine and cysteine, precursors of H2S and GSH, respectively, are higher in ESRD patients than in healthy people [11,12]. Plasma GSH level is lower in patients with moderate and severe CKD, and the GSH level is positively correlated with creatinine clearance [13]. These clinical studies suggest that H2S, GSH, and the CSE-transsulfuration pathway are deeply associated with the progression of kidney fibrosis. Supporting this, many investigators, including us, have recently reported that kidney fibrosis induced by 5/6 of nephrectomy, unilateral ureteral obstruction (UUO), or streptozotocin injection impair the functions of all three H2S-producing enzymes in the kidneys, and exogenous supplements of H2S attenuate kidney fibrosis and GSH reduction, while inhibition of H2S production worsens them [2,14–19]. These studies have suggested that the antifibrotic effects of H2S are mediated by inhibition of oxidative stress, inflammation, and fibrogenic pathways including TGF-β1/SMAD3, MAPK, and NF-κB [2,14,19–23]. Mitochondria are most susceptible organelle to ROS/oxidative damage and mitochondrial oxidative damage is critical for the progression of fibrosis by inducing apoptosis and inflammation [10,24]. It was recently reported that CSE regulates mitochondrial H2S and GSH levels [25,26]. We also recently found that a reduction in mitochondrial GSH level exacerbates kidney fibrosis by enhancing mitochondrial oxidative stress, thereby inducing mitochondrial damage and apoptosis of tubule cells [27]. Exogenous supplementation of H2S ameliorates renal fibrosis by downregulating ROS, oxidative damage, and the inflammatory responses, and upregulating GSH in kidneys after UUO or ischemic injuries [2,14]. I n the kidney, CSE is abundantly expressed, 20-fold more than the CBS expression, and is mainly responsible for H2S production in combination with CBS [6,21,22]. Therefore, we have investigated the role of CSE in fibrotic changes of kidneys after UUO and its underlying mechanisms using Cse knockout (Cse–/–) mice.
2.2. Cse gene deletion impairs H2S and GSH production in the kidney To investigate whether increased fibrosis in Cse gene-deleted mice after UUO is associated with impaired transsulfuration pathways, which are linked to H2S and glutathione (GSH) production, [4,25] we examined expressions of CSE, CBS, and 3-MST proteins, and levels of H2S and GSH in the kidneys. After UUO of Cse+/+ mice, kidney levels of H2S decreased in a time-dependent manner (Fig. 2A), which were accompanied by time-dependent decreases in renal CSE, CBS, and 3-MST expression (Fig. 2B-E). Similarly, renal GSH levels were decreased a time-time-dependent manner after UUO (Fig. 2F), accompanied by time-dependent decreases in renal glutathione peroxide 1 (GPx1) and glutathione reductase (GR) expression (Fig. 2G-I). Levels of H2S and GSH and these enzymes (CSE, CBS, and 3-MST) were negatively correlated with α-SMA expression and collagen deposition (Fig. 2A-L). These results indicate that CSE/CBS/3-MST-mediated H2S/GSH production is intimately linked with progression of kidney fibrosis. Next, we examined whether the systemic Cse gene deletion in mice affects H2S, GSH production, the ratio of GSSG to tGSH, and CBS and 3MST expression. After sham-operation, H2S and GSH levels in the Cse–/– mouse kidneys were significantly lower than those in Cse+/+ kidneys, about 60% and 70%, respectively (Fig. 3A, B). These data indicate that Cse gene deletion impairs H2S and GSH production in the kidney. At 5 days after UUO, H2S and GSH levels significantly decreased in both Cse–/– and Cse+/+ kidneys but both decreases were significantly greater in the Cse–/– kidneys than in Cse+/+ kidneys (Fig. 3A, B). In addition the ratio of GSSG to tGSH was significantly greater in the Cse–/– kidneys than in Cse+/+ kidneys after UUO (Fig. 3C). Consistent with the levels of H2S and GSH, expressions of CSE, CBS, and 3-MST decreased after UUO (Fig. 3D-G). In contrast, renal CBS and 3-MST expression was comparable between Cse+/+ and Cse–/– mice at either pre- or post-UUO (Fig. 3D, E, G). These results suggest that CSE plays a pivotal role in the maintenance of renal H2S/GSH levels, thereby protecting kidney against fibrosis via their anti-oxidative activities.
2. Result 2.1. Cse gene deletion exacerbates UUO-induced fibrosis in the kidney
2.3. Cse gene deletion exacerbates ROS production and oxidative stress after UUO
First, we determined the effect of the systemic Cse gene deletion on kidney fibrosis after UUO. UUO induced tubular damage and expansion of the interstitial area; these phenotypes were much more apparent in Cse–/– mice than in Cse+/+ mice (Fig. 1A [top panel], B). Collagen deposition, as evaluated by Sirius red staining, also increased in the interstitium after UUO and this increase was significantly greater in Cse–/– kidneys than in Cse+/+ kidneys (Fig. 1A [middle panel], C). Furthermore, UUO-induced increases in F4/80-positive cells, monocytes/macrophages, were greater in the interstitium of Cse–/– kidneys than Cse+/+ kidneys. (Fig. 1A [bottom panel], D). In contrast, there were no significant differences in tubular damage, collagen deposition, and number of F4/80-positive cells between sham-operated Cse–/– and Cse+/+ mouse kidneys (Fig. 1A-D). Expressions of TNF-α, a pro-inflammatory cytokine, and Ly6G, a neutrophil marker, increased after UUO and these increases were significantly greater in Cse–/– kidneys than in Cse+/+ kidneys (Fig. 1E-G). There were no significant differences in TNF-α or Ly6G expression between sham-operated Cse–/– kidneys and Cse+/+ kidneys (Fig. 1E-G). TGF-β1 acts as a main mediator in fibrosis development and progression in CKD via Smad-3 phosphorylation (p-Smad3) and the subsequent activation of collagen-producing myofibroblasts [9]. Post-UUO increases of TGF-β1, p-Smad3, and α-smooth muscle actin (α-SMA; a myofibroblast marker) expression were all significantly greater in Cse–/– kidneys than in Cse+/+ kidneys (Fig. 1H-K). Taken together, the systemic Cse gene deletion in mice exacerbates kidney fibrosis, tubular damage, and inflammation by promoting greater activation of TGF- β1
H2S and GSH play important roles in maintaining redox balance, which is critical for the progression of renal fibrosis and tubular damage [6]. Expression of 4-HNE, oxidized peroxiredoxin (Prx-SO3), and 8hydroxy-2'-deoxyguanosine (8-OHdG), indicators of lipid peroxidation, peroxiredoxin oxidation, and DNA oxidation, [28,29] respectively, significantly increased in both Cse–/– and Cse+/+ mouse kidneys after UUO and those increases were greater in Cse–/– kidneys (Fig. 4A-E). There were no significant differences in expression of 4-HNE, Prx-SO3, or 8-OHdG between sham-operated Cse–/– and Cse+/+ mouse kidneys (Fig. 4A-E). These results indicate that Cse gene deletion in mice exacerbates oxidative stress in the ureteral obstructed kidneys after UUO, and this increased oxidative stress may be associated with a decreased antioxidative effect in kidneys due to reduced levels of H2S and GSH by Cse gene deletion. GSH is required for the GSH-mediated antioxidative system, which contains enzymes such as glutathione peroxidase 1 (GPx1) and glutathione reductase (GR). Since GPx1 is a major enzyme for removal of H2O2 using reduced GSH, which consequently produces oxidized glutathione (GSSG) [30], we determined GPx1 levels. UUO significantly decreased GPx1 levels in the kidneys and this decrease was greater in the Cse–/– kidneys than in Cse+/+ kidneys (Fig. 5A, B). Furthermore, the expression of GR, which reduces GSSG [30] to GSH, was lowered in the kidneys from both groups after UUO, and this reduction was greater in the Cse–/– kidneys than in the Cse+/+ kidneys (Fig. 5A, C). There was no significant difference in GPx1 or GR expression between sham-operated Cse–/– and Cse+/+ mouse kidneys (Fig. 5A-C). These data 424
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Fig. 1. Fibrosis, tubular damage, and inflammation in Cse–/– and Cse+/+ mouse kidneys after unilateral ureteral obstruction (UUO). Cse–/– and Cse+/+ mice were subjected to UUO by tying the right ureter. (A) Five days after surgery, kidney sections were subjected to staining with PAS (top panel), Sirius red (middle panel), and immunohistochemical stains using antiF4/80 antibody (bottom panel). Arrowheads indicate F4/80-positive cells. (B) Kidney damage was scored as described in Section 4. ND; not detected. (C) Collagen deposition was measured as described in the Materials and Methods. (D) Number of F4/80-positive cells in the interstitial area are shown. (E, H) Kidney samples were subjected to western blotting using anti-TNF-α (E), -Ly6G (E), -TGF-β1 (H), -p-SMAD3 (H), and -α-SMA (H) antibodies. GAPDH was used as a loading control. (F, G, I-K) Bands densities were measured using ImageJ software developed by the NIH. Results were expressed as the mean ± SEM (n = 6). *, P < 0.05 vs. Cse WT sham. †, P < 0.05 vs. Cse WT UUO (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.).
redox balance by regulating H2S and GSH levels [25,26]. In the present study, UUO increased levels of fission 1 protein (Fis1), a regulator of mitochondrial fission, in wild-type mice, whereas UUO decreased Opa1, a mitochondrial fusion protein, in a time-dependent manner (Fig. 6AC), resulting in mitochondrial fragmentation (Fig. 6D). The change of Fis1 expression was greater in Cse–/– kidneys than in Cse+/+ kidneys after UUO, whereas there was no difference in Opa1 expression between Cse–/– and Cse+/+ kidneys (Fig. 6E-G). This indicates that Cse gene deletion exacerbates UUO-induced mitochondrial fragmentation.
indicate that Cse gene deletion impairs the GSH-mediated antioxidant system after UUO. Furthermore, Cse gene deletion exacerbated UUOinduced decreases of catalase, manganese superoxide dismutase (MnSOD), and cupper zinc SOD (CuZnSOD) (Fig. 5D-G). Taken together, these results indicate that Cse gene deletion reduces H2S and GSH production, and exacerbates ROS/oxidative stress and antioxidant enzyme impairment following UUO.
2.4. Cse gene deletion exacerbates mitochondrial damage after UUO Under normal conditions, mitochondria dynamically undergo fission and fusion, and this process is balanced appropriately [31]. Accumulating evidence has demonstrated that fibrosis is associated with a shift toward mitochondrial fission, which leads to mitochondrial fragmentation, damage, and subsequent apoptotic cell death [6,24,32]. Furthermore, recent studies reported that CSE regulates mitochondrial
2.5. Cse gene deletion exacerbates tubule cell apoptosis after UUO Since H2S and GSH exert a protective role in tubular cell apoptosis, which is a major pathway of kidney fibrosis and mitochondrial damage after UUO, [6,32] we investigated whether Cse gene deletion affected kidney tubule cell apoptosis after UUO. UUO significantly increased the 425
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Fig. 2. H2S levels and H2S-producing enzyme expression in kidneys after unilateral ureteral obstruction (UUO). C57BL/6J female wild-type mice were subjected to UUO. (A) At the indicated day after surgery, H2S levels in kidneys tissues were measured. (B) Kidney samples were subjected to western blotting using anti-CSE, -CBS, and −3-MST antibodies. GAPDH was used as a loading control. (C-E) The densities of bands were measured using the ImageJ software. (F) GSH level in kidney tissues was measured as described in Section 4. (G) Kidney samples were subjected to western blotting using anti-GPx1, -GR, and -α-SMA antibodies. GAPDH was used as a loading control. (H-J) The densities of bands were measured using ImageJ software. (K) Kidney sections were subjected to Sirius red staining. (L) Collagen deposition was measured. Results were expressed as the mean ± SEM (n = 6). *, P < 0.05 vs. sham.
UUO significantly increased the number of TUNEL-positive tubule cells and those increases were greater in Cse–/– kidneys than in Cse+/+ kidneys (Fig. 7G, H). These results indicated that Cse gene deficiency exacerbates UUO-induced apoptosis of tubule cells.
expression of pro-apoptotic factors active caspase-3 and Bax, whereas UUO decreased the expression of anti-apoptotic factors Bcl-2 and XIAP (Fig. 7A-F). Those post-UUO changes in pro-apoptotic and anti-apoptotic factors were greater in Cse–/– kidneys than in Cse+/+ kidneys (Fig. 7A-F). Consistent with changes in levels of apoptosis regulators,
Fig. 3. H2S and GSH levels and H2S producing-enzyme expressions in the Cse–/– and Cse+/+ mice kidneys after unilateral ureteral obstruction (UUO). Cse knockout (Cse–/–) and wild-type mice (Cse+/+) mice were subjected to UUO. Five days after surgery, kidneys were harvested then H2S (A), GSH and GSSG (B, C) levels in the kidney tissue were measured (Note, although the H2S determining method used this study is widely used, the method may not determine the amount of free H2S) (D) Kidney samples were subjected to western blotting using anti-CSE, -CBS, and −3-MST antibodies. GAPDH was used as a loading control. (E-G) Band densities were measured using ImageJ software. Results were expressed as the mean ± SEM (n = 6). *, P < 0.05 vs. Cse WT sham. †, P < 0.05 vs. Cse WT UUO.
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Fig. 4. Oxidative damages in the Cse–/– and Cse+/+ mice kidneys after unilateral ureteral obstruction (UUO). Cse–/– and Cse+/+ mice were subjected to UUO and five days after surgery kidneys were harvested. (A) Kidney samples were subjected western blotting using anti-4-HNE and -Prx-SO3 antibodies. GAPDH was used as a loading control. (B, C) Band densities were measured using ImageJ software. (D) Kidney sections were subjected to immunohistochemical staining using an anti-8-OHdG antibody. Hematoxylin was used to visualize cell nuclei. (E) Intensity of 8-OHdG stains in tubule cells was determined. Results were expressed as the mean ± SEM (n = 6). *, P < 0.05 vs. Cse WT sham. †, P < 0.05 vs. Cse WT UUO.
MST, and that Cse gene deletion exacerbated UUO-induced kidney fibrosis along with greater reductions of H2S in the kidney when compared to wild-type kidneys. These results indicate that the reduced H2S production that occurs upon Cse gene deletion may increase oxidative stress and lead to greater fibrosis. The expression of CSE, CBS and 3MST are transcriptionally and/or post-transcriptionally regulated by various factors [33]. Although currently little information is available regarding to regulation of H2S-producing enzyme expressions, we can speculate that UUO may influence various transcriptional and translational factors including Sp1 and consequently lead to down-regulation in the expression of H2S-producing enzymes. Recently Wu et al. reported that ischemia/reperfusion reduced Cbs gene expression by increased Sp1 phosphorylation via ERK activation in the kidney, and consequently led to decreased H2S levels [34]. Therefore, our results indicate that reduced H2S level and H2S-producing enzyme expression may increase oxidative stress and lead to greater fibrosis. In addition, the increased oxidative stress and fibrosis in Cse gene deleted mice may
3. Discussion This study demonstrates, for the first time, that ureteral obstruction reduced H2S and GSH levels together with downregulation of H2S- and GSH-producing transsulfuration pathway enzymes. We also found that Cse gene deletion in mice reduced levels of H2S and GSH in the kidneys, and exacerbates UUO-induced oxidative stress, mitochondrial fragmentation, apoptosis, and inflammation by promoting a greater reduction of H2S and GSH levels in the kidney, consequently resulting in greater kidney fibrosis. These results indicate that CSE plays a crucial role in the progression of kidney fibrosis, suggesting that CSE could be a potential protein to target in the development of therapeutics for fibrotic tissue diseases, including CKD. Accumulating evidence has demonstrated that H2S inhibits the progression of tissue fibrosis by reducing oxidative stress [6,7,25]. In the present study, we found that UUO reduced tissue H2S levels together with reductions in the H2S-producing enzymes CSE, CBS and 3-
Fig. 5. Expressions of GR, GPx1, catalase, and SODs in the Cse–/– and Cse+/+ mice kidneys after unilateral ureteral obstruction (UUO). Cse–/– and Cse+/+ mice were subjected to UUO. (A) Five days after surgery, kidney samples were subjected western blotting using anti-GPx1 and -GR antibodies. GAPDH was used as a loading control. (B, C) Bands densities were measured using ImageJ software. (D) Kidney samples were subjected to western blotting using anti-catalase, -MnSOD, and -CuZnSOD antibodies. (E-G) Band densities were measured using ImageJ software. Results were expressed as the mean ± SEM (n = 6). *, P < 0.05 vs. Cse WT CLT. †, P < 0.05 vs. Cse WT UO.
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Fig. 6. Expression of mitochondrial dynamics-regulatory proteins in the Cse–/– and Cse+/+ mice kidneys after unilateral ureteral obstruction (UUO). (A-D) C57BL/6J female wild-type mice were subjected to UUO. On the indicated days after surgery, kidneys samples were subjected western blotting using anti-Fis1 and -Opa1 antibodies. GAPDH was used as a loading control. (D) On the indicated days after surgery, mitochondrial structures were evaluated using transmission electron microscopy (TEM). M, mitochondria. N, nucleus. (E-G) Cse–/– and Cse+/+ mice were subjected to UUO. (E) Five days after surgeries, kidneys were harvested and then subjected western blotting using anti-Fis1, and -Opa1 antibodies. GAPDH was used as a loading control. (F, G) Bands densities were measured using ImageJ software. Results were expressed as the mean ± SEM (n = 6). *, P < 0.05 vs. Cse WT sham. †, P < 0.05 vs. Cse WT UUO.
reported that CSE overexpression protected cells against H2O2-mediated cell death [36]. Therefore, we speculate that the exacerbation of fibrosis in Cse-deficient mice may be caused by the reduced ability of cells to cope with oxidative stress. In the present study, we found, for the first time, that Cse gene deletion alone dramatically reduces levels of H2S and GSH in kidneys without any significant reduction of CBS and 3MST expressions. However, deletion of this gene alone did not induce significant tissue oxidative damages like lipid peroxidation, DNA oxidation, or protein oxidation. Cse gene deletion exacerbated UUO-induced decreases of H2S and GSH. These results indicate that CSE is a critical enzyme for production or maintenance of cellular H2S and GSH levels under both normal and pathological conditions. Yang et al. found that Cse gene deletion alone reduces H2S and GSH levels in vascular tissues including the mesenteric artery, aorta, and smooth muscle cells isolated from mesenteric arteries, yet Cse gene deletion did not significantly change ROS levels in those tissues [37]. Lee et al. found that inhibiting CSE using the CSE inhibitor propargylglycine (PAG) or Cse siRNA reduces GSH levels [5] and that PAG and Cse siRNA treatment increase sensitivity to H2O2-induced cytotoxicity [5]. Furthermore, Mani et al.
be associated with reduced H2S production. H2S has been shown to directly scavenge ROS including superoxide anions, hydroxyl radicals, and hydrogen peroxide in various disease models [1,6,7]. Furthermore, H2S regulates levels of the most abundant antioxidant molecule, GSH, by reducing cystine to cysteine, a critical component for GSH production, and the entry of extracellular cysteine into cells, leading to increased GSH [25]. In previous studies, we found that administering NaHS, a H2S donor, reduces ROS levels such as superoxide, hydrogen peroxide, and lipid peroxidation in kidney tissue and inhibits the progression of renal fibrosis after UUO and ischemia/ reperfusion injury [2,14]. Furthermore, we found that deleting methionine sulfoxide reductase A (MsrA), which is a critical enzyme for producing homocysteine, augments kidney fibrosis after UUO along with reductions in H2S and GSH, the major intracellular antioxidant molecules [35]. Ida and colleagues recently reported that CSE can also catalyze the production of cysteine persulfide (CysSSH) using cysteine as the substrate [36]. CysSSH, in turn, produces other reactive persulfide/polysulfide species such as GSH-based per-and polysulfides (e. g., GSSH, GSSSH, GSSSG, etc) [36]. In this study they showed that GSSH readily reacts with H2O2 and scavenges peroxides. Furthermore, they
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Fig. 7. Expression of apoptosis-regulatory proteins and apoptosis in the Cse–/– and Cse+/+ mice kidneys after unilateral ureteral obstruction (UUO). Cse–/– and Cse+/+ mice were subjected to UUO. (A) Five days after surgery, kidney samples were subjected to western blotting using anti-Bcl2, -Bax, -XIAP, and -cleaved caspase 3 antibodies. GAPDH was used as a loading control. (B-F) Band densities were measured using ImageJ software. (G) The number of apoptotic cells in kidney tissue was evaluated by a terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay. Arrowheads indicate TUNEL-positive cells. Images were obtained from the Cortex. (H) TUNEL-positive cells were counted. Results were expressed as the mean ± SEM (n = 6). *, P < 0.05 vs. Cse WT sham. †, P < 0.05 vs. Cse WT UUO.
lipids stimulates the immune system, leading to tissue damage, kidney cell apoptosis, and renal fibrosis [6,7]. It has been reported that increased ROS levels can directly induce mitochondrial fragmentation and permeability, resulting in apoptotic cell death via caspase activation [24,32]. Many studies have demonstrated that inhibiting mitochondrial oxidative stress prevents kidney fibrosis by reducing apoptotic cell death [24,32]. In this present study, we found that Cse gene deficiency increased mitochondrial fission and DNA oxidative damage with a greater activation of mitochondrial apoptotic signals after UUO. These results suggested that lowered GSH and H2S levels that occur upon Cse gene deletion might exacerbate apoptosis and fibrosis progression by increasing mitochondrial oxidative damage and activating apoptosis. Emerging evidence has indicated an anti-inflammatory effect of H2S and the transsulfuration pathway in various organs [2,14]. We previously found that NaHS treatment reduces leukocyte infiltration into the interstitium after UUO or ischemic insults and reduces fibrotic lesions [2,14]. Song et al. reported that NaHS administration prevents post-UUO increases in proinflammatory cytokines such as TNF-α, MCP1, and IL-1β and decreases macrophage infiltration, whereas PAG administration exacerbates inflammation [20]. In the present study, we found that increases in macrophages, neutrophils, and TNF-α following UUO were greater in Cse–/– than in Cse+/+ mouse kidneys. Results of previous studies and the current study indicate that H2S/H2S-producing enzymes protect against fibrosis progression, at least in part, by their anti-inflammatory actions. Among profibrotic signaling pathways, TGFβ/Smad pathway plays a pivotal role in extracellular matrix accumulation via myofibroblast proliferation, fibroblast activation, and inhibition of ECM degradation [8,9]. In previous study, we and others have found that the anti-fibrotic effect of H2S is associated with
reported that low GSH levels in Cse-KO mice were due to a shortage of intracellular cysteine rather than impairment of the GSH biosynthetic pathways [38]. Wang et al. reported that reduced production of cysteine, an essential substrate for GSH synthesis, in CSE deficiency lowered GSH levels [39]. A dysregulated redox balance due to the excessive production of ROS and/or dysfunction of the antioxidant system results in various cellular and tissue damage including mitochondrial damage, apoptosis, inflammation, and increased extracellular matrix production by myofibroblasts, consequently leading to fibrosis [6,8,24,40]. Mitochondria are the major ROS-producing intracellular organelle, yet are also the most susceptible to ROS damage. Mitochondria do not contain catalase which detoxifies H2O2, implying that mitochondrial GSH (mGSH) acts as a major H2O2-detoxifying molecule in mitochondria [41]. Furthermore, mGSH also is critical for removing lipid peroxides through GPx4 and GSH-S-transferase in the mitochondria [41]. A recent study demonstrated that H2S increases mitochondrial GSH levels by redistributing cytosolic GSH to mitochondria [25]. Furthermore, CSE enters the mitochondria after injury where it promotes the production of H2S, but this does not occur under normal physiological conditions [26]. GSH plays a critical role in maintaining cellular redox balance; GSH reduces oxidative stress by counteracting with H2O2 or lipid peroxides and also acts as an essential constituent molecule in the functional (CXXC) motif of several antioxidant enzymes including GPx, peroxiredoxins, glutaredoxins, and thioredoxins [4]. Many studies have demonstrated that GSH supplements such as N-acetylcysteine (NAC), a stable form of cysteine for GSH synthesis, alleviate angiotensin-II-, UUO-, or diabetes-induced kidney fibrosis by reducing ROS/oxidative stress [42–44]. ROS-induced oxidative damage to proteins, DNA, mitochondria, and 429
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lumen; and 3, severe damage with flat epithelial cells lacking nuclear staining and the congestion of lumen as described previously [47].
downregulation of the TGF-β/Smad pathway [2,20]. Additionally, administration of NaHS reduces the post-UUO increase in TGF-β/Smad signals and reduces renal fibrosis [2]. Similarly, Li and Yuan et al. recently reported that NaHS treatment delays renal fibrosis by attenuating increases in TGF-β1 in kidneys of streptozotocin-induced diabetic rats [23,45]. In the present study, Cse gene deletion accelerated increases of TGF-β1/p-Smad3 and myofibroblast accumulation after UUO. This indicated that the worsened fibrosis observed upon Cse gene deletion was also associated with increased activation of the TGF-β/ Smad pathway. Taken together, the results presented in this study indicate that H2S and CSE have a crucial role in the progression of kidney fibrosis after UUO, suggesting that H2S and CSE may be potential targets for developing treatments for chronic kidney diseases.
4.4. Western blot analysis Western blotting was performed as described previously [48]. Western blotting was performed using antibodies directed against cystathionine γ-lyase (CSE; Abnova, Taipei City, Taiwan) and cystathionine β-synthase (CBS; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), 3-mercaptopyruvate sulfurtransferase (3-MST; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), transforming growth factor β1 (TGF-β1; Abcam, Cambridge, MA, USA), phosphorylated SMAD3 (pSMAD3; Cell Signaling Technology, Beverly, MA, USA), α-smooth muscle actin (α-SMA, Sigma-Aldrich, St Louis, MO), 4-hydroxynonenal (4-HNE; Abcam, Cambridge, UK), hyperoxidized peroxiredoxin-SO3 (Prx-SO3, Abcam, Cambridge, MA, USA), glutathione reductase (GR, Abcam, Cambridge, MA, USA) glutathione peroxidase 1 (GPx1, Santa Cruz Biotechnology, Santa Cruz, CA, USA), catalase (Fitzgerald, Concord, MA, USA), manganese superoxide dismutase (MnSOD; Calbiochem, La Jolla, CA, USA), copper-zinc superoxide dismutase (CuZnSOD; Chemicon, Temecula, CA, USA), tumor necrosis factor α (TNF-α; Thermo Scientific, MA, USA), Ly6G (eBioscience, San Diego, CA, USA), fission 1 (Fis1; Sigma-Aldrich), optic atrophy 1 (Opa1; BD Bioscience, San Diego, CA, USA), Bcl-2 (EMD Millipore), Bax (5B7) (EMD Millipore, MA, USA), X-linked inhibitor-of-apoptosis protein (XIAP, AnaSpec, San Jose, CA, USA), cleaved caspase-3 (Cell Signaling, Danvers, MA, USA), and GAPDH (Novus Biologicals, Littleton, CO, USA).
4. Materials and methods 4.1. Animal preparation All experiments were conducted using 20- to 21-week-old Cse genedeleted (Cse–/–) and wild-type (Cse+/+) female mice weighing 22–25 g. Cse–/– and Cse+/+ mice were bred by intercross of Cse+/– (hetero type) mice. The generation and characterization of Cse–/– and Cse+/+ mice (C57BL/6J background) has been described previously [46]. This study was conducted in accordance with the guidelines of the Institutional Animal Care and Use Committee of Kyungpook National University, Republic of Korea, and the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85–23, revised 2011). The mice were provided free access to water and standard chow. The mice were anesthetized by intraperitoneal injection of pentobarbital sodium before surgery (50 mg/ kg body weight; Sigma-Aldrich, St Louis, MO). To induce ureteral obstruction, the right kidney was exposed via flank incision, the right ureter was completely obstructed using a 6–0 silk thread, and the incision was subsequently sutured. The same procedure except for the ureteral obstruction was performed for the sham operation. Body temperature was maintained at 36.5–37 °C throughout all surgical procedures using a temperature-controlled heating device (FHC, Bowdonham, ME, USA). Five days after surgery, the kidneys were excised and were either snap-frozen in liquid nitrogen for molecular analysis or perfusion-fixed in PLP (4% paraformaldehyde, 75 mM L-lysine, and 10 mM sodium periodate; Sigma-Aldrich) for immunostaining and histological studies.
4.5. Immunohistochemical staining Immunohistochemical staining was performed using antibodies against 8-hydroxy-2'-deoxyguanosine (8-OHdG; Abcam, Cambridge, MA, USA) and anti-F4/80 (Serotec, Oxford, UK) antibodies, as described previously [40]. The 8-OHdG-antibody binds to DNA damaged by oxidation in mitochondria and nuclei [29]. Sections were viewed under a Leica microscope (DM2500, Wetzlar, Germany). Photomicrographs were obtained randomly from the cortex region. F4/80and 8-OHdG-positive cells were counted and intensity of 8-OHdG stains were measured under 400× magnification using an image analysis program (i-solution, IMT, Korea).
4.2. Picrosirius red staining
4.6. Measurement of H2S levels and glutathione in the kidney
Paraffin-embedded kidney tissue sections were stained with Picrosirius red (PSR) according to the standard protocol. Dewaxed kidney tissue sections were exposed to PSR stain for 1 h, and then washed twice with acidified water (0.5% glacial acetic acid). Sections were then serially dehydrated in alcohol solutions of different concentrations. Photomicrographs (200× magnification) were obtained randomly from the kidney cortex region using a Leica microscope (DM2500, Wetzlar, Germany). Regions of collagen deposition in the PSR-stained kidney sections were measured using an image analysis program (i-solution, IMT, Korea).
H2S levels in kidney tissue were determined as previously described [2,14]. Briefly, kidney tissues were homogenized in ice-cold 100 mM potassium phosphate buffer (pH 7.4) with protease inhibitors. One hundred microliters of homogenate was mixed with 100 µL of 1% zinc acetate, 100 µL of borate buffer (pH 10.0), 200 µL of 20 mM N,N-dimethyl-p-phenylenediamine dihydrochloride in 7.2 M HCl, and 200 µL of 30 mM FeCl3 in 1.2 M HCl and incubated in the dark for 15 min at 37 °C. The sample was centrifuged for 5 min at 10,000 rpm at 4 °C. The supernatant was taken and the optical density was measured at 670 nm. H2S concentration was calculated against a standard curve created using NaHS solution. Total glutathione (tGSH) and oxidized GSH (GSSG) levels were measured using an enzymatic recycling method, as described previously [30,49]. Given that GSH and related thiols are sensitive to oxidation and degradation during sampling and analysis, samples were harvested quickly in liquid nitrogen, stored at −70 °C until use, and analyzed rapidly. The amount of glutathione was determined by the formation of 5-thio-2-nitrobenzoic acid (TBA) from 5,5-dithiobis (2-nitrobenzoic acid; DTNB), as described by Akerboom and Sies [49]. GSH levels were defined as the change in optical density at 412 nm over 1 min at 37 °C.
4.3. Histology Kidney paraffin-sections were stained with Periodic Acid Schiff (PAS, Muto Pure Chemicals, Tokyo, Japan) stain according to a standard protocol. To determine morphological damage to tubular cells, 50 tubules in the cortex region of the kidneys were analyzed using the following scoring method: 0, no damage; 1, mild damage with the rounding of epithelial cells and dilated tubular lumen; 2, moderate damage with flattened epithelial cells, dilated lumen, and congestion of 430
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Original article
Hydrogen sulfide-producing cystathionine γ-lyase is critical in the progression of kidney fibrosis
MARK
Sang Jun Hana, Mi Ra Noha, Jung-Min Jungb, Isao Ishiic, Jeongsoo Yoob, Jee In Kimd, ⁎ Kwon Moo Parka, a Department of Anatomy, Cardiovascular Research Institute and BK21 Plus, Kyungpook National University School of Medicine, 680 Gukchaebosang-ro, Junggu, Daegu 41944, Republic of Korea b Department of Molecular Medicine, BK21 Plus, Kyungpook National University School of Medicine, 680 Gukchaebosang-ro, Junggu, Daegu 41944, Republic of Korea c Laboratory of Health Chemistry, Showa Pharmaceutical University, Machida, Tokyo 194-8543, Japan d Department of Molecular Medicine and MRC, College of Medicine, Keimyung University, 1095 Dalgubeol-daero 250-gil, Dalseogu, Daegu 42601, Republic of Korea
A R T I C L E I N F O
A B S T R A C T
Keywords: Chronic kidney disease Cystathionine γ-lyase Reactive oxygen species Kidney fibrosis Hydrogen sulfide
Cystathionine γ-lyase (CSE), the last key enzyme of the transsulfuration pathway, is involved in the production of hydrogen sulfide (H2S) and glutathione (GSH), which regulate redox balance and act as important antioxidant molecules. Impairment of the H2S- and GSH-mediated antioxidant system is associated with the progression of chronic kidney disease (CKD), characterized by kidney fibrosis and dysfunction. Here, we evaluated the role of CSE in the progression of kidney fibrosis after unilateral ureteral obstruction (UUO) using mice deficient in the Cse gene. UUO of wild-type mice reduced the expression of H2S-producing enzymes, CSE, cystathionine βsynthase, and 3-mercaptopyruvate sulfurtransferase in the obstructed kidneys, resulting in decreased H2S and GSH levels. Cse gene deletion lowered H2S and GSH levels in the kidneys. Deleting the Cse gene exacerbated the decrease in H2S and GSH levels and increase in superoxide formation and oxidative damage to proteins, lipids, and DNA in the kidneys after UUO, which were accompanied by greater kidney fibrosis, deposition of extracellular matrixes, expression of α-smooth muscle actin, tubular damage, and infiltration of inflammatory cells. Furthermore, Cse gene deletion exacerbated mitochondrial fragmentation and apoptosis of renal tubule cells after UUO. The data provided herein constitute in vivo evidence that Cse deficiency impairs renal the H2S- and GSH-producing activity and exacerbates UUO-induced kidney fibrosis. These data propose a novel therapeutic approach against CKD by regulating CSE and the transsulfuration pathway.
1. Introduction Pyridoxal-5′-phosphate (P5P)-dependent cystathionine γ-lyase (CSE), cystathionine β-synthase (CBS), and P5P-independent 3-mercaptopyruvate sulfurtransferase (3-MST) produce hydrogen sulfide (H2S) [1]. CSE and CBS are involved in the synthesis of glutathione (GSH) by suppling endogenous cysteine, a component of GSH. These two molecules, H2S and GSH, produced by those enzymes, play important roles in the progression and development of various fibrogenic diseases, which are associated with oxidative tissue damage [1]. H2S controls cellular redox status by regulating NF–E2-related factor 2 (Nrf2)/antioxidant responsive element (ARE) signaling and directly
scavenging free radicals [2,3]. GSH is the most abundant antioxidant molecule and plays a critical role in maintaining cellular redox balance [4,5]. Emerging evidence has demonstrated that H2S and GSH are associated with the progression of fibrosis in various organs [6,7]. However, the roles of these enzymes in kidney fibrosis remain to be defined. Kidney fibrosis is a major cause in the development and progression of chronic kidney disease (CKD), which evokes severe clinical problems [8]. Kidney fibrosis is characterized by increased numbers of myofibroblasts, infiltration and accumulation of inflammatory cells, and excessive accumulation of extracellular matrix components such as collagen and fibronectin [9,10]. Recently, it was reported that
List of abbreviations: CSE, Cystathionine γ-lyase; H2S, Hydrogen sulfide; GSH, Glutathione; CKD, Chronic kidney disease; UUO, Unilateral ureteral obstruction; P5P, Pyridoxal-5′phosphate; CBS, Cystathionine β-synthase; 3-MST, 3-mercaptopyruvate sulfurtransferase; Nrf2, NF–E2-related factor 2; ARE, Antioxidant responsive element; ESRD, End-stage renal disease; α-SMA, α-smooth muscle actin; Gpx, Glutathione peroxide; 8-OHdG, 8-hydroxy-2'-deoxyguanosine; Fis1, Fission 1 protein; PAG, Propargylglycine; NAC, N-acetylcysteine; TGFβ1, Transforming growth factor β1; 4-HNE, 4-hydroxynonenal; MnSOD, Manganese superoxide dismutase; CuZnSOD, Copper-zinc superoxide dismutase; TNF-α, Tumor necrosis factor α; Opa1, Optic atrophy 1; XIAP, X-linked inhibitor-of-apoptosis protein ⁎ Corresponding author. E-mail address: [email protected] (K.M. Park). http://dx.doi.org/10.1016/j.freeradbiomed.2017.08.017 Received 8 June 2017; Received in revised form 29 July 2017; Accepted 21 August 2017 Available online 24 August 2017 0891-5849/ © 2017 Elsevier Inc. All rights reserved.
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signals and myofibroblast activation after UUO.
impairments in H2S production and GSH-associated antioxidant system play a critical role in the development and progression of kidney fibrosis; plasma H2S and CSE mRNA levels in end-stage renal disease (ESRD) patients are lower than levels observed in healthy people. Furthermore, levels of plasma homocysteine and cysteine, precursors of H2S and GSH, respectively, are higher in ESRD patients than in healthy people [11,12]. Plasma GSH level is lower in patients with moderate and severe CKD, and the GSH level is positively correlated with creatinine clearance [13]. These clinical studies suggest that H2S, GSH, and the CSE-transsulfuration pathway are deeply associated with the progression of kidney fibrosis. Supporting this, many investigators, including us, have recently reported that kidney fibrosis induced by 5/6 of nephrectomy, unilateral ureteral obstruction (UUO), or streptozotocin injection impair the functions of all three H2S-producing enzymes in the kidneys, and exogenous supplements of H2S attenuate kidney fibrosis and GSH reduction, while inhibition of H2S production worsens them [2,14–19]. These studies have suggested that the antifibrotic effects of H2S are mediated by inhibition of oxidative stress, inflammation, and fibrogenic pathways including TGF-β1/SMAD3, MAPK, and NF-κB [2,14,19–23]. Mitochondria are most susceptible organelle to ROS/oxidative damage and mitochondrial oxidative damage is critical for the progression of fibrosis by inducing apoptosis and inflammation [10,24]. It was recently reported that CSE regulates mitochondrial H2S and GSH levels [25,26]. We also recently found that a reduction in mitochondrial GSH level exacerbates kidney fibrosis by enhancing mitochondrial oxidative stress, thereby inducing mitochondrial damage and apoptosis of tubule cells [27]. Exogenous supplementation of H2S ameliorates renal fibrosis by downregulating ROS, oxidative damage, and the inflammatory responses, and upregulating GSH in kidneys after UUO or ischemic injuries [2,14]. I n the kidney, CSE is abundantly expressed, 20-fold more than the CBS expression, and is mainly responsible for H2S production in combination with CBS [6,21,22]. Therefore, we have investigated the role of CSE in fibrotic changes of kidneys after UUO and its underlying mechanisms using Cse knockout (Cse–/–) mice.
2.2. Cse gene deletion impairs H2S and GSH production in the kidney To investigate whether increased fibrosis in Cse gene-deleted mice after UUO is associated with impaired transsulfuration pathways, which are linked to H2S and glutathione (GSH) production, [4,25] we examined expressions of CSE, CBS, and 3-MST proteins, and levels of H2S and GSH in the kidneys. After UUO of Cse+/+ mice, kidney levels of H2S decreased in a time-dependent manner (Fig. 2A), which were accompanied by time-dependent decreases in renal CSE, CBS, and 3-MST expression (Fig. 2B-E). Similarly, renal GSH levels were decreased a time-time-dependent manner after UUO (Fig. 2F), accompanied by time-dependent decreases in renal glutathione peroxide 1 (GPx1) and glutathione reductase (GR) expression (Fig. 2G-I). Levels of H2S and GSH and these enzymes (CSE, CBS, and 3-MST) were negatively correlated with α-SMA expression and collagen deposition (Fig. 2A-L). These results indicate that CSE/CBS/3-MST-mediated H2S/GSH production is intimately linked with progression of kidney fibrosis. Next, we examined whether the systemic Cse gene deletion in mice affects H2S, GSH production, the ratio of GSSG to tGSH, and CBS and 3MST expression. After sham-operation, H2S and GSH levels in the Cse–/– mouse kidneys were significantly lower than those in Cse+/+ kidneys, about 60% and 70%, respectively (Fig. 3A, B). These data indicate that Cse gene deletion impairs H2S and GSH production in the kidney. At 5 days after UUO, H2S and GSH levels significantly decreased in both Cse–/– and Cse+/+ kidneys but both decreases were significantly greater in the Cse–/– kidneys than in Cse+/+ kidneys (Fig. 3A, B). In addition the ratio of GSSG to tGSH was significantly greater in the Cse–/– kidneys than in Cse+/+ kidneys after UUO (Fig. 3C). Consistent with the levels of H2S and GSH, expressions of CSE, CBS, and 3-MST decreased after UUO (Fig. 3D-G). In contrast, renal CBS and 3-MST expression was comparable between Cse+/+ and Cse–/– mice at either pre- or post-UUO (Fig. 3D, E, G). These results suggest that CSE plays a pivotal role in the maintenance of renal H2S/GSH levels, thereby protecting kidney against fibrosis via their anti-oxidative activities.
2. Result 2.1. Cse gene deletion exacerbates UUO-induced fibrosis in the kidney
2.3. Cse gene deletion exacerbates ROS production and oxidative stress after UUO
First, we determined the effect of the systemic Cse gene deletion on kidney fibrosis after UUO. UUO induced tubular damage and expansion of the interstitial area; these phenotypes were much more apparent in Cse–/– mice than in Cse+/+ mice (Fig. 1A [top panel], B). Collagen deposition, as evaluated by Sirius red staining, also increased in the interstitium after UUO and this increase was significantly greater in Cse–/– kidneys than in Cse+/+ kidneys (Fig. 1A [middle panel], C). Furthermore, UUO-induced increases in F4/80-positive cells, monocytes/macrophages, were greater in the interstitium of Cse–/– kidneys than Cse+/+ kidneys. (Fig. 1A [bottom panel], D). In contrast, there were no significant differences in tubular damage, collagen deposition, and number of F4/80-positive cells between sham-operated Cse–/– and Cse+/+ mouse kidneys (Fig. 1A-D). Expressions of TNF-α, a pro-inflammatory cytokine, and Ly6G, a neutrophil marker, increased after UUO and these increases were significantly greater in Cse–/– kidneys than in Cse+/+ kidneys (Fig. 1E-G). There were no significant differences in TNF-α or Ly6G expression between sham-operated Cse–/– kidneys and Cse+/+ kidneys (Fig. 1E-G). TGF-β1 acts as a main mediator in fibrosis development and progression in CKD via Smad-3 phosphorylation (p-Smad3) and the subsequent activation of collagen-producing myofibroblasts [9]. Post-UUO increases of TGF-β1, p-Smad3, and α-smooth muscle actin (α-SMA; a myofibroblast marker) expression were all significantly greater in Cse–/– kidneys than in Cse+/+ kidneys (Fig. 1H-K). Taken together, the systemic Cse gene deletion in mice exacerbates kidney fibrosis, tubular damage, and inflammation by promoting greater activation of TGF- β1
H2S and GSH play important roles in maintaining redox balance, which is critical for the progression of renal fibrosis and tubular damage [6]. Expression of 4-HNE, oxidized peroxiredoxin (Prx-SO3), and 8hydroxy-2'-deoxyguanosine (8-OHdG), indicators of lipid peroxidation, peroxiredoxin oxidation, and DNA oxidation, [28,29] respectively, significantly increased in both Cse–/– and Cse+/+ mouse kidneys after UUO and those increases were greater in Cse–/– kidneys (Fig. 4A-E). There were no significant differences in expression of 4-HNE, Prx-SO3, or 8-OHdG between sham-operated Cse–/– and Cse+/+ mouse kidneys (Fig. 4A-E). These results indicate that Cse gene deletion in mice exacerbates oxidative stress in the ureteral obstructed kidneys after UUO, and this increased oxidative stress may be associated with a decreased antioxidative effect in kidneys due to reduced levels of H2S and GSH by Cse gene deletion. GSH is required for the GSH-mediated antioxidative system, which contains enzymes such as glutathione peroxidase 1 (GPx1) and glutathione reductase (GR). Since GPx1 is a major enzyme for removal of H2O2 using reduced GSH, which consequently produces oxidized glutathione (GSSG) [30], we determined GPx1 levels. UUO significantly decreased GPx1 levels in the kidneys and this decrease was greater in the Cse–/– kidneys than in Cse+/+ kidneys (Fig. 5A, B). Furthermore, the expression of GR, which reduces GSSG [30] to GSH, was lowered in the kidneys from both groups after UUO, and this reduction was greater in the Cse–/– kidneys than in the Cse+/+ kidneys (Fig. 5A, C). There was no significant difference in GPx1 or GR expression between sham-operated Cse–/– and Cse+/+ mouse kidneys (Fig. 5A-C). These data 424
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Fig. 1. Fibrosis, tubular damage, and inflammation in Cse–/– and Cse+/+ mouse kidneys after unilateral ureteral obstruction (UUO). Cse–/– and Cse+/+ mice were subjected to UUO by tying the right ureter. (A) Five days after surgery, kidney sections were subjected to staining with PAS (top panel), Sirius red (middle panel), and immunohistochemical stains using antiF4/80 antibody (bottom panel). Arrowheads indicate F4/80-positive cells. (B) Kidney damage was scored as described in Section 4. ND; not detected. (C) Collagen deposition was measured as described in the Materials and Methods. (D) Number of F4/80-positive cells in the interstitial area are shown. (E, H) Kidney samples were subjected to western blotting using anti-TNF-α (E), -Ly6G (E), -TGF-β1 (H), -p-SMAD3 (H), and -α-SMA (H) antibodies. GAPDH was used as a loading control. (F, G, I-K) Bands densities were measured using ImageJ software developed by the NIH. Results were expressed as the mean ± SEM (n = 6). *, P < 0.05 vs. Cse WT sham. †, P < 0.05 vs. Cse WT UUO (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.).
redox balance by regulating H2S and GSH levels [25,26]. In the present study, UUO increased levels of fission 1 protein (Fis1), a regulator of mitochondrial fission, in wild-type mice, whereas UUO decreased Opa1, a mitochondrial fusion protein, in a time-dependent manner (Fig. 6AC), resulting in mitochondrial fragmentation (Fig. 6D). The change of Fis1 expression was greater in Cse–/– kidneys than in Cse+/+ kidneys after UUO, whereas there was no difference in Opa1 expression between Cse–/– and Cse+/+ kidneys (Fig. 6E-G). This indicates that Cse gene deletion exacerbates UUO-induced mitochondrial fragmentation.
indicate that Cse gene deletion impairs the GSH-mediated antioxidant system after UUO. Furthermore, Cse gene deletion exacerbated UUOinduced decreases of catalase, manganese superoxide dismutase (MnSOD), and cupper zinc SOD (CuZnSOD) (Fig. 5D-G). Taken together, these results indicate that Cse gene deletion reduces H2S and GSH production, and exacerbates ROS/oxidative stress and antioxidant enzyme impairment following UUO.
2.4. Cse gene deletion exacerbates mitochondrial damage after UUO Under normal conditions, mitochondria dynamically undergo fission and fusion, and this process is balanced appropriately [31]. Accumulating evidence has demonstrated that fibrosis is associated with a shift toward mitochondrial fission, which leads to mitochondrial fragmentation, damage, and subsequent apoptotic cell death [6,24,32]. Furthermore, recent studies reported that CSE regulates mitochondrial
2.5. Cse gene deletion exacerbates tubule cell apoptosis after UUO Since H2S and GSH exert a protective role in tubular cell apoptosis, which is a major pathway of kidney fibrosis and mitochondrial damage after UUO, [6,32] we investigated whether Cse gene deletion affected kidney tubule cell apoptosis after UUO. UUO significantly increased the 425
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Fig. 2. H2S levels and H2S-producing enzyme expression in kidneys after unilateral ureteral obstruction (UUO). C57BL/6J female wild-type mice were subjected to UUO. (A) At the indicated day after surgery, H2S levels in kidneys tissues were measured. (B) Kidney samples were subjected to western blotting using anti-CSE, -CBS, and −3-MST antibodies. GAPDH was used as a loading control. (C-E) The densities of bands were measured using the ImageJ software. (F) GSH level in kidney tissues was measured as described in Section 4. (G) Kidney samples were subjected to western blotting using anti-GPx1, -GR, and -α-SMA antibodies. GAPDH was used as a loading control. (H-J) The densities of bands were measured using ImageJ software. (K) Kidney sections were subjected to Sirius red staining. (L) Collagen deposition was measured. Results were expressed as the mean ± SEM (n = 6). *, P < 0.05 vs. sham.
UUO significantly increased the number of TUNEL-positive tubule cells and those increases were greater in Cse–/– kidneys than in Cse+/+ kidneys (Fig. 7G, H). These results indicated that Cse gene deficiency exacerbates UUO-induced apoptosis of tubule cells.
expression of pro-apoptotic factors active caspase-3 and Bax, whereas UUO decreased the expression of anti-apoptotic factors Bcl-2 and XIAP (Fig. 7A-F). Those post-UUO changes in pro-apoptotic and anti-apoptotic factors were greater in Cse–/– kidneys than in Cse+/+ kidneys (Fig. 7A-F). Consistent with changes in levels of apoptosis regulators,
Fig. 3. H2S and GSH levels and H2S producing-enzyme expressions in the Cse–/– and Cse+/+ mice kidneys after unilateral ureteral obstruction (UUO). Cse knockout (Cse–/–) and wild-type mice (Cse+/+) mice were subjected to UUO. Five days after surgery, kidneys were harvested then H2S (A), GSH and GSSG (B, C) levels in the kidney tissue were measured (Note, although the H2S determining method used this study is widely used, the method may not determine the amount of free H2S) (D) Kidney samples were subjected to western blotting using anti-CSE, -CBS, and −3-MST antibodies. GAPDH was used as a loading control. (E-G) Band densities were measured using ImageJ software. Results were expressed as the mean ± SEM (n = 6). *, P < 0.05 vs. Cse WT sham. †, P < 0.05 vs. Cse WT UUO.
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Fig. 4. Oxidative damages in the Cse–/– and Cse+/+ mice kidneys after unilateral ureteral obstruction (UUO). Cse–/– and Cse+/+ mice were subjected to UUO and five days after surgery kidneys were harvested. (A) Kidney samples were subjected western blotting using anti-4-HNE and -Prx-SO3 antibodies. GAPDH was used as a loading control. (B, C) Band densities were measured using ImageJ software. (D) Kidney sections were subjected to immunohistochemical staining using an anti-8-OHdG antibody. Hematoxylin was used to visualize cell nuclei. (E) Intensity of 8-OHdG stains in tubule cells was determined. Results were expressed as the mean ± SEM (n = 6). *, P < 0.05 vs. Cse WT sham. †, P < 0.05 vs. Cse WT UUO.
MST, and that Cse gene deletion exacerbated UUO-induced kidney fibrosis along with greater reductions of H2S in the kidney when compared to wild-type kidneys. These results indicate that the reduced H2S production that occurs upon Cse gene deletion may increase oxidative stress and lead to greater fibrosis. The expression of CSE, CBS and 3MST are transcriptionally and/or post-transcriptionally regulated by various factors [33]. Although currently little information is available regarding to regulation of H2S-producing enzyme expressions, we can speculate that UUO may influence various transcriptional and translational factors including Sp1 and consequently lead to down-regulation in the expression of H2S-producing enzymes. Recently Wu et al. reported that ischemia/reperfusion reduced Cbs gene expression by increased Sp1 phosphorylation via ERK activation in the kidney, and consequently led to decreased H2S levels [34]. Therefore, our results indicate that reduced H2S level and H2S-producing enzyme expression may increase oxidative stress and lead to greater fibrosis. In addition, the increased oxidative stress and fibrosis in Cse gene deleted mice may
3. Discussion This study demonstrates, for the first time, that ureteral obstruction reduced H2S and GSH levels together with downregulation of H2S- and GSH-producing transsulfuration pathway enzymes. We also found that Cse gene deletion in mice reduced levels of H2S and GSH in the kidneys, and exacerbates UUO-induced oxidative stress, mitochondrial fragmentation, apoptosis, and inflammation by promoting a greater reduction of H2S and GSH levels in the kidney, consequently resulting in greater kidney fibrosis. These results indicate that CSE plays a crucial role in the progression of kidney fibrosis, suggesting that CSE could be a potential protein to target in the development of therapeutics for fibrotic tissue diseases, including CKD. Accumulating evidence has demonstrated that H2S inhibits the progression of tissue fibrosis by reducing oxidative stress [6,7,25]. In the present study, we found that UUO reduced tissue H2S levels together with reductions in the H2S-producing enzymes CSE, CBS and 3-
Fig. 5. Expressions of GR, GPx1, catalase, and SODs in the Cse–/– and Cse+/+ mice kidneys after unilateral ureteral obstruction (UUO). Cse–/– and Cse+/+ mice were subjected to UUO. (A) Five days after surgery, kidney samples were subjected western blotting using anti-GPx1 and -GR antibodies. GAPDH was used as a loading control. (B, C) Bands densities were measured using ImageJ software. (D) Kidney samples were subjected to western blotting using anti-catalase, -MnSOD, and -CuZnSOD antibodies. (E-G) Band densities were measured using ImageJ software. Results were expressed as the mean ± SEM (n = 6). *, P < 0.05 vs. Cse WT CLT. †, P < 0.05 vs. Cse WT UO.
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Fig. 6. Expression of mitochondrial dynamics-regulatory proteins in the Cse–/– and Cse+/+ mice kidneys after unilateral ureteral obstruction (UUO). (A-D) C57BL/6J female wild-type mice were subjected to UUO. On the indicated days after surgery, kidneys samples were subjected western blotting using anti-Fis1 and -Opa1 antibodies. GAPDH was used as a loading control. (D) On the indicated days after surgery, mitochondrial structures were evaluated using transmission electron microscopy (TEM). M, mitochondria. N, nucleus. (E-G) Cse–/– and Cse+/+ mice were subjected to UUO. (E) Five days after surgeries, kidneys were harvested and then subjected western blotting using anti-Fis1, and -Opa1 antibodies. GAPDH was used as a loading control. (F, G) Bands densities were measured using ImageJ software. Results were expressed as the mean ± SEM (n = 6). *, P < 0.05 vs. Cse WT sham. †, P < 0.05 vs. Cse WT UUO.
reported that CSE overexpression protected cells against H2O2-mediated cell death [36]. Therefore, we speculate that the exacerbation of fibrosis in Cse-deficient mice may be caused by the reduced ability of cells to cope with oxidative stress. In the present study, we found, for the first time, that Cse gene deletion alone dramatically reduces levels of H2S and GSH in kidneys without any significant reduction of CBS and 3MST expressions. However, deletion of this gene alone did not induce significant tissue oxidative damages like lipid peroxidation, DNA oxidation, or protein oxidation. Cse gene deletion exacerbated UUO-induced decreases of H2S and GSH. These results indicate that CSE is a critical enzyme for production or maintenance of cellular H2S and GSH levels under both normal and pathological conditions. Yang et al. found that Cse gene deletion alone reduces H2S and GSH levels in vascular tissues including the mesenteric artery, aorta, and smooth muscle cells isolated from mesenteric arteries, yet Cse gene deletion did not significantly change ROS levels in those tissues [37]. Lee et al. found that inhibiting CSE using the CSE inhibitor propargylglycine (PAG) or Cse siRNA reduces GSH levels [5] and that PAG and Cse siRNA treatment increase sensitivity to H2O2-induced cytotoxicity [5]. Furthermore, Mani et al.
be associated with reduced H2S production. H2S has been shown to directly scavenge ROS including superoxide anions, hydroxyl radicals, and hydrogen peroxide in various disease models [1,6,7]. Furthermore, H2S regulates levels of the most abundant antioxidant molecule, GSH, by reducing cystine to cysteine, a critical component for GSH production, and the entry of extracellular cysteine into cells, leading to increased GSH [25]. In previous studies, we found that administering NaHS, a H2S donor, reduces ROS levels such as superoxide, hydrogen peroxide, and lipid peroxidation in kidney tissue and inhibits the progression of renal fibrosis after UUO and ischemia/ reperfusion injury [2,14]. Furthermore, we found that deleting methionine sulfoxide reductase A (MsrA), which is a critical enzyme for producing homocysteine, augments kidney fibrosis after UUO along with reductions in H2S and GSH, the major intracellular antioxidant molecules [35]. Ida and colleagues recently reported that CSE can also catalyze the production of cysteine persulfide (CysSSH) using cysteine as the substrate [36]. CysSSH, in turn, produces other reactive persulfide/polysulfide species such as GSH-based per-and polysulfides (e. g., GSSH, GSSSH, GSSSG, etc) [36]. In this study they showed that GSSH readily reacts with H2O2 and scavenges peroxides. Furthermore, they
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Fig. 7. Expression of apoptosis-regulatory proteins and apoptosis in the Cse–/– and Cse+/+ mice kidneys after unilateral ureteral obstruction (UUO). Cse–/– and Cse+/+ mice were subjected to UUO. (A) Five days after surgery, kidney samples were subjected to western blotting using anti-Bcl2, -Bax, -XIAP, and -cleaved caspase 3 antibodies. GAPDH was used as a loading control. (B-F) Band densities were measured using ImageJ software. (G) The number of apoptotic cells in kidney tissue was evaluated by a terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay. Arrowheads indicate TUNEL-positive cells. Images were obtained from the Cortex. (H) TUNEL-positive cells were counted. Results were expressed as the mean ± SEM (n = 6). *, P < 0.05 vs. Cse WT sham. †, P < 0.05 vs. Cse WT UUO.
lipids stimulates the immune system, leading to tissue damage, kidney cell apoptosis, and renal fibrosis [6,7]. It has been reported that increased ROS levels can directly induce mitochondrial fragmentation and permeability, resulting in apoptotic cell death via caspase activation [24,32]. Many studies have demonstrated that inhibiting mitochondrial oxidative stress prevents kidney fibrosis by reducing apoptotic cell death [24,32]. In this present study, we found that Cse gene deficiency increased mitochondrial fission and DNA oxidative damage with a greater activation of mitochondrial apoptotic signals after UUO. These results suggested that lowered GSH and H2S levels that occur upon Cse gene deletion might exacerbate apoptosis and fibrosis progression by increasing mitochondrial oxidative damage and activating apoptosis. Emerging evidence has indicated an anti-inflammatory effect of H2S and the transsulfuration pathway in various organs [2,14]. We previously found that NaHS treatment reduces leukocyte infiltration into the interstitium after UUO or ischemic insults and reduces fibrotic lesions [2,14]. Song et al. reported that NaHS administration prevents post-UUO increases in proinflammatory cytokines such as TNF-α, MCP1, and IL-1β and decreases macrophage infiltration, whereas PAG administration exacerbates inflammation [20]. In the present study, we found that increases in macrophages, neutrophils, and TNF-α following UUO were greater in Cse–/– than in Cse+/+ mouse kidneys. Results of previous studies and the current study indicate that H2S/H2S-producing enzymes protect against fibrosis progression, at least in part, by their anti-inflammatory actions. Among profibrotic signaling pathways, TGFβ/Smad pathway plays a pivotal role in extracellular matrix accumulation via myofibroblast proliferation, fibroblast activation, and inhibition of ECM degradation [8,9]. In previous study, we and others have found that the anti-fibrotic effect of H2S is associated with
reported that low GSH levels in Cse-KO mice were due to a shortage of intracellular cysteine rather than impairment of the GSH biosynthetic pathways [38]. Wang et al. reported that reduced production of cysteine, an essential substrate for GSH synthesis, in CSE deficiency lowered GSH levels [39]. A dysregulated redox balance due to the excessive production of ROS and/or dysfunction of the antioxidant system results in various cellular and tissue damage including mitochondrial damage, apoptosis, inflammation, and increased extracellular matrix production by myofibroblasts, consequently leading to fibrosis [6,8,24,40]. Mitochondria are the major ROS-producing intracellular organelle, yet are also the most susceptible to ROS damage. Mitochondria do not contain catalase which detoxifies H2O2, implying that mitochondrial GSH (mGSH) acts as a major H2O2-detoxifying molecule in mitochondria [41]. Furthermore, mGSH also is critical for removing lipid peroxides through GPx4 and GSH-S-transferase in the mitochondria [41]. A recent study demonstrated that H2S increases mitochondrial GSH levels by redistributing cytosolic GSH to mitochondria [25]. Furthermore, CSE enters the mitochondria after injury where it promotes the production of H2S, but this does not occur under normal physiological conditions [26]. GSH plays a critical role in maintaining cellular redox balance; GSH reduces oxidative stress by counteracting with H2O2 or lipid peroxides and also acts as an essential constituent molecule in the functional (CXXC) motif of several antioxidant enzymes including GPx, peroxiredoxins, glutaredoxins, and thioredoxins [4]. Many studies have demonstrated that GSH supplements such as N-acetylcysteine (NAC), a stable form of cysteine for GSH synthesis, alleviate angiotensin-II-, UUO-, or diabetes-induced kidney fibrosis by reducing ROS/oxidative stress [42–44]. ROS-induced oxidative damage to proteins, DNA, mitochondria, and 429
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lumen; and 3, severe damage with flat epithelial cells lacking nuclear staining and the congestion of lumen as described previously [47].
downregulation of the TGF-β/Smad pathway [2,20]. Additionally, administration of NaHS reduces the post-UUO increase in TGF-β/Smad signals and reduces renal fibrosis [2]. Similarly, Li and Yuan et al. recently reported that NaHS treatment delays renal fibrosis by attenuating increases in TGF-β1 in kidneys of streptozotocin-induced diabetic rats [23,45]. In the present study, Cse gene deletion accelerated increases of TGF-β1/p-Smad3 and myofibroblast accumulation after UUO. This indicated that the worsened fibrosis observed upon Cse gene deletion was also associated with increased activation of the TGF-β/ Smad pathway. Taken together, the results presented in this study indicate that H2S and CSE have a crucial role in the progression of kidney fibrosis after UUO, suggesting that H2S and CSE may be potential targets for developing treatments for chronic kidney diseases.
4.4. Western blot analysis Western blotting was performed as described previously [48]. Western blotting was performed using antibodies directed against cystathionine γ-lyase (CSE; Abnova, Taipei City, Taiwan) and cystathionine β-synthase (CBS; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), 3-mercaptopyruvate sulfurtransferase (3-MST; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), transforming growth factor β1 (TGF-β1; Abcam, Cambridge, MA, USA), phosphorylated SMAD3 (pSMAD3; Cell Signaling Technology, Beverly, MA, USA), α-smooth muscle actin (α-SMA, Sigma-Aldrich, St Louis, MO), 4-hydroxynonenal (4-HNE; Abcam, Cambridge, UK), hyperoxidized peroxiredoxin-SO3 (Prx-SO3, Abcam, Cambridge, MA, USA), glutathione reductase (GR, Abcam, Cambridge, MA, USA) glutathione peroxidase 1 (GPx1, Santa Cruz Biotechnology, Santa Cruz, CA, USA), catalase (Fitzgerald, Concord, MA, USA), manganese superoxide dismutase (MnSOD; Calbiochem, La Jolla, CA, USA), copper-zinc superoxide dismutase (CuZnSOD; Chemicon, Temecula, CA, USA), tumor necrosis factor α (TNF-α; Thermo Scientific, MA, USA), Ly6G (eBioscience, San Diego, CA, USA), fission 1 (Fis1; Sigma-Aldrich), optic atrophy 1 (Opa1; BD Bioscience, San Diego, CA, USA), Bcl-2 (EMD Millipore), Bax (5B7) (EMD Millipore, MA, USA), X-linked inhibitor-of-apoptosis protein (XIAP, AnaSpec, San Jose, CA, USA), cleaved caspase-3 (Cell Signaling, Danvers, MA, USA), and GAPDH (Novus Biologicals, Littleton, CO, USA).
4. Materials and methods 4.1. Animal preparation All experiments were conducted using 20- to 21-week-old Cse genedeleted (Cse–/–) and wild-type (Cse+/+) female mice weighing 22–25 g. Cse–/– and Cse+/+ mice were bred by intercross of Cse+/– (hetero type) mice. The generation and characterization of Cse–/– and Cse+/+ mice (C57BL/6J background) has been described previously [46]. This study was conducted in accordance with the guidelines of the Institutional Animal Care and Use Committee of Kyungpook National University, Republic of Korea, and the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85–23, revised 2011). The mice were provided free access to water and standard chow. The mice were anesthetized by intraperitoneal injection of pentobarbital sodium before surgery (50 mg/ kg body weight; Sigma-Aldrich, St Louis, MO). To induce ureteral obstruction, the right kidney was exposed via flank incision, the right ureter was completely obstructed using a 6–0 silk thread, and the incision was subsequently sutured. The same procedure except for the ureteral obstruction was performed for the sham operation. Body temperature was maintained at 36.5–37 °C throughout all surgical procedures using a temperature-controlled heating device (FHC, Bowdonham, ME, USA). Five days after surgery, the kidneys were excised and were either snap-frozen in liquid nitrogen for molecular analysis or perfusion-fixed in PLP (4% paraformaldehyde, 75 mM L-lysine, and 10 mM sodium periodate; Sigma-Aldrich) for immunostaining and histological studies.
4.5. Immunohistochemical staining Immunohistochemical staining was performed using antibodies against 8-hydroxy-2'-deoxyguanosine (8-OHdG; Abcam, Cambridge, MA, USA) and anti-F4/80 (Serotec, Oxford, UK) antibodies, as described previously [40]. The 8-OHdG-antibody binds to DNA damaged by oxidation in mitochondria and nuclei [29]. Sections were viewed under a Leica microscope (DM2500, Wetzlar, Germany). Photomicrographs were obtained randomly from the cortex region. F4/80and 8-OHdG-positive cells were counted and intensity of 8-OHdG stains were measured under 400× magnification using an image analysis program (i-solution, IMT, Korea).
4.2. Picrosirius red staining
4.6. Measurement of H2S levels and glutathione in the kidney
Paraffin-embedded kidney tissue sections were stained with Picrosirius red (PSR) according to the standard protocol. Dewaxed kidney tissue sections were exposed to PSR stain for 1 h, and then washed twice with acidified water (0.5% glacial acetic acid). Sections were then serially dehydrated in alcohol solutions of different concentrations. Photomicrographs (200× magnification) were obtained randomly from the kidney cortex region using a Leica microscope (DM2500, Wetzlar, Germany). Regions of collagen deposition in the PSR-stained kidney sections were measured using an image analysis program (i-solution, IMT, Korea).
H2S levels in kidney tissue were determined as previously described [2,14]. Briefly, kidney tissues were homogenized in ice-cold 100 mM potassium phosphate buffer (pH 7.4) with protease inhibitors. One hundred microliters of homogenate was mixed with 100 µL of 1% zinc acetate, 100 µL of borate buffer (pH 10.0), 200 µL of 20 mM N,N-dimethyl-p-phenylenediamine dihydrochloride in 7.2 M HCl, and 200 µL of 30 mM FeCl3 in 1.2 M HCl and incubated in the dark for 15 min at 37 °C. The sample was centrifuged for 5 min at 10,000 rpm at 4 °C. The supernatant was taken and the optical density was measured at 670 nm. H2S concentration was calculated against a standard curve created using NaHS solution. Total glutathione (tGSH) and oxidized GSH (GSSG) levels were measured using an enzymatic recycling method, as described previously [30,49]. Given that GSH and related thiols are sensitive to oxidation and degradation during sampling and analysis, samples were harvested quickly in liquid nitrogen, stored at −70 °C until use, and analyzed rapidly. The amount of glutathione was determined by the formation of 5-thio-2-nitrobenzoic acid (TBA) from 5,5-dithiobis (2-nitrobenzoic acid; DTNB), as described by Akerboom and Sies [49]. GSH levels were defined as the change in optical density at 412 nm over 1 min at 37 °C.
4.3. Histology Kidney paraffin-sections were stained with Periodic Acid Schiff (PAS, Muto Pure Chemicals, Tokyo, Japan) stain according to a standard protocol. To determine morphological damage to tubular cells, 50 tubules in the cortex region of the kidneys were analyzed using the following scoring method: 0, no damage; 1, mild damage with the rounding of epithelial cells and dilated tubular lumen; 2, moderate damage with flattened epithelial cells, dilated lumen, and congestion of 430
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