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Methionine sulfoxide reductase A deficiency exacerbates progression of kidney fibrosis induced by unilateral ureteral obstruction
Free Radical Biology and Medicine 89 (2015) 201–208
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Original Contribution
Methionine sulfoxide reductase A deficiency exacerbates progression of kidney fibrosis induced by unilateral ureteral obstruction Jee In Kim a, Mi Ra Noh b, Ki Young Kim c, Hee-Seong Jang b, Hwa-Young Kim c,n, Kwon Moo Park b,nn a
Department of Molecular Medicine and MRC, Keimyung University School of Medicine, Daegu 705-717, Republic of Korea Department of Anatomy and BK21 Plus Program, Kyungpook National University School of Medicine, Daegu 700-422, Republic of Korea c Department of Biochemistry and Molecular Biology, Yeungnam University College of Medicine, Daegu 705-717, Republic of Korea b
art ic l e i nf o
a b s t r a c t
Article history: Received 16 March 2015 Received in revised form 13 July 2015 Accepted 18 July 2015 Available online 22 July 2015
Methionine sulfoxide reductase A (MsrA), which stereospecifically catalyzes the reduction of methionine-S-sulfoxide, is an important reactive oxygen species (ROS) scavenger. Tissue fibrosis is a maladaptive repair process following injury, associated with oxidative stress. In this study, we investigated the role of MsrA in unilateral ureteral obstruction (UUO)-induced kidney fibrosis and its underlying mechanisms by using MsrA gene-deleted mice (MsrA / ). MsrA deletion increased collagen deposition in the interstitium and the expression of collagen III and α-smooth muscle actin in the UUO kidneys, indicating that MsrA deficiency exacerbated the progression of UUO-induced kidney fibrosis. UUO reduced the kidney expression of MsrA, MsrB1, and MsrB2, thereby decreasing MsrA and MsrB activity. UUO increased hydrogen peroxide and lipid peroxidation levels and the ratio of oxidized glutathione (GSSG) to total glutathione (GSH) in the kidneys. The UUO-induced elevations in the levels of these oxidative stress markers and leukocyte markers were much higher in the MsrA / than in the MsrA þ / þ kidneys, the latter suggesting that the exacerbated kidney fibrosis in MsrA / mice was associated with enhanced inflammatory responses. Collectively, our data suggest that MsrA plays a protective role in the progression of UUO-induced kidney fibrosis via suppression of fibrotic responses caused by oxidative stress and inflammation. & 2015 Elsevier Inc. All rights reserved.
Keywords: Methionine sulfoxide reductase MsrA Ureteral obstruction Fibrosis Oxidative stress Inflammation
1. Introduction The sulfur-containing amino acid methionine is highly sensitive to oxidation, yielding free and protein-based methionine sulfoxides. Methionine oxidation may damage protein structure and function [1]. To cope with methionine oxidation-induced protein damage, cells have evolved a reductase system consisting of two stereospecific enzymes [2,3]. Methionine sulfoxide reductases (Msrs) catalyze the reduction of methionine sulfoxide to methionine. MsrA and MsrB specifically reduce the S- and R-form of methionine sulfoxide, respectively. In addition to their role in protein repair, Msrs act as antioxidants to protect against oxidative stress because of their ability to scavenge reactive oxygen species n Correspondence to: Department of Biochemistry and Molecular Biology, Yeungnam University College of Medicine, 170 Hyeonchungno, Namgu, Daegu 705717, Republic of Korea. fax: þ 82 53 629 7093 nn Correspondence to: Department of Anatomy, Kyungpook National University School of Medicine, 101 Dongindong-2ga, Junggu, Daegu 700-422, Republic of Korea; fax: 82 53 427 1468 E-mail addresses: [email protected] (H.-Y. Kim), [email protected] (K.M. Park).
http://dx.doi.org/10.1016/j.freeradbiomed.2015.07.018 0891-5849/& 2015 Elsevier Inc. All rights reserved.
(ROS) via cyclic oxidation/reduction of methionine [4,5]. Furthermore, Msrs regulate the biological activity of proteins by modulating specific methionine sulfoxide residues that are involved in the activation or inactivation of certain protein functions. For example, MsrA regulates the activity of calcium/calmodulin-dependent protein kinase II [6], while MsrB controls actin dynamics [7]. Hence, Msr dysfunction may cause the accumulation of faulty proteins and affect the antioxidant defense system and signal transduction pathways [8]. Mammals have a single MsrA protein type and three MsrB isoforms (MsrB1, B2, and B3) [9]. MsrA and MsrB1 are abundantly expressed in the kidneys [10,11]. Fibrosis is a pivotal feature in progressive renal disease and is a pathway common to end-stage renal disease. The fibrotic changes are caused by various factors including oxidative stress and inflammatory responses. Oxidative stress is associated with the excessive production of ROS and/or disturbances in cellular antioxidant systems. Increased ROS production and oxidative stress stimulate fibrogenic intracellular signaling, leading to an increase in the production of extracellular matrix components, leukocyte infiltration in the interstitium, and the proliferation of interstitial fibroblasts [12,13]. Unilateral ureteral obstruction (UUO) is a
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common cause of renal dysfunction in children and adults, and is widely used in rodent models to study the pathophysiology of kidney fibrosis [13–15]. We previously found that MsrA deficiency worsened ischemia/reperfusion (I/R)-induced acute kidney injury [16]. Since MsrA deficiency leads to increased oxidative stress and inflammatory responses in the kidneys, it is interesting to know whether MsrA is involved in the progression of kidney fibrosis. To the best of our knowledge, the present study is the first to report the role of MsrA in UUO-induced kidney fibrosis. Our study suggests that MsrA may represent a novel therapeutic target for fibrotic kidney diseases for which effective therapeutic strategies have not yet been developed.
2. Materials and methods 2.1. Animal preparation All experiments were conducted using 8- to 10-week-old MsrA gene-deleted (MsrA–/–) and wild-type (MsrA þ / þ ) male mice weighing 22–24 g. The generation of MsrA–/– mice has been described elsewhere [17]. The breeding lines for MsrA–/– mice were obtained by backcrossing into the C57BL/6 genetic background for 48 generations. The study was conducted in accordance with the guidelines of the Institutional Animal Care and Use Committee of Kyungpook National University and of Yeungnam University, Republic of Korea. The mice were provided free access to water and standard chow. The mice were anesthetized by intraperitoneal injection of pentobarbital sodium (50 mg/kg body weight; Sigma– Aldrich) before surgery. For UUO surgery, the right kidney was exposed via flank incision, the right ureter was completely obstructed near the renal pelvis by using a 6–0 nylon thread, and the incision was subsequently sutured. Sham surgeries were conducted using the same surgical procedure except for ureter ligation. Body temperature was maintained at 36.5–37.5 °C throughout all surgical procedures by using a temperature-controlled heating device (FHC, Bowdonham, ME, USA). After surgery, the plasma creatinine concentrations were determined to evaluate renal function by using a Vitros 250 Chemistry Analyzer (Johnson & Johnson). The kidneys were excised at 5 days after UUO and were either snap-frozen in liquid nitrogen for Western blot analysis or perfusion-fixed in PLP (4% paraformaldehyde, 75 mM L-lysine, and 10 mM sodium periodate; Sigma–Aldrich) for histological studies. 2.2. Immunoblot analysis Immunoblot analyses were performed using antibodies against
α-smooth muscle actin (α-SMA; Sigma–Aldrich), catalase (Fitz-
gerald), manganese superoxide dismutase (MnSOD; Calbiochem), 4-hydroxynonenal (HNE; Abcam), lymphocyte antigen 6 complex (Ly6G; eBioscience), MsrA [16], MsrB1 [9], MsrB2 [18], glyceraldehyde 3-phosphate dehydrogenase (GAPDH; Novus), glutathione peroxidase 1 (Gpx1; Santa Cruz Biotechnologies), thioredoxin 1 (Trx1; Santa Cruz Biotechnologies), and thioredoxin 2 (Trx2; Bioworld). Protein densities were measured using the ImageJ program (National Institutes of Health, Bethesda, MD, USA).
2.4. Immunohistochemical staining Immunohistochemistry staining was performed using anticollagen III (Abcam) and anti-F4/80 [12] antibodies. Goat serum was used instead of the primary antibodies to determine nonspecific staining. Images were captured using a Leica DM2500 microscope (Leica). Collagen III-positive areas were quantified using the i-Solution DT image acquisition and analysis program (iMT i-Solution) incorporating a Nikon Fx35 digital camera (Nikon). 2.5. MsrA and MsrB activity The reaction mixture (100 μL) contained 50 mM sodium phosphate (pH 7.5), 50 mM NaCl, 20 mM dithiothreitol, 200 μM dabsylated methionine-S-sulfoxide (for MsrA) or methionine-Rsulfoxide (for MsrB), and 200 μg of crude protein. The reaction was conducted at 37 °C for 30 min, after which the reaction product, dabsyl-Met, was analyzed via HPLC as previously described [19]. 2.6. Measurement of hydrogen peroxide in the kidneys A ferric-sensitive dye, xylenol orange (Sigma–Aldrich), was used to determine hydrogen peroxide (H2O2) levels in tissue samples as previously described [20]. 2.7. Measurement of lipid peroxidation in the kidneys Thiobarbituric acid-reactive substance (TBARS; Sigma–Aldrich) levels were measured as an indicator of lipid peroxidation as previously described [21]. Malondialdehyde (MDA), an end product of lipid peroxidation, reacts with thiobarbituric acid to produce an MDA-thiobarbituric acid adduct that is measured in the assay. An extinction coefficient of 153000 M 1 cm 1 at 532 nm for the chromophore was used to calculate TBARS levels. 2.8. Measurement of glutathione levels in the kidneys The ratio of oxidized glutathione (GSSG) to total glutathione (GSH, reduced GSH þ GSSG) was measured using an enzymatic recycling method as previously described [22,23]. The amount of total glutathione was determined by the formation of 5-thio-2nitrobenzoic acid (TBA) from 5,5-dithiobis (2-nitrobenzoic acid, DTNB) [16,22]. GSSG was measured by adding 2-vinylpyridine, which inhibits TBA formation when DTNB and glutathione react with each other [23]. The total GSH and GSSG levels were defined as the change in absorbance at 412 nm for 1 min at 37 °C. 2.9. Measurement of catalase activity in the kidneys Frozen kidney tissues were homogenized in phosphate-buffered saline (pH 7.4) on ice. The kidney lysate was centrifuged at 15,000g for 20 min at 4 °C. The catalase activity in the supernatant was measured in a reaction mixture containing 10 mM sodium phosphate buffer (pH 7.0) and 1.5 M hydrogen peroxide (Sigma– Aldrich) as described previously [24]. The catalase activity was defined as the change in absorbance at 240 nm for 1 min. 2.10. Statistical analysis
2.3. Histology PLP-fixed kidneys were embedded in paraffin and were cut into 4-μm sections. The sections were stained with Sirius red (0.05% in saturated picric acid solution) to detect collagen fibers. Images were captured, and the area of collagen deposition was determined using the i-Solution software (iMT i-Solution, Inc.).
The results were expressed as the means 7SEM. The statistical differences between the groups were analyzed using one-way analysis of variance (ANOVA) followed by Tukey’s least significant difference post hoc test using the SPSS software (version 12.0; SPSS, Inc., Chicago, IL, USA). The between-group differences were considered statistically significant at Po 0.05.
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3. Results 3.1. MsrA gene deletion exacerbates UUO-induced kidney fibrosis To determine the effect of MsrA on the progression of fibrosis, UUO-induced kidney fibrosis was evaluated using Sirius red
MsrA KO
MsrA WT
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Sham UUO
p<0.01 p<0.001
UUO
Sham UUO
Sham UUO
40
p<0.05
Collagen III-positive area (%)
40 Sirius red-positive area (%)
staining for collagen deposition, and immunohistochemical staining for collagen III expression. Sirius red-positive signals markedly increased in the interstitium of the ureterally obstructed kidneys (Fig. 1A). The Sirius red-positive area after UUO was profoundly larger in the MsrA–/– than in the MsrA þ / þ kidneys (Fig. 1B). Consistent with this result, the UUO-induced collagen III expression was
Sham
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30 20 p<0.01
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GAPDH Sham UUO 5D MsrA WT
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Sham UUO 5D
10
p<0.01 p<0.001
8 6
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p<0.001
4 2 0
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14
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12
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α-SMA
10
Sham UUO 7D
p<0.001
p<0.01
8 6 4 2 0
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Fig. 1. Collagen deposition and expression, and ɑ-smooth muscle actin expression in the kidneys after unilateral ureteral obstruction. MsrA þ / þ and MsrA / male mice were subjected to either unilateral ureteral obstruction (UUO) or a sham surgery (n ¼6–7 for each group). The kidneys were excised at 5 or 7 days after the surgery. (A and C) Paraffin-embedded kidney sections were stained with Sirius red (A) or immunohistochemically using an anti-collagen III antibody (C), and images of the cortex were acquired. (B, D) Sirius red- (B) or collagen III-positive areas (D) were quantified as described under Materials and methods. (E and F) The ɑ-smooth muscle actin (ɑ-SMA) protein level, a marker of fibrosis, in the kidneys excised at 5 (E) or 7 (F) days after UUO was determined using Western blotting. The protein band densities were quantified using the ImageJ program (National Institutes of Health, Bethesda, MD, USA). Each protein band density was normalized to that of the loading control GAPDH. The data represent the means 7 SE (n¼6–7).
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mainly detected in the interstitium (Fig. 1C), and the area of collagen III expression was also significantly larger in the MsrA–/– than in the MsrA þ / þ kidneys (Fig. 1D). Increased expression levels of αSMA, a myofibroblast marker protein, were observed in the kidneys at 5 and 7 days after UUO in both mouse groups (Fig. 1E and F). However, the increases in the α-SMA expression were more profound in the MsrA–/– than in the MsrA þ / þ kidneys (Fig. 1E and F). These data indicate that MsrA deficiency exacerbates UUO-induced kidney fibrosis. The plasma creatinine concentrations were not significantly altered after UUO (0.1570.02, 0.1870.01, 0.1770.01, and 0.1570.02 mg/dL in MsrA þ / þ sham, MsrA þ / þ UUO, MsrA–/– sham, and MsrA–/– UUO kidneys, respectively). Consistent with this finding, the contralateral, normal functioning kidney is known to prevent significant UUO-induced renal functional impairment. Body weight/tibia length (g/mm) was similar in the MsrA þ / þ and MsrA–/– kidneys and was not significantly changed after UUO in all mouse groups (1.1070.01, 1.0470.02, 1.2470.06, and 1.1470.03 g/mm in MsrA þ / þ sham, MsrA þ / þ UUO, MsrA–/– sham, and MsrA–/– UUO mice, respectively). 3.2. UUO reduces MsrA and MsrB expression and activity in the kidneys We next investigated whether UUO affected the expression and activity of Msrs in the kidneys. The MsrA protein levels in the MsrA þ / þ kidneys were significantly reduced at 5 days after UUO (Fig. 2A and B). Accordingly, the MsrA activity was significantly decreased after UUO (Fig. 3A). The MsrB1 and MsrB2 protein expression levels were similar in the MsrA–/– and MsrA þ / þ kidneys (Fig. 2A, C, and D). Notably, no MsrB3 protein expression was detected in the kidneys of all mouse groups. The MsrB1 and MsrB2 expression levels were significantly reduced after UUO, similar to those of MsrA (Fig. 2C and D). Furthermore, the reduction of the MsrB1 protein expression level was more profound in the MsrA–/– than in the MsrA þ / þ kidneys (Fig. 2A and C), while no significant difference in MsrB2 reduction was detected between the two kidney groups (Fig. 2A and D). Consistent with the reduced MsrB1 and MsrB2 protein expression levels in the UUO kidneys, the MsrB
MsrA WT Sham UUO
activity significantly decreased in both the MsrA–/– and the MsrA þ / kidneys (Fig. 3B). The MsrA–/– kidneys showed a slightly lower level of MsrB activity than that observed in the MsrA þ / þ kidneys in both the sham and UUO groups. Collectively, these data suggest that UUO reduces the protein expression of MsrA, MsrB1, and MsrB2, thereby reducing Msr activity.
þ
3.3. MsrA gene deletion elevates ROS production and oxidative stress after UUO ROS and oxidative stress levels were measured in MsrA þ / þ and MsrA–/– kidneys to determine whether aggravation of UUO-induced fibrosis by MsrA gene deletion was linked to reduced MsrA antioxidant activity. UUO significantly increased the H2O2 levels in the MsrA þ / þ and MsrA–/– kidneys (Fig. 4A). The UUO-induced elevation in H2O2 levels was more profound in the MsrA–/– than in the MsrA þ / þ kidneys. H2O2 levels were similar in the MsrA þ / þ sham and MsrA / sham kidneys (Fig. 4A). The TBARS level, an index of lipid peroxidation, significantly increased in both the MsrA þ / þ and the MsrA / kidneys after UUO, with the increase in MsrA / being larger than that in the MsrA þ / þ kidneys (Fig. 4B). Consistent with the increase in the TBARS levels, the HNE (a lipid peroxidation product) kidney levels increased after UUO (Fig. 4C). They were significantly higher in the MsrA / than in the MsrA þ / þ kidneys (Fig. 4C and D), while they were not significantly different between the MsrA þ / þ sham and the MsrA / sham kidneys (Fig. 4C and D). Next, we determined the total GSH and GSSG levels in the kidneys. Although UUO significantly reduced the total GSH levels in the kidneys compared to those in the kidneys of the mice that underwent sham surgery (Fig. 4E), the UUO-induced reductions in the total GSH levels were similar in the MsrA / and MsrA þ / þ kidneys. The ratio of GSSG to total GSH also significantly increased after UUO (Fig. 4F), and was more profound in the MsrA / than in the MsrA þ / þ kidneys (Fig. 4F). The total GSH levels and the GSSG/total GSH ratio were similar in the MsrA / sham and MsrA þ / þ sham kidneys (Fig. 4F). Together, these data suggest that MsrA deficiency accelerates GSH oxidation in the kidneys following UUO.
Sham
MsrA KO UUO MsrA MsrB1 MsrB2
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Fig. 2. The expression of MsrA, MsrB1, and MsrB2 in the kidneys after unilateral ureteral obstruction. MsrA þ / þ and MsrA / male mice were subjected to either unilateral ureteral obstruction (UUO) or a sham surgery (n¼ 6–7 for each group). The kidneys were excised at 5 days after the surgery. (A) The protein levels of MsrA, MsrB1, and MsrB2 were determined using Western blotting. GAPDH was used as a loading control. (B–D) The protein band densities were quantified using the ImageJ program (National Institutes of Health, Bethesda, MD, USA). Each protein band density was normalized to that of the loading control GAPDH. The data represent the means7 SE (n¼6–7). ND, not detectable; NS, not significant.
400 UUO
300 200 100 0
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2000 Sham
p<0.05
MsrB activity (pmol/mg protein/min)
MsrA activity (pmol/mg protein/min)
J.I. Kim et al. / Free Radical Biology and Medicine 89 (2015) 201–208
ND
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p<0.05
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Fig. 3. Activity of MsrA and MsrB in the kidneys after unilateral ureteral obstruction. MsrA þ / þ and MsrA / male mice were subjected to either unilateral ureteral obstruction (UUO) or a sham surgery (n¼ 6–7 for each group). The kidneys were excised at 5 days after the surgery. The MsrA and MsrB activities were determined as described in the Materials and Methods. The data represent the means 7 SE (n ¼4). ND, not detectable; NS, not significant.
3.0 2.5
Sham UUO
p<0.01
p<0.05
2.0 1.5 1.0 0.5 0.0
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/
Fig. 4. The levels of oxidative stress markers in the kidneys after unilateral ureteral obstruction. MsrA and MsrA male mice were subjected to either unilateral ureteral obstruction (UUO) or a sham surgery (n¼ 6–7 for each group). The kidneys were excised at 5 days after the surgery. The levels of hydrogen peroxide (H2O2, A), malondialdehyde (MDA, B), and 4-hydroxynonenal (HNE, C) in the kidneys were determined as described under Materials and methods. (D) The protein band density of HNE in (C) was quantified using the ImageJ program (National Institutes of Health, Bethesda, MD, USA), and was normalized to that of the loading control GAPDH. (E and F) The total glutathione (GSH) levels and the ratio of oxidized GSH (GSSG) to total GSH were determined as described under Materials and methods. The data represent the means7 SE (n¼ 6–7). NS, not significant.
In addition, we determined the levels of catalase and MnSOD in the kidneys. In the sham-operated kidneys, the catalase and MnSOD levels were similar in the MsrA þ / þ and MsrA / kidneys (Fig. 5A). UUO reduced the expression of catalase and MnSOD in both the MsrA þ / þ and the MsrA / kidneys (Fig. 5A). The reduction in catalase expression was more profound in the MsrA / than in the MsrA þ / þ kidneys, while the reduction in MnSOD expression after UUO was similar in both kidney types (Fig. 5A). The
catalase activity reduced more profoundly in the MsrA / than in the MsrA þ / þ kidneys after UUO (Fig. 5B), which was consistent with the respective protein expression data (Fig. 5A). We also determined the Gpx1 protein expression level in the kidneys (Fig. 5C), and found that it was similar in the MsrA þ / þ sham and MsrA / sham kidneys. In contrast, it significantly decreased after UUO in both the MsrA / and the MsrA þ / þ kidneys without showing a significant difference between the two kidney types.
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Fig. 5. The expression levels of catalase, manganese superoxide dismutase, thioredoxin 1/2, and glutathione peroxidase 1, and catalase activity in the kidneys after unilateral ureteral obstruction. MsrA þ / þ and MsrA / male mice were subjected to either unilateral ureteral obstruction (UUO) or a sham surgery (n¼ 5–7 for each group). The kidneys were excised at 5 days after the surgery. (A) The protein levels of catalase and manganese superoxide dismutase (MnSOD) were determined using Western blotting. (B) The catalase activity was determined as described under Materials and methods. (C) The protein levels of thioredoxin 1 (Trx1), thioredoxin 2 (Trx2), and glutathione peroxidase 1 (Gpx1) were analyzed using Western blotting. The protein band densities were quantified using the ImageJ program (National Institutes of Health, Bethesda, MD, USA). Each protein band density was normalized to that of the loading control GAPDH. The data represent the means 7 SE (n ¼5–7). NS, not significant.
Since Trx is the in vivo reductant of Msrs [18], we further determined the Trx expression levels in the kidneys. UUO significantly reduced Trx1 and Trx2 expression in both the MsrA / and the MsrA þ / þ kidneys without showing a significant difference between the two kidney types (Fig. 5C).
fibrosis observed in the MsrA / kidneys is associated with an elevated inflammatory response.
3.4. MsrA gene deletion accelerates the inflammatory response in the kidneys after UUO
Renal fibrosis is characterized by kidney-specific phenotypic changes, such as a loss of epithelial cell polarity causing cell dysfunction, expansion of the interstitial area due to the accumulation of extracellular matrix components, including collagens, and leukocyte infiltration [12,15,25,26]. A growing body of evidence suggests that decreasing oxidative stress and the inflammatory response retards kidney fibrosis in various clinical settings and experimental models [13–15,27]. In the present study, we have demonstrated the involvement of MsrA in the progression of kidney fibrosis, as MsrA deficiency aggravated UUO-induced kidney fibrosis by enhancing oxidative stress and inflammation. Therefore, our data support the findings that oxidative stress and inflammation are pivotal factors in the progression of kidney fibrosis, and suggest that MsrA plays an important role in protecting kidney function in fibrosis-related chronic kidney diseases. A better understanding of the protective role of MsrA in fibrosis may
Since leukocyte infiltration into injured tissue is a pivotal process of fibrosis and is a consequence or cause of oxidative stress [13,20], we examined the infiltration of leukocytes in the kidneys after UUO by immunohistochemical and immunoblot analyses using an antibody against F4/80, a macrophage marker, and against Ly6G, a leukocyte marker, respectively. UUO dramatically increased the number of F4/80-positive cells in the interstitium of the obstructed kidneys (Fig. 6A). The increase in F4/80-positive cells was more profound in the MsrA / than in the MsrA þ / þ kidneys (Fig. 6A). The protein expression level of Ly6G also significantly increased in the kidneys after UUO (Fig. 6B), and was higher in the MsrA / than in the MsrA þ / þ kidneys (Fig. 6B). These results indicate that the increased UUO-induced kidney MsrA WT
4. Discussion
MsrA KO Ly6G
Ly6G (vs. WT sham)
UUO
Sham
GAPDH Sham UUO MsrA WT 6
Sham UUO MsrA KO
Sham
UUO p<0.05
5 4 3
p<0.001
p<0.05
2 1 0
MsrA WT þ/þ
MsrA KO
Fig. 6. The analyses of the inflammatory responses in the kidneys after unilateral ureteral obstruction. MsrA and MsrA / male mice were subjected to either unilateral ureteral obstruction (UUO) or a sham surgery. The kidneys were excised at 5 days after the surgery. (A) Paraffin-embedded kidney sections were immunohistochemically stained using the F4/80 antibody, and images of the cortex were acquired. (B) The protein levels of Ly6G were determined using Western blotting. The protein band densities were quantified using the ImageJ program (National Institutes of Health, Bethesda, MD, USA). Each protein band density was normalized to that of the loading control GAPDH. The data represent the means 7SE (n¼ 6–7).
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further the development of fibrosis treatment strategies. Msr enzymes repair proteins damaged by ROS by reversing the oxidation of methionine residues and scavenging cellular ROS via catalyzing the cyclic oxidation/reduction process of methionine [5,28,29]. In the present study, UUO decreased the expression and, consequently, the activity of MsrA, MsrB1, and MsrB2 in the kidneys. In parallel, UUO elevated the H2O2, TBARS, and HNE levels and the GSSG/GSH ratio in the kidneys. Furthermore, all of these oxidative stress markers were more profoundly elevated in the MsrA / than in the MsrA þ / þ kidneys. These data suggest that UUO impairs the cellular ROS-scavenging capacity and redox balance by affecting the methionine sulfoxide reduction system, particularly MsrA function, leading to increased fibrosis. Consistent with this notion, Maier et al. reported that patients with idiopathic pulmonary fibrosis had a higher level of methionine sulfoxide in their bronchoalveolar lavage fluid [30,31]. In addition, Ogawa et al. reported that the inhibition of MsrA activity by an anti-MsrA antibody elevated systemic sclerosis characterized by fibrosis via increased oxidative stress [32]. We recently reported that MsrA / mice were more susceptible to acute kidney I/R injury than MsrA þ / þ mice [16]. Based on the findings of the present and previous studies, it appears that methionine-S-sulfoxide reduction plays a pivotal role in kidney I/R injury and fibrosis. Therefore, it will be interesting to investigate whether methionine-R-sulfoxide reduction (i.e., MsrB activity) is involved in kidney function. In this study, we found that catalase expression and activity were similar in the MsrA / and MsrA þ / þ sham kidneys. However, UUO reduced the catalase expression and activity in the kidneys, and these reductions were more profound in the MsrA / than in the MsrA þ / þ kidneys. These data suggest that the exacerbated UUO-induced fibrosis in the MsrA / kidneys is associated with decreased catalase activity, and that MsrA is involved in the regulation of catalase expression and activity after UUO. It has been reported that Msr together with GroEL enhanced the recovery of bacterial catalase activity impaired by the oxidation of methionine residues [33]. MsrA may influence GSH production since it is involved in the regulation of the transsulfuration pathway under oxidative stress [16]. Cysteine is a biosynthetic product of the transsulfuration pathway, and is a component of GSH. However, in the present study, the total GSH levels were similar in the MsrA / and the MsrA þ / þ kidneys even after UUO, although the GSSG/total GSH ratio, a representative redox status marker, was much higher in the MsrA / than in the MsrA þ / þ kidneys. These data suggest that increased UUO-induced oxidative stress in the MsrA / kidneys is associated with the antioxidant scavenging by MsrA but not with MsrA’s modulation of GSH biosynthesis. We further found that MsrA deficiency did not affect the UUO-induced Gpx1 protein expression, suggesting that the elevated UUO-induced oxidative stress in MsrA / kidneys might not be associated with Gpx1’s antioxidant function. Oxidative stress induces kidney tissue damage and accelerates leukocyte infiltration into the kidney tissue. This triggers ROS production, thereby causing further tissue and cell damage [20,34,35]. A number of reports have demonstrated that the infiltration of inflammatory cells, including T-cells and macrophages, plays a causative role in UUO-induced kidney fibrosis [14,36–38]. In our previous studies, we confirmed that inhibitors of ROSproducing enzymes reduced leukocyte infiltration into I/R- and UUO-injured kidneys and prevented kidney fibrosis [12,13,20]. We also recently reported that I/R-induced inflammatory responses in the kidneys were accelerated by MsrA gene depletion [16]. In addition, the present study showed that UUO elevated Ly6G protein expression and the number of F4/80-positive cells in the kidneys, and that these inflammatory responses were enhanced by MsrA deficiency.
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In conclusion, to the best of our knowledge, the present study was the first to demonstrate that UUO caused a reduction in Msr enzyme function, which increased oxidative stress, inflammatory responses, and fibrosis. Furthermore, MsrA deficiency induced an increase in inflammatory responses in the kidneys under oxidative stress, leading to aggravation of kidney fibrosis. These data suggest that MsrA plays a protective role in the progression of UUO-induced kidney fibrosis via suppression of fibrotic responses caused by oxidative stress and inflammation.
Author disclosure statement No competing financial interests exist.
Acknowledgments This study was supported by the National Research Foundation of Korea (NRF) grants funded by the Korean government (NRF2014R1A11049549 to K.M.P. and MSIP 2014R1A5A2010008 to J.I.K.).
References [1] H.Y. Kim, The methionine sulfoxide reduction system: selenium utilization and methionine sulfoxide reductase enzymes and their functions, Antioxid. Redox Signal. 19 (2013) 958–969. [2] N. Brot, L. Weissbach, J. Werth, H. Weissbach, Enzymatic reduction of proteinbound methionine sulfoxide, Proc. Natl. Acad. Sci. USA 78 (1981) 2155–2158. [3] R. Grimaud, B. Ezraty, J.K. Mitchell, D. Lafitte, C. Briand, Derrick, P. J.; Barras, F. Repair of oxidized proteins. Identification of a new methionine sulfoxide reductase, J. Biol. Chem. 276 (2001) 48915–48920. [4] J. Moskovitz, B.S. Berlett, J.M. Poston, E.R. Stadtman, The yeast peptide-methionine sulfoxide reductase functions as an antioxidant in vivo, . Proc. Natl. Acad. Sci. USA 94 (1997) 9585–9589. [5] R.L. Levine, L. Mosoni, B.S. Berlett, E.R. Stadtman, Methionine residues as endogenous antioxidants in proteins, Proc. Natl. Acad. Sci. USA 93 (1996) 15036–15040. [6] J.R. Erickson, M.L. Joiner, X. Guan, W. Kutschke, J. Yang, C.V. Oddis, R.K. Bartlett, J.S. Lowe, S.E. O'Donnell, N. Aykin-Burns, M.C. Zimmerman, K. Zimmerman, A. J. Ham, R.M. Weiss, D.R. Spitz, M.A. Shea, R.J. Colbran, P.J. Mohler, M. E. Anderson, A dynamic pathway for calcium-independent activation of CaMKII by methionine oxidation, Cell 133 (2008) 462–474. [7] B.C. Lee, Z. Peterfi, F.W. Hoffmann, R.E. Moore, A. Kaya, A. Avanesov, L. Tarrago, Y. Zhou, E. Weerapana, D.E. Fomenko, P.R. Hoffmann, V.N. Gladyshev, MsrB1 and MICALs regulate actin assembly and macrophage function via reversible stereoselective methionine oxidation, Mol. Cell 51 (2013) 397–404. [8] J. Moskovitz, Methionine sulfoxide reductases: ubiquitous enzymes involved in antioxidant defense, protein regulation, and prevention of aging-associated diseases, Biochim. Biophys. Acta 1703 (2005) 213–219. [9] Y. Kimura, H. Kimura, Hydrogen sulfide protects neurons from oxidative stress, FASEB J 18 (2004) 1165–1167. [10] J. Moskovitz, N.A. Jenkins, D.J. Gilbert, N.G. Copeland, F. Jursky, H. Weissbach, N. Brot, Chromosomal localization of the mammalian peptide-methionine sulfoxide reductase gene and its differential expression in various tissues, Proc. Natl. Acad. Sci. USA 93 (1996) 3205–3208. [11] D.E. Fomenko, S.V. Novoselov, S.K. Natarajan, B.C. Lee, A. Koc, B.A. Carlson, T. H. Lee, H.Y. Kim, D.L. Hatfield, V.N. Gladyshev, MsrB1 (methionine-R-sulfoxide reductase 1) knock-out mice: roles of MsrB1 in redox regulation and identification of a novel selenoprotein form, J. Biol. Chem. 284 (2009) 5986–5993. [12] H.S. Jang, J.I. Kim, K.J. Jung, J. Kim, K.H. Han, K.M. Park, Bone marrow-derived cells play a major role in kidney fibrosis via proliferation and differentiation in the infiltrated site, Biochim. Biophys. Acta 1832 (2013) 817–825. [13] H.S. Jang, J.I. Kim, J. Kim, Y.K. Na, J.W. Park, K.M. Park, Bone marrow derived cells and reactive oxygen species in hypertrophy of contralateral kidney of transient unilateral renal ischemia-induced mouse, Free Radic. Res. 46 (2012) 903–911. [14] R.L. Chevalier, M.S. Forbes, B.A. Thornhill, Ureteral obstruction as a model of renal interstitial fibrosis and obstructive nephropathy, Kidney Int. 75 (2009) 1145–1152. [15] R.L. Chevalier, B.A. Thornhill, M.S. Forbes, S.C. Kiley, Mechanisms of renal injury and progression of renal disease in congenital obstructive nephropathy, Pediatr. Nephrol. 25 (2010) 687–697. [16] J.I. Kim, S.H. Choi, K.J. Jung, E. Lee, H.Y. Kim, K.M. Park, Protective role of methionine sulfoxide reductase A against ischemia/reperfusion injury in mouse kidney and its involvement in the regulation of trans-sulfuration pathway, Antioxid. Redox Signal. 18 (2013) 2241–2250.
208
J.I. Kim et al. / Free Radical Biology and Medicine 89 (2015) 201–208
[17] J. Moskovitz, S. Bar-Noy, W.M. Williams, J. Requena, B.S. Berlett, E.R. Stadtman, Methionine sulfoxide reductase (MsrA) is a regulator of antioxidant defense and lifespan in mamma, Proc. Natl. Acad. Sci. USA 98 (2001) 12920–12925. [18] H.Y. Kim, J.R. Kim, Thioredoxin as a reducing agent for mammalian methionine sulfoxide reductases B lacking resolving cysteine, Biochem. Biophys. Res. Commun. 371 (2008) 490–494. [19] H.Y. Kim, Glutaredoxin serves as a reductant for methionine sulfoxide reductases with or without resolving cysteine, Acta Biochim. Biophys. Sin. (Shangha 44 (2012) 623–627. [20] J. Kim, Y.M. Seok, K.J. Jung, K.M. Park, Reactive oxygen species/oxidative stress contributes to progression of kidney fibrosis following transient ischemic injury in mice, Am. J. Physiol. Renal Physiol 297 (2009) F461–F470. [21] J.A. Buege, S.D. Aust, Microsomal lipid peroxidation, Methods Enzymol. 52 (1978) 302–310. [22] T.P. Akerboom, H. Sies, Assay of glutathione, glutathione disulfide, and glutathione mixed disulfides in biological samples, Methods Enzymol. 77 (1981) 373–382. [23] M.E. Anderson, Determination of glutathione and glutathione disulfide in biological samples, Methods Enzymol. 113 (1985) 548–555. [24] R.F. Beers Jr, I.W. Sizer, A spectrophotometric method for measuring the breakdown of hydrogen peroxide by catalase, J. Biol. Chem. 195 (1952) 133–140. [25] O. Skalli, P. Ropraz, A. Trzeciak, G. Benzonana, D. Gillessen, G. Gabbiani, A monoclonal antibody against alpha-smooth muscle actin: a new probe for smooth muscle differentiation, J. Cell Biol. 103 (1986) 2787–2796. [26] H.S. Jang, J. Kim, K.Y. Kim, J.I. Kim, M.H. Cho, K.M. Park, Previous ischemia and reperfusion injury results in resistance of the kidney against subsequent ischemia and reperfusion insult in mice; a role for the Akt signal pathway, Nephrol. Dial. Transplant. 27 (2012) 3762–3770. [27] K.J. Jung, H.S. Jang, J.I. Kim, S.J. Han, J.W. Park, K.M. Park, Involvement of hydrogen sulfide and homocysteine transsulfuration pathway in the progression of kidney fibrosis after ureteral obstruction, Biochim. Biophys. Acta 1832 (2013) 1989–1997. [28] E.R. Stadtman, J. Moskovitz, B.S. Berlett, R.L. Levine, Cyclic oxidation and reduction of protein methionine residues is an important antioxidant
mechanism, Mol. Cell. Biochem. 234-235 (2002) 3–9. [29] S. Luo, R.L. Levine, Methionine in proteins defends against oxidative stress, FASEB J 23 (2009) 464–472. [30] K. Maier, L. Leuschel, U. Costabel, Increased levels of oxidized methionine residues in bronchoalveolar lavage fluid proteins from patients with idiopathic pulmonary fibrosis, Am. Rev. Respir. Dis. 143 (1991) 271–274. [31] K.L. Maier, L. Leuschel, U. Costabel, Increased oxidized methionine residues in BAL fluid proteins in acute or chronic bronchitis, Eur. Respir. J. 5 (1992) 651–658. [32] F. Ogawa, K. Shimizu, T. Hara, E. Muroi, K. Komura, M. Takenaka, M. Hasegawa, M. Fujimoto, K. Takehara, S. Sato, Autoantibody against one of the antioxidant repair enzymes, methionine sulfoxide reductase A, in systemic sclerosis: association with pulmonary fibrosis and vascular damage, Arch. Dermatol. Res. 302 (2010) 27–35. [33] M. Mahawar, V. Tran, J.S. Sharp, R.J. Maier, Synergistic roles of Helicobacter pylori methionine sulfoxide reductase and GroEL in repairing oxidant-damaged catalase, J. Biol. Chem. 286 (2011) 19159–19169. [34] J. Kim, H.S. Jang, K.M. Park, Reactive oxygen species generated by renal ischemia and reperfusion trigger protection against subsequent renal ischemia and reperfusion injury in mice, Am. J. Physiol. Renal Physiol 298 (2010) F158–F166. [35] K.M. Park, J.I. Kim, Y. Ahn, A.J. Bonventre, J.V. Bonventre, Testosterone is responsible for enhanced susceptibility of males to ischemic renal injury, J. Biol. Chem. 279 (2004) 52282–52292. [36] T.A. Wynn, Cellular and molecular mechanisms of fibrosis, J. Pathol. 214 (2008) 199–210. [37] K. Kitagawa, T. Wada, K. Furuichi, H. Hashimoto, Y. Ishiwata, M. Asano, M. Takeya, W.A. Kuziel, K. Matsushima, N. Mukaida, H. Yokoyama, Blockade of CCR2 ameliorates progressive fibrosis in kidney, Am. J. Pathol. 165 (2004) 237–246. [38] M. Niedermeier, B. Reich, M. Rodriguez Gomez, A. Denzel, K. Schmidbauer, N. Gobel, Y. Talke, F. Schweda, M. Mack, CD4 þ T cells control the differentiation of Gr1 þ monocytes into fibrocytes, Proc. Natl. Acad. Sci. USA 106 (2009) 17892–17897.
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Original Contribution
Methionine sulfoxide reductase A deficiency exacerbates progression of kidney fibrosis induced by unilateral ureteral obstruction Jee In Kim a, Mi Ra Noh b, Ki Young Kim c, Hee-Seong Jang b, Hwa-Young Kim c,n, Kwon Moo Park b,nn a
Department of Molecular Medicine and MRC, Keimyung University School of Medicine, Daegu 705-717, Republic of Korea Department of Anatomy and BK21 Plus Program, Kyungpook National University School of Medicine, Daegu 700-422, Republic of Korea c Department of Biochemistry and Molecular Biology, Yeungnam University College of Medicine, Daegu 705-717, Republic of Korea b
art ic l e i nf o
a b s t r a c t
Article history: Received 16 March 2015 Received in revised form 13 July 2015 Accepted 18 July 2015 Available online 22 July 2015
Methionine sulfoxide reductase A (MsrA), which stereospecifically catalyzes the reduction of methionine-S-sulfoxide, is an important reactive oxygen species (ROS) scavenger. Tissue fibrosis is a maladaptive repair process following injury, associated with oxidative stress. In this study, we investigated the role of MsrA in unilateral ureteral obstruction (UUO)-induced kidney fibrosis and its underlying mechanisms by using MsrA gene-deleted mice (MsrA / ). MsrA deletion increased collagen deposition in the interstitium and the expression of collagen III and α-smooth muscle actin in the UUO kidneys, indicating that MsrA deficiency exacerbated the progression of UUO-induced kidney fibrosis. UUO reduced the kidney expression of MsrA, MsrB1, and MsrB2, thereby decreasing MsrA and MsrB activity. UUO increased hydrogen peroxide and lipid peroxidation levels and the ratio of oxidized glutathione (GSSG) to total glutathione (GSH) in the kidneys. The UUO-induced elevations in the levels of these oxidative stress markers and leukocyte markers were much higher in the MsrA / than in the MsrA þ / þ kidneys, the latter suggesting that the exacerbated kidney fibrosis in MsrA / mice was associated with enhanced inflammatory responses. Collectively, our data suggest that MsrA plays a protective role in the progression of UUO-induced kidney fibrosis via suppression of fibrotic responses caused by oxidative stress and inflammation. & 2015 Elsevier Inc. All rights reserved.
Keywords: Methionine sulfoxide reductase MsrA Ureteral obstruction Fibrosis Oxidative stress Inflammation
1. Introduction The sulfur-containing amino acid methionine is highly sensitive to oxidation, yielding free and protein-based methionine sulfoxides. Methionine oxidation may damage protein structure and function [1]. To cope with methionine oxidation-induced protein damage, cells have evolved a reductase system consisting of two stereospecific enzymes [2,3]. Methionine sulfoxide reductases (Msrs) catalyze the reduction of methionine sulfoxide to methionine. MsrA and MsrB specifically reduce the S- and R-form of methionine sulfoxide, respectively. In addition to their role in protein repair, Msrs act as antioxidants to protect against oxidative stress because of their ability to scavenge reactive oxygen species n Correspondence to: Department of Biochemistry and Molecular Biology, Yeungnam University College of Medicine, 170 Hyeonchungno, Namgu, Daegu 705717, Republic of Korea. fax: þ 82 53 629 7093 nn Correspondence to: Department of Anatomy, Kyungpook National University School of Medicine, 101 Dongindong-2ga, Junggu, Daegu 700-422, Republic of Korea; fax: 82 53 427 1468 E-mail addresses: [email protected] (H.-Y. Kim), [email protected] (K.M. Park).
http://dx.doi.org/10.1016/j.freeradbiomed.2015.07.018 0891-5849/& 2015 Elsevier Inc. All rights reserved.
(ROS) via cyclic oxidation/reduction of methionine [4,5]. Furthermore, Msrs regulate the biological activity of proteins by modulating specific methionine sulfoxide residues that are involved in the activation or inactivation of certain protein functions. For example, MsrA regulates the activity of calcium/calmodulin-dependent protein kinase II [6], while MsrB controls actin dynamics [7]. Hence, Msr dysfunction may cause the accumulation of faulty proteins and affect the antioxidant defense system and signal transduction pathways [8]. Mammals have a single MsrA protein type and three MsrB isoforms (MsrB1, B2, and B3) [9]. MsrA and MsrB1 are abundantly expressed in the kidneys [10,11]. Fibrosis is a pivotal feature in progressive renal disease and is a pathway common to end-stage renal disease. The fibrotic changes are caused by various factors including oxidative stress and inflammatory responses. Oxidative stress is associated with the excessive production of ROS and/or disturbances in cellular antioxidant systems. Increased ROS production and oxidative stress stimulate fibrogenic intracellular signaling, leading to an increase in the production of extracellular matrix components, leukocyte infiltration in the interstitium, and the proliferation of interstitial fibroblasts [12,13]. Unilateral ureteral obstruction (UUO) is a
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common cause of renal dysfunction in children and adults, and is widely used in rodent models to study the pathophysiology of kidney fibrosis [13–15]. We previously found that MsrA deficiency worsened ischemia/reperfusion (I/R)-induced acute kidney injury [16]. Since MsrA deficiency leads to increased oxidative stress and inflammatory responses in the kidneys, it is interesting to know whether MsrA is involved in the progression of kidney fibrosis. To the best of our knowledge, the present study is the first to report the role of MsrA in UUO-induced kidney fibrosis. Our study suggests that MsrA may represent a novel therapeutic target for fibrotic kidney diseases for which effective therapeutic strategies have not yet been developed.
2. Materials and methods 2.1. Animal preparation All experiments were conducted using 8- to 10-week-old MsrA gene-deleted (MsrA–/–) and wild-type (MsrA þ / þ ) male mice weighing 22–24 g. The generation of MsrA–/– mice has been described elsewhere [17]. The breeding lines for MsrA–/– mice were obtained by backcrossing into the C57BL/6 genetic background for 48 generations. The study was conducted in accordance with the guidelines of the Institutional Animal Care and Use Committee of Kyungpook National University and of Yeungnam University, Republic of Korea. The mice were provided free access to water and standard chow. The mice were anesthetized by intraperitoneal injection of pentobarbital sodium (50 mg/kg body weight; Sigma– Aldrich) before surgery. For UUO surgery, the right kidney was exposed via flank incision, the right ureter was completely obstructed near the renal pelvis by using a 6–0 nylon thread, and the incision was subsequently sutured. Sham surgeries were conducted using the same surgical procedure except for ureter ligation. Body temperature was maintained at 36.5–37.5 °C throughout all surgical procedures by using a temperature-controlled heating device (FHC, Bowdonham, ME, USA). After surgery, the plasma creatinine concentrations were determined to evaluate renal function by using a Vitros 250 Chemistry Analyzer (Johnson & Johnson). The kidneys were excised at 5 days after UUO and were either snap-frozen in liquid nitrogen for Western blot analysis or perfusion-fixed in PLP (4% paraformaldehyde, 75 mM L-lysine, and 10 mM sodium periodate; Sigma–Aldrich) for histological studies. 2.2. Immunoblot analysis Immunoblot analyses were performed using antibodies against
α-smooth muscle actin (α-SMA; Sigma–Aldrich), catalase (Fitz-
gerald), manganese superoxide dismutase (MnSOD; Calbiochem), 4-hydroxynonenal (HNE; Abcam), lymphocyte antigen 6 complex (Ly6G; eBioscience), MsrA [16], MsrB1 [9], MsrB2 [18], glyceraldehyde 3-phosphate dehydrogenase (GAPDH; Novus), glutathione peroxidase 1 (Gpx1; Santa Cruz Biotechnologies), thioredoxin 1 (Trx1; Santa Cruz Biotechnologies), and thioredoxin 2 (Trx2; Bioworld). Protein densities were measured using the ImageJ program (National Institutes of Health, Bethesda, MD, USA).
2.4. Immunohistochemical staining Immunohistochemistry staining was performed using anticollagen III (Abcam) and anti-F4/80 [12] antibodies. Goat serum was used instead of the primary antibodies to determine nonspecific staining. Images were captured using a Leica DM2500 microscope (Leica). Collagen III-positive areas were quantified using the i-Solution DT image acquisition and analysis program (iMT i-Solution) incorporating a Nikon Fx35 digital camera (Nikon). 2.5. MsrA and MsrB activity The reaction mixture (100 μL) contained 50 mM sodium phosphate (pH 7.5), 50 mM NaCl, 20 mM dithiothreitol, 200 μM dabsylated methionine-S-sulfoxide (for MsrA) or methionine-Rsulfoxide (for MsrB), and 200 μg of crude protein. The reaction was conducted at 37 °C for 30 min, after which the reaction product, dabsyl-Met, was analyzed via HPLC as previously described [19]. 2.6. Measurement of hydrogen peroxide in the kidneys A ferric-sensitive dye, xylenol orange (Sigma–Aldrich), was used to determine hydrogen peroxide (H2O2) levels in tissue samples as previously described [20]. 2.7. Measurement of lipid peroxidation in the kidneys Thiobarbituric acid-reactive substance (TBARS; Sigma–Aldrich) levels were measured as an indicator of lipid peroxidation as previously described [21]. Malondialdehyde (MDA), an end product of lipid peroxidation, reacts with thiobarbituric acid to produce an MDA-thiobarbituric acid adduct that is measured in the assay. An extinction coefficient of 153000 M 1 cm 1 at 532 nm for the chromophore was used to calculate TBARS levels. 2.8. Measurement of glutathione levels in the kidneys The ratio of oxidized glutathione (GSSG) to total glutathione (GSH, reduced GSH þ GSSG) was measured using an enzymatic recycling method as previously described [22,23]. The amount of total glutathione was determined by the formation of 5-thio-2nitrobenzoic acid (TBA) from 5,5-dithiobis (2-nitrobenzoic acid, DTNB) [16,22]. GSSG was measured by adding 2-vinylpyridine, which inhibits TBA formation when DTNB and glutathione react with each other [23]. The total GSH and GSSG levels were defined as the change in absorbance at 412 nm for 1 min at 37 °C. 2.9. Measurement of catalase activity in the kidneys Frozen kidney tissues were homogenized in phosphate-buffered saline (pH 7.4) on ice. The kidney lysate was centrifuged at 15,000g for 20 min at 4 °C. The catalase activity in the supernatant was measured in a reaction mixture containing 10 mM sodium phosphate buffer (pH 7.0) and 1.5 M hydrogen peroxide (Sigma– Aldrich) as described previously [24]. The catalase activity was defined as the change in absorbance at 240 nm for 1 min. 2.10. Statistical analysis
2.3. Histology PLP-fixed kidneys were embedded in paraffin and were cut into 4-μm sections. The sections were stained with Sirius red (0.05% in saturated picric acid solution) to detect collagen fibers. Images were captured, and the area of collagen deposition was determined using the i-Solution software (iMT i-Solution, Inc.).
The results were expressed as the means 7SEM. The statistical differences between the groups were analyzed using one-way analysis of variance (ANOVA) followed by Tukey’s least significant difference post hoc test using the SPSS software (version 12.0; SPSS, Inc., Chicago, IL, USA). The between-group differences were considered statistically significant at Po 0.05.
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3. Results 3.1. MsrA gene deletion exacerbates UUO-induced kidney fibrosis To determine the effect of MsrA on the progression of fibrosis, UUO-induced kidney fibrosis was evaluated using Sirius red
MsrA KO
MsrA WT
MsrA KO
Sham UUO
p<0.01 p<0.001
UUO
Sham UUO
Sham UUO
40
p<0.05
Collagen III-positive area (%)
40 Sirius red-positive area (%)
staining for collagen deposition, and immunohistochemical staining for collagen III expression. Sirius red-positive signals markedly increased in the interstitium of the ureterally obstructed kidneys (Fig. 1A). The Sirius red-positive area after UUO was profoundly larger in the MsrA–/– than in the MsrA þ / þ kidneys (Fig. 1B). Consistent with this result, the UUO-induced collagen III expression was
Sham
MsrA WT
203
p<0.001
30 20
p<0.01
10 0
MsrA WT
MsrA KO
30 20 p<0.01
10 0
MsrA WT 14 12
GAPDH Sham UUO 5D MsrA WT
Sham UUO 5D MsrA KO
α-SMA (vs. WT sham)
α-SMA
Sham UUO 5D
10
p<0.01 p<0.001
8 6
MsrA KO
p<0.001
4 2 0
MsrA WT
MsrA KO p<0.05
14
GAPDH Sham
UUO 7D
MsrA WT
Sham
UUO 7D
MsrA KO
12
α-SMA (vs. WT sham)
α-SMA
10
Sham UUO 7D
p<0.001
p<0.01
8 6 4 2 0
MsrA WT
MsrA KO
Fig. 1. Collagen deposition and expression, and ɑ-smooth muscle actin expression in the kidneys after unilateral ureteral obstruction. MsrA þ / þ and MsrA / male mice were subjected to either unilateral ureteral obstruction (UUO) or a sham surgery (n ¼6–7 for each group). The kidneys were excised at 5 or 7 days after the surgery. (A and C) Paraffin-embedded kidney sections were stained with Sirius red (A) or immunohistochemically using an anti-collagen III antibody (C), and images of the cortex were acquired. (B, D) Sirius red- (B) or collagen III-positive areas (D) were quantified as described under Materials and methods. (E and F) The ɑ-smooth muscle actin (ɑ-SMA) protein level, a marker of fibrosis, in the kidneys excised at 5 (E) or 7 (F) days after UUO was determined using Western blotting. The protein band densities were quantified using the ImageJ program (National Institutes of Health, Bethesda, MD, USA). Each protein band density was normalized to that of the loading control GAPDH. The data represent the means 7 SE (n¼6–7).
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mainly detected in the interstitium (Fig. 1C), and the area of collagen III expression was also significantly larger in the MsrA–/– than in the MsrA þ / þ kidneys (Fig. 1D). Increased expression levels of αSMA, a myofibroblast marker protein, were observed in the kidneys at 5 and 7 days after UUO in both mouse groups (Fig. 1E and F). However, the increases in the α-SMA expression were more profound in the MsrA–/– than in the MsrA þ / þ kidneys (Fig. 1E and F). These data indicate that MsrA deficiency exacerbates UUO-induced kidney fibrosis. The plasma creatinine concentrations were not significantly altered after UUO (0.1570.02, 0.1870.01, 0.1770.01, and 0.1570.02 mg/dL in MsrA þ / þ sham, MsrA þ / þ UUO, MsrA–/– sham, and MsrA–/– UUO kidneys, respectively). Consistent with this finding, the contralateral, normal functioning kidney is known to prevent significant UUO-induced renal functional impairment. Body weight/tibia length (g/mm) was similar in the MsrA þ / þ and MsrA–/– kidneys and was not significantly changed after UUO in all mouse groups (1.1070.01, 1.0470.02, 1.2470.06, and 1.1470.03 g/mm in MsrA þ / þ sham, MsrA þ / þ UUO, MsrA–/– sham, and MsrA–/– UUO mice, respectively). 3.2. UUO reduces MsrA and MsrB expression and activity in the kidneys We next investigated whether UUO affected the expression and activity of Msrs in the kidneys. The MsrA protein levels in the MsrA þ / þ kidneys were significantly reduced at 5 days after UUO (Fig. 2A and B). Accordingly, the MsrA activity was significantly decreased after UUO (Fig. 3A). The MsrB1 and MsrB2 protein expression levels were similar in the MsrA–/– and MsrA þ / þ kidneys (Fig. 2A, C, and D). Notably, no MsrB3 protein expression was detected in the kidneys of all mouse groups. The MsrB1 and MsrB2 expression levels were significantly reduced after UUO, similar to those of MsrA (Fig. 2C and D). Furthermore, the reduction of the MsrB1 protein expression level was more profound in the MsrA–/– than in the MsrA þ / þ kidneys (Fig. 2A and C), while no significant difference in MsrB2 reduction was detected between the two kidney groups (Fig. 2A and D). Consistent with the reduced MsrB1 and MsrB2 protein expression levels in the UUO kidneys, the MsrB
MsrA WT Sham UUO
activity significantly decreased in both the MsrA–/– and the MsrA þ / kidneys (Fig. 3B). The MsrA–/– kidneys showed a slightly lower level of MsrB activity than that observed in the MsrA þ / þ kidneys in both the sham and UUO groups. Collectively, these data suggest that UUO reduces the protein expression of MsrA, MsrB1, and MsrB2, thereby reducing Msr activity.
þ
3.3. MsrA gene deletion elevates ROS production and oxidative stress after UUO ROS and oxidative stress levels were measured in MsrA þ / þ and MsrA–/– kidneys to determine whether aggravation of UUO-induced fibrosis by MsrA gene deletion was linked to reduced MsrA antioxidant activity. UUO significantly increased the H2O2 levels in the MsrA þ / þ and MsrA–/– kidneys (Fig. 4A). The UUO-induced elevation in H2O2 levels was more profound in the MsrA–/– than in the MsrA þ / þ kidneys. H2O2 levels were similar in the MsrA þ / þ sham and MsrA / sham kidneys (Fig. 4A). The TBARS level, an index of lipid peroxidation, significantly increased in both the MsrA þ / þ and the MsrA / kidneys after UUO, with the increase in MsrA / being larger than that in the MsrA þ / þ kidneys (Fig. 4B). Consistent with the increase in the TBARS levels, the HNE (a lipid peroxidation product) kidney levels increased after UUO (Fig. 4C). They were significantly higher in the MsrA / than in the MsrA þ / þ kidneys (Fig. 4C and D), while they were not significantly different between the MsrA þ / þ sham and the MsrA / sham kidneys (Fig. 4C and D). Next, we determined the total GSH and GSSG levels in the kidneys. Although UUO significantly reduced the total GSH levels in the kidneys compared to those in the kidneys of the mice that underwent sham surgery (Fig. 4E), the UUO-induced reductions in the total GSH levels were similar in the MsrA / and MsrA þ / þ kidneys. The ratio of GSSG to total GSH also significantly increased after UUO (Fig. 4F), and was more profound in the MsrA / than in the MsrA þ / þ kidneys (Fig. 4F). The total GSH levels and the GSSG/total GSH ratio were similar in the MsrA / sham and MsrA þ / þ sham kidneys (Fig. 4F). Together, these data suggest that MsrA deficiency accelerates GSH oxidation in the kidneys following UUO.
Sham
MsrA KO UUO MsrA MsrB1 MsrB2
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1.6 1.4 1.2
Sham UUO p<0.01
NS p<0.01
1.0 0.8 0.6 0.4 0.2 0.0
MsrA WT
MsrA KO
Fig. 2. The expression of MsrA, MsrB1, and MsrB2 in the kidneys after unilateral ureteral obstruction. MsrA þ / þ and MsrA / male mice were subjected to either unilateral ureteral obstruction (UUO) or a sham surgery (n¼ 6–7 for each group). The kidneys were excised at 5 days after the surgery. (A) The protein levels of MsrA, MsrB1, and MsrB2 were determined using Western blotting. GAPDH was used as a loading control. (B–D) The protein band densities were quantified using the ImageJ program (National Institutes of Health, Bethesda, MD, USA). Each protein band density was normalized to that of the loading control GAPDH. The data represent the means7 SE (n¼6–7). ND, not detectable; NS, not significant.
400 UUO
300 200 100 0
205
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2000 Sham
p<0.05
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ND
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1500 1000 500 0
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0.30
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3.5
GSSG/total GSH
Total GSH (vs. WT sham)
MsrA WT
UUO
HNE (vs. WT sham)
Hydrogen Peroxide (μmole/mg protein)
Fig. 3. Activity of MsrA and MsrB in the kidneys after unilateral ureteral obstruction. MsrA þ / þ and MsrA / male mice were subjected to either unilateral ureteral obstruction (UUO) or a sham surgery (n¼ 6–7 for each group). The kidneys were excised at 5 days after the surgery. The MsrA and MsrB activities were determined as described in the Materials and Methods. The data represent the means 7 SE (n ¼4). ND, not detectable; NS, not significant.
3.0 2.5
Sham UUO
p<0.01
p<0.05
2.0 1.5 1.0 0.5 0.0
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MsrA KO
MsrA WT þ/þ
MsrA KO
/
Fig. 4. The levels of oxidative stress markers in the kidneys after unilateral ureteral obstruction. MsrA and MsrA male mice were subjected to either unilateral ureteral obstruction (UUO) or a sham surgery (n¼ 6–7 for each group). The kidneys were excised at 5 days after the surgery. The levels of hydrogen peroxide (H2O2, A), malondialdehyde (MDA, B), and 4-hydroxynonenal (HNE, C) in the kidneys were determined as described under Materials and methods. (D) The protein band density of HNE in (C) was quantified using the ImageJ program (National Institutes of Health, Bethesda, MD, USA), and was normalized to that of the loading control GAPDH. (E and F) The total glutathione (GSH) levels and the ratio of oxidized GSH (GSSG) to total GSH were determined as described under Materials and methods. The data represent the means7 SE (n¼ 6–7). NS, not significant.
In addition, we determined the levels of catalase and MnSOD in the kidneys. In the sham-operated kidneys, the catalase and MnSOD levels were similar in the MsrA þ / þ and MsrA / kidneys (Fig. 5A). UUO reduced the expression of catalase and MnSOD in both the MsrA þ / þ and the MsrA / kidneys (Fig. 5A). The reduction in catalase expression was more profound in the MsrA / than in the MsrA þ / þ kidneys, while the reduction in MnSOD expression after UUO was similar in both kidney types (Fig. 5A). The
catalase activity reduced more profoundly in the MsrA / than in the MsrA þ / þ kidneys after UUO (Fig. 5B), which was consistent with the respective protein expression data (Fig. 5A). We also determined the Gpx1 protein expression level in the kidneys (Fig. 5C), and found that it was similar in the MsrA þ / þ sham and MsrA / sham kidneys. In contrast, it significantly decreased after UUO in both the MsrA / and the MsrA þ / þ kidneys without showing a significant difference between the two kidney types.
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Fig. 5. The expression levels of catalase, manganese superoxide dismutase, thioredoxin 1/2, and glutathione peroxidase 1, and catalase activity in the kidneys after unilateral ureteral obstruction. MsrA þ / þ and MsrA / male mice were subjected to either unilateral ureteral obstruction (UUO) or a sham surgery (n¼ 5–7 for each group). The kidneys were excised at 5 days after the surgery. (A) The protein levels of catalase and manganese superoxide dismutase (MnSOD) were determined using Western blotting. (B) The catalase activity was determined as described under Materials and methods. (C) The protein levels of thioredoxin 1 (Trx1), thioredoxin 2 (Trx2), and glutathione peroxidase 1 (Gpx1) were analyzed using Western blotting. The protein band densities were quantified using the ImageJ program (National Institutes of Health, Bethesda, MD, USA). Each protein band density was normalized to that of the loading control GAPDH. The data represent the means 7 SE (n ¼5–7). NS, not significant.
Since Trx is the in vivo reductant of Msrs [18], we further determined the Trx expression levels in the kidneys. UUO significantly reduced Trx1 and Trx2 expression in both the MsrA / and the MsrA þ / þ kidneys without showing a significant difference between the two kidney types (Fig. 5C).
fibrosis observed in the MsrA / kidneys is associated with an elevated inflammatory response.
3.4. MsrA gene deletion accelerates the inflammatory response in the kidneys after UUO
Renal fibrosis is characterized by kidney-specific phenotypic changes, such as a loss of epithelial cell polarity causing cell dysfunction, expansion of the interstitial area due to the accumulation of extracellular matrix components, including collagens, and leukocyte infiltration [12,15,25,26]. A growing body of evidence suggests that decreasing oxidative stress and the inflammatory response retards kidney fibrosis in various clinical settings and experimental models [13–15,27]. In the present study, we have demonstrated the involvement of MsrA in the progression of kidney fibrosis, as MsrA deficiency aggravated UUO-induced kidney fibrosis by enhancing oxidative stress and inflammation. Therefore, our data support the findings that oxidative stress and inflammation are pivotal factors in the progression of kidney fibrosis, and suggest that MsrA plays an important role in protecting kidney function in fibrosis-related chronic kidney diseases. A better understanding of the protective role of MsrA in fibrosis may
Since leukocyte infiltration into injured tissue is a pivotal process of fibrosis and is a consequence or cause of oxidative stress [13,20], we examined the infiltration of leukocytes in the kidneys after UUO by immunohistochemical and immunoblot analyses using an antibody against F4/80, a macrophage marker, and against Ly6G, a leukocyte marker, respectively. UUO dramatically increased the number of F4/80-positive cells in the interstitium of the obstructed kidneys (Fig. 6A). The increase in F4/80-positive cells was more profound in the MsrA / than in the MsrA þ / þ kidneys (Fig. 6A). The protein expression level of Ly6G also significantly increased in the kidneys after UUO (Fig. 6B), and was higher in the MsrA / than in the MsrA þ / þ kidneys (Fig. 6B). These results indicate that the increased UUO-induced kidney MsrA WT
4. Discussion
MsrA KO Ly6G
Ly6G (vs. WT sham)
UUO
Sham
GAPDH Sham UUO MsrA WT 6
Sham UUO MsrA KO
Sham
UUO p<0.05
5 4 3
p<0.001
p<0.05
2 1 0
MsrA WT þ/þ
MsrA KO
Fig. 6. The analyses of the inflammatory responses in the kidneys after unilateral ureteral obstruction. MsrA and MsrA / male mice were subjected to either unilateral ureteral obstruction (UUO) or a sham surgery. The kidneys were excised at 5 days after the surgery. (A) Paraffin-embedded kidney sections were immunohistochemically stained using the F4/80 antibody, and images of the cortex were acquired. (B) The protein levels of Ly6G were determined using Western blotting. The protein band densities were quantified using the ImageJ program (National Institutes of Health, Bethesda, MD, USA). Each protein band density was normalized to that of the loading control GAPDH. The data represent the means 7SE (n¼ 6–7).
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further the development of fibrosis treatment strategies. Msr enzymes repair proteins damaged by ROS by reversing the oxidation of methionine residues and scavenging cellular ROS via catalyzing the cyclic oxidation/reduction process of methionine [5,28,29]. In the present study, UUO decreased the expression and, consequently, the activity of MsrA, MsrB1, and MsrB2 in the kidneys. In parallel, UUO elevated the H2O2, TBARS, and HNE levels and the GSSG/GSH ratio in the kidneys. Furthermore, all of these oxidative stress markers were more profoundly elevated in the MsrA / than in the MsrA þ / þ kidneys. These data suggest that UUO impairs the cellular ROS-scavenging capacity and redox balance by affecting the methionine sulfoxide reduction system, particularly MsrA function, leading to increased fibrosis. Consistent with this notion, Maier et al. reported that patients with idiopathic pulmonary fibrosis had a higher level of methionine sulfoxide in their bronchoalveolar lavage fluid [30,31]. In addition, Ogawa et al. reported that the inhibition of MsrA activity by an anti-MsrA antibody elevated systemic sclerosis characterized by fibrosis via increased oxidative stress [32]. We recently reported that MsrA / mice were more susceptible to acute kidney I/R injury than MsrA þ / þ mice [16]. Based on the findings of the present and previous studies, it appears that methionine-S-sulfoxide reduction plays a pivotal role in kidney I/R injury and fibrosis. Therefore, it will be interesting to investigate whether methionine-R-sulfoxide reduction (i.e., MsrB activity) is involved in kidney function. In this study, we found that catalase expression and activity were similar in the MsrA / and MsrA þ / þ sham kidneys. However, UUO reduced the catalase expression and activity in the kidneys, and these reductions were more profound in the MsrA / than in the MsrA þ / þ kidneys. These data suggest that the exacerbated UUO-induced fibrosis in the MsrA / kidneys is associated with decreased catalase activity, and that MsrA is involved in the regulation of catalase expression and activity after UUO. It has been reported that Msr together with GroEL enhanced the recovery of bacterial catalase activity impaired by the oxidation of methionine residues [33]. MsrA may influence GSH production since it is involved in the regulation of the transsulfuration pathway under oxidative stress [16]. Cysteine is a biosynthetic product of the transsulfuration pathway, and is a component of GSH. However, in the present study, the total GSH levels were similar in the MsrA / and the MsrA þ / þ kidneys even after UUO, although the GSSG/total GSH ratio, a representative redox status marker, was much higher in the MsrA / than in the MsrA þ / þ kidneys. These data suggest that increased UUO-induced oxidative stress in the MsrA / kidneys is associated with the antioxidant scavenging by MsrA but not with MsrA’s modulation of GSH biosynthesis. We further found that MsrA deficiency did not affect the UUO-induced Gpx1 protein expression, suggesting that the elevated UUO-induced oxidative stress in MsrA / kidneys might not be associated with Gpx1’s antioxidant function. Oxidative stress induces kidney tissue damage and accelerates leukocyte infiltration into the kidney tissue. This triggers ROS production, thereby causing further tissue and cell damage [20,34,35]. A number of reports have demonstrated that the infiltration of inflammatory cells, including T-cells and macrophages, plays a causative role in UUO-induced kidney fibrosis [14,36–38]. In our previous studies, we confirmed that inhibitors of ROSproducing enzymes reduced leukocyte infiltration into I/R- and UUO-injured kidneys and prevented kidney fibrosis [12,13,20]. We also recently reported that I/R-induced inflammatory responses in the kidneys were accelerated by MsrA gene depletion [16]. In addition, the present study showed that UUO elevated Ly6G protein expression and the number of F4/80-positive cells in the kidneys, and that these inflammatory responses were enhanced by MsrA deficiency.
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In conclusion, to the best of our knowledge, the present study was the first to demonstrate that UUO caused a reduction in Msr enzyme function, which increased oxidative stress, inflammatory responses, and fibrosis. Furthermore, MsrA deficiency induced an increase in inflammatory responses in the kidneys under oxidative stress, leading to aggravation of kidney fibrosis. These data suggest that MsrA plays a protective role in the progression of UUO-induced kidney fibrosis via suppression of fibrotic responses caused by oxidative stress and inflammation.
Author disclosure statement No competing financial interests exist.
Acknowledgments This study was supported by the National Research Foundation of Korea (NRF) grants funded by the Korean government (NRF2014R1A11049549 to K.M.P. and MSIP 2014R1A5A2010008 to J.I.K.).
References [1] H.Y. Kim, The methionine sulfoxide reduction system: selenium utilization and methionine sulfoxide reductase enzymes and their functions, Antioxid. Redox Signal. 19 (2013) 958–969. [2] N. Brot, L. Weissbach, J. Werth, H. Weissbach, Enzymatic reduction of proteinbound methionine sulfoxide, Proc. Natl. Acad. Sci. USA 78 (1981) 2155–2158. [3] R. Grimaud, B. Ezraty, J.K. Mitchell, D. Lafitte, C. Briand, Derrick, P. J.; Barras, F. Repair of oxidized proteins. Identification of a new methionine sulfoxide reductase, J. Biol. Chem. 276 (2001) 48915–48920. [4] J. Moskovitz, B.S. Berlett, J.M. Poston, E.R. Stadtman, The yeast peptide-methionine sulfoxide reductase functions as an antioxidant in vivo, . Proc. Natl. Acad. Sci. USA 94 (1997) 9585–9589. [5] R.L. Levine, L. Mosoni, B.S. Berlett, E.R. Stadtman, Methionine residues as endogenous antioxidants in proteins, Proc. Natl. Acad. Sci. USA 93 (1996) 15036–15040. [6] J.R. Erickson, M.L. Joiner, X. Guan, W. Kutschke, J. Yang, C.V. Oddis, R.K. Bartlett, J.S. Lowe, S.E. O'Donnell, N. Aykin-Burns, M.C. Zimmerman, K. Zimmerman, A. J. Ham, R.M. Weiss, D.R. Spitz, M.A. Shea, R.J. Colbran, P.J. Mohler, M. E. Anderson, A dynamic pathway for calcium-independent activation of CaMKII by methionine oxidation, Cell 133 (2008) 462–474. [7] B.C. Lee, Z. Peterfi, F.W. Hoffmann, R.E. Moore, A. Kaya, A. Avanesov, L. Tarrago, Y. Zhou, E. Weerapana, D.E. Fomenko, P.R. Hoffmann, V.N. Gladyshev, MsrB1 and MICALs regulate actin assembly and macrophage function via reversible stereoselective methionine oxidation, Mol. Cell 51 (2013) 397–404. [8] J. Moskovitz, Methionine sulfoxide reductases: ubiquitous enzymes involved in antioxidant defense, protein regulation, and prevention of aging-associated diseases, Biochim. Biophys. Acta 1703 (2005) 213–219. [9] Y. Kimura, H. Kimura, Hydrogen sulfide protects neurons from oxidative stress, FASEB J 18 (2004) 1165–1167. [10] J. Moskovitz, N.A. Jenkins, D.J. Gilbert, N.G. Copeland, F. Jursky, H. Weissbach, N. Brot, Chromosomal localization of the mammalian peptide-methionine sulfoxide reductase gene and its differential expression in various tissues, Proc. Natl. Acad. Sci. USA 93 (1996) 3205–3208. [11] D.E. Fomenko, S.V. Novoselov, S.K. Natarajan, B.C. Lee, A. Koc, B.A. Carlson, T. H. Lee, H.Y. Kim, D.L. Hatfield, V.N. Gladyshev, MsrB1 (methionine-R-sulfoxide reductase 1) knock-out mice: roles of MsrB1 in redox regulation and identification of a novel selenoprotein form, J. Biol. Chem. 284 (2009) 5986–5993. [12] H.S. Jang, J.I. Kim, K.J. Jung, J. Kim, K.H. Han, K.M. Park, Bone marrow-derived cells play a major role in kidney fibrosis via proliferation and differentiation in the infiltrated site, Biochim. Biophys. Acta 1832 (2013) 817–825. [13] H.S. Jang, J.I. Kim, J. Kim, Y.K. Na, J.W. Park, K.M. Park, Bone marrow derived cells and reactive oxygen species in hypertrophy of contralateral kidney of transient unilateral renal ischemia-induced mouse, Free Radic. Res. 46 (2012) 903–911. [14] R.L. Chevalier, M.S. Forbes, B.A. Thornhill, Ureteral obstruction as a model of renal interstitial fibrosis and obstructive nephropathy, Kidney Int. 75 (2009) 1145–1152. [15] R.L. Chevalier, B.A. Thornhill, M.S. Forbes, S.C. Kiley, Mechanisms of renal injury and progression of renal disease in congenital obstructive nephropathy, Pediatr. Nephrol. 25 (2010) 687–697. [16] J.I. Kim, S.H. Choi, K.J. Jung, E. Lee, H.Y. Kim, K.M. Park, Protective role of methionine sulfoxide reductase A against ischemia/reperfusion injury in mouse kidney and its involvement in the regulation of trans-sulfuration pathway, Antioxid. Redox Signal. 18 (2013) 2241–2250.
208
J.I. Kim et al. / Free Radical Biology and Medicine 89 (2015) 201–208
[17] J. Moskovitz, S. Bar-Noy, W.M. Williams, J. Requena, B.S. Berlett, E.R. Stadtman, Methionine sulfoxide reductase (MsrA) is a regulator of antioxidant defense and lifespan in mamma, Proc. Natl. Acad. Sci. USA 98 (2001) 12920–12925. [18] H.Y. Kim, J.R. Kim, Thioredoxin as a reducing agent for mammalian methionine sulfoxide reductases B lacking resolving cysteine, Biochem. Biophys. Res. Commun. 371 (2008) 490–494. [19] H.Y. Kim, Glutaredoxin serves as a reductant for methionine sulfoxide reductases with or without resolving cysteine, Acta Biochim. Biophys. Sin. (Shangha 44 (2012) 623–627. [20] J. Kim, Y.M. Seok, K.J. Jung, K.M. Park, Reactive oxygen species/oxidative stress contributes to progression of kidney fibrosis following transient ischemic injury in mice, Am. J. Physiol. Renal Physiol 297 (2009) F461–F470. [21] J.A. Buege, S.D. Aust, Microsomal lipid peroxidation, Methods Enzymol. 52 (1978) 302–310. [22] T.P. Akerboom, H. Sies, Assay of glutathione, glutathione disulfide, and glutathione mixed disulfides in biological samples, Methods Enzymol. 77 (1981) 373–382. [23] M.E. Anderson, Determination of glutathione and glutathione disulfide in biological samples, Methods Enzymol. 113 (1985) 548–555. [24] R.F. Beers Jr, I.W. Sizer, A spectrophotometric method for measuring the breakdown of hydrogen peroxide by catalase, J. Biol. Chem. 195 (1952) 133–140. [25] O. Skalli, P. Ropraz, A. Trzeciak, G. Benzonana, D. Gillessen, G. Gabbiani, A monoclonal antibody against alpha-smooth muscle actin: a new probe for smooth muscle differentiation, J. Cell Biol. 103 (1986) 2787–2796. [26] H.S. Jang, J. Kim, K.Y. Kim, J.I. Kim, M.H. Cho, K.M. Park, Previous ischemia and reperfusion injury results in resistance of the kidney against subsequent ischemia and reperfusion insult in mice; a role for the Akt signal pathway, Nephrol. Dial. Transplant. 27 (2012) 3762–3770. [27] K.J. Jung, H.S. Jang, J.I. Kim, S.J. Han, J.W. Park, K.M. Park, Involvement of hydrogen sulfide and homocysteine transsulfuration pathway in the progression of kidney fibrosis after ureteral obstruction, Biochim. Biophys. Acta 1832 (2013) 1989–1997. [28] E.R. Stadtman, J. Moskovitz, B.S. Berlett, R.L. Levine, Cyclic oxidation and reduction of protein methionine residues is an important antioxidant
mechanism, Mol. Cell. Biochem. 234-235 (2002) 3–9. [29] S. Luo, R.L. Levine, Methionine in proteins defends against oxidative stress, FASEB J 23 (2009) 464–472. [30] K. Maier, L. Leuschel, U. Costabel, Increased levels of oxidized methionine residues in bronchoalveolar lavage fluid proteins from patients with idiopathic pulmonary fibrosis, Am. Rev. Respir. Dis. 143 (1991) 271–274. [31] K.L. Maier, L. Leuschel, U. Costabel, Increased oxidized methionine residues in BAL fluid proteins in acute or chronic bronchitis, Eur. Respir. J. 5 (1992) 651–658. [32] F. Ogawa, K. Shimizu, T. Hara, E. Muroi, K. Komura, M. Takenaka, M. Hasegawa, M. Fujimoto, K. Takehara, S. Sato, Autoantibody against one of the antioxidant repair enzymes, methionine sulfoxide reductase A, in systemic sclerosis: association with pulmonary fibrosis and vascular damage, Arch. Dermatol. Res. 302 (2010) 27–35. [33] M. Mahawar, V. Tran, J.S. Sharp, R.J. Maier, Synergistic roles of Helicobacter pylori methionine sulfoxide reductase and GroEL in repairing oxidant-damaged catalase, J. Biol. Chem. 286 (2011) 19159–19169. [34] J. Kim, H.S. Jang, K.M. Park, Reactive oxygen species generated by renal ischemia and reperfusion trigger protection against subsequent renal ischemia and reperfusion injury in mice, Am. J. Physiol. Renal Physiol 298 (2010) F158–F166. [35] K.M. Park, J.I. Kim, Y. Ahn, A.J. Bonventre, J.V. Bonventre, Testosterone is responsible for enhanced susceptibility of males to ischemic renal injury, J. Biol. Chem. 279 (2004) 52282–52292. [36] T.A. Wynn, Cellular and molecular mechanisms of fibrosis, J. Pathol. 214 (2008) 199–210. [37] K. Kitagawa, T. Wada, K. Furuichi, H. Hashimoto, Y. Ishiwata, M. Asano, M. Takeya, W.A. Kuziel, K. Matsushima, N. Mukaida, H. Yokoyama, Blockade of CCR2 ameliorates progressive fibrosis in kidney, Am. J. Pathol. 165 (2004) 237–246. [38] M. Niedermeier, B. Reich, M. Rodriguez Gomez, A. Denzel, K. Schmidbauer, N. Gobel, Y. Talke, F. Schweda, M. Mack, CD4 þ T cells control the differentiation of Gr1 þ monocytes into fibrocytes, Proc. Natl. Acad. Sci. USA 106 (2009) 17892–17897.