NDT Advance Access published July 3, 2015 Nephrol Dial Transplant (2015) 0: 1–10 doi: 10.1093/ndt/gfv226
Original Article Hydrogen sulfide accelerates the recovery of kidney tubules after renal ischemia/reperfusion injury
1
Department of Anatomy, Cardiovascular Research Institute and BK21 Plus, Kyungpook National University School of Medicine, Daegu 700-422,
Republic of Korea, 2Department of Molecular Medicine and MRC, College of Medicine, Keimyung University, Daegu 704-701, Republic of Korea and 3School of Life Sciences and Biotechnology, College of Natural Sciences, Kyungpook National University, Taegu 702-701, Republic of Korea
Correspondence and offprint requests to: Kwon Moo Park; E-mail: [email protected]
A B S T R AC T Background. Progression of acute kidney injury to chronic kidney disease (CKD) is associated with inadequate recovery of damaged kidney. Hydrogen sulfide (H2S) regulates a variety of cellular signals involved in cell death, differentiation and proliferation. This study aimed to identify the role of H2S and its producing enzymes in the recovery of kidney following ischemia/reperfusion (I/R) injury. Methods. Mice were subjected to 30 min of bilateral renal ischemia. Some mice were administered daily NaHS, an H2S donor, and propargylglycine (PAG), an inhibitor of the H2Sproducing enzyme cystathionine gamma-lyase (CSE), during the recovery phase. Cell proliferation was assessed via 50 -bromo20 -deoxyuridine (BrdU) incorporation assay. Results. Ischemia resulted in decreases in CSE and cystathionine beta-synthase (CBS) expression and activity, and H2S level in the kidney. These decreases did not return to sham level until 8 days after ischemia when kidney had fibrotic lesions. NaHS administration to I/R-injured mice accelerated the recovery of renal function and tubule morphology, whereas PAG delayed that. Furthermore, PAG increased mortality after ischemia. NaHS administration to I/R-injured mice accelerated tubular cell proliferation, whereas it inhibited interstitial cell proliferation. In addition, NaHS treatment reduced post-I/R superoxide formation, lipid peroxidation, level of GSSG/GSH and Nox4 expression, whereas it increased catalase and MnSOD expression. Conclusions. Our findings demonstrate that H2S accelerates the recovery of I/R-induced kidney damage, suggesting that the H2S-producing transsulfuration pathway plays an important role in kidney repair after acute injury. © The Author 2015. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved.
Keywords: CBS, CSE, hydrogen sulfide, ischemia, reactive oxygen species, regeneration
INTRODUCTION Hydrogen sulfide (H2S) plays many important roles as a signaling molecule and antioxidant in mammalian cells, including kidney tubular epithelial cells [1, 2]. H2S is synthesized together with cysteine, a component of glutathione, by transsulfuration pathways; two pyridoxal-50 -phosphate-dependent enzymes, cystathionine β-synthase (CBS) and cystathionine γ-lyase (CSE), and a pyridoxal-50 -phosphate-independent enzyme, 3-mercaptopyruvate sulfurtransferase (3-MST) [3]. CBS and CSE are enriched in the kidney [4, 5]. Defects in transsulfuration pathways result in reduced glutathione, which is the most abundant antioxidant molecule in mammalian cells [6]. It has been reported that CSE gene deficiency in mice increases kidney susceptibility to ischemia and reperfusion (I/R) injury [7]. Ischemia/reperfusion in the kidney induces tubular epithelial cell disruption, resulting in renal functional impairment. Kidney tubular epithelial cells injured by I/R can be restored through proliferation within several days after renal blood flow recovery. If recovery from kidney damage is delayed or insufficient, the kidney progresses to chronic kidney disease (CKD), which is characterized by fibrotic lesions, so it is very important to find recovery accelerating-factor from ischemic injury. The inadequate recovery after injury is associated with various factors, including reactive oxygen species (ROS). Recently, we found that tissue ROS generated by I/R are sustained for at least 16 days after I/R in the mouse kidney, whereas plasma creatinine (PCr) and blood urea nitrogen levels returned to 1
Downloaded from http://ndt.oxfordjournals.org/ at Univ. of Massachusetts/Amherst Library on July 5, 2015
Sang Jun Han1, Jee In Kim2, Jeen-Woo Park3 and Kwon Moo Park1
ORIGINAL ARTICLE
M AT E R I A L S A N D M E T H O D S Animal preparation Eight-week-old C57BL/6 male mice (Koatech, Gyounggido, Korea) were used in experiments. All studies were conducted in accordance with the guidelines provided by the Animal Care and Use Committee of Kyungpook National University. For the induction of ischemia, kidneys were exposed through flank incisions, and the renal pedicles were clamped completely for 30 min using microaneurysm clamps while the mice were anesthetized with pentobarbital sodium (50 mg/kg BW). The same procedure, except for the clamping of the renal pedicle, was used during the sham operation. Mice received daily intraperitoneal administration of sodium hydrosulfide hydrate (NaHS; 500 µg/kg BW: Sigma, St. Louis, MO), an H2S donor, DL-Propargylglycine (PAG; 50 mg/kg BW: Sigma), an inhibitor of CSE, or 0.9% NaCl (vehicle), beginning 2 days after ischemia until the end of the experiment. Chosen NaHS and PAG dosages were based on our and other previous studies [1, 10, 11]. There were no significant changes of body weight, behavior and fur condition after NaHS and PAG administrations compared with vehicle-treated animals. NaHS and PAG were prepared freshly before use. Each experimental animal group included more than five mice. For use in biochemical and histological studies, kidneys were frozen in liquid nitrogen immediately after extraction or perfusion fixing, respectively. Plasma creatinine concentration Blood was taken from the retrobulbar vein plexus at the times indicated in the figures. Plasma creatinine concentrations were measured using a Vitros 250 (Johnson & Johnson, Rochester, NY, USA). BrdU-incorporation experiment To detect proliferating cells, 5-bromo-20 -deoxyuridine (BrdU; 50 mg/kg body weight; Sigma) was intraperitoneally
2
administered daily into mice beginning 2 days after either ischemia or sham operation until mice were sacrificed. The BrdU-incorporation experiment is commonly utilized to detect proliferating cells in vivo, as it is incorporated into the newly synthesized DNA of replicating cells (during the S phase of the cell cycle) instead of thymidine [12]. Histology Kidneys were perfusion-fixed with PLP (4% paraformaldehyde, 75 mM L-lysine, 10 mM sodium periodate; Sigma). Kidney paraffin sections were stained with PAS stain according to a standard protocol. To determine morphological tubular damage, kidney damage in a PAS-stained kidney section was scored at 10 fields in the outer medulla per kidney, as previously described [13]. Western blot analysis Western blot was performed as previously described [14]. Western blot was performed using proliferating cell nuclear antigen (PCNA; 1:10 000; DAKO, Glostrup, Denmark), catalase (1:10 000; Fitzgerald, Concord, MA, USA), manganese superoxide dismutase (MnSOD; 1:2000; Calbiochem, La Jolla, CA, USA), cystathionine β-synthase (CBS; 1:2000; Santa Cruz Biotechnology, Santa Cruz, CA, USA), cystathionine γ-lyase (CSE; 1:1000; Santa Cruz Biotechnology), 4-hydroxynonenal (HNE; 1:10 000; Abcam, Cambridge, UK), phospho-ERK ( p-ERK; 1:5000; Cell Signaling, Danvers, MA, USA), total-ERK (t-ERK; 1:5000; Cell Signaling), NADPH oxidase 4 (Nox4; 1:4000; Santa Cruz Biotechnology) and GAPDH (1:20 000; NOVUS, Littleton, CO, USA) antibodies. Sirius red staining To evaluate collagen deposition in the kidney, kidney paraffin sections were Sirius red-stained according to a standard protocol. Briefly, de-waxing paraffin kidney sections were incubated with picrosirius red for 1 h, washed by acidified water (0.5% glacial acetic acid) two times and rehydrated in alcohol. Sirius-red-positive area was measured using an image analysis program (i-solution, IMT, Korea). Immunohistochemical staining Immunohistochemical staining was performed using antiBrdU (1:100; Abcam) antibody as described previously [8]. Sections were observed under a Leica microscope (DM2500; Wetzlar, Germany). Measurement of H2S levels and H2S-producing capacity in the kidney H2S levels in kidney tissue were determined as previously described with slight modifications [1]. Briefly, kidney was 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 r.p.m. at 4°C. The supernatant was taken, and
S. J. Han et al.
Downloaded from http://ndt.oxfordjournals.org/ at Univ. of Massachusetts/Amherst Library on July 5, 2015
almost normal within the same time frame [8]. In addition, we found that the removal of ROS in recovering I/R-injured kidney retarded the progression of fibrosis and that ROS performed different roles in the restoration of damaged tubular epithelial cells and interstitial cells depending on cell type and ROS concentration [9]. Recently, we found that I/R injury in the kidney resulted in the dysfunction of H2S-producing transsulfuration pathways [2] and fibrotic changes of kidney after unilateral ureteral obstruction [1] was improved by supplementation with an H2S donor. However, the role of the transsulfuration system in the recovery process of the kidney after I/R injury remains to be defined. Therefore, in this present study, we investigated the role of H2S and its synthetic pathway, the transsulfuration pathway, in the repair of kidney tubular epithelial cells after I/R injury. Here, we report that H2S accelerates the recovery of I/R-injured kidney by accelerating the regeneration of tubular epithelial cells with an increased antioxidant effect. These findings indicate that H2S plays a critical role in the recovery from acute kidney injury (AKI), suggesting that H2S pathway is highly associated with the progression of AKI to CKD.
Measurement of the oxidized glutathione (GSSG) to total glutathione ratio in the kidney The ratio of oxidized glutathione (GSSG) to total glutathione (GSH; reduced glutathione + GSSG) was measured using an
Statistical analysis Results were expressed as means ± SEM. Statistical differences among groups were calculated using Students t-tests and one-way analysis of variance for comparison between two groups. A P-value of
Original Article Hydrogen sulfide accelerates the recovery of kidney tubules after renal ischemia/reperfusion injury
1
Department of Anatomy, Cardiovascular Research Institute and BK21 Plus, Kyungpook National University School of Medicine, Daegu 700-422,
Republic of Korea, 2Department of Molecular Medicine and MRC, College of Medicine, Keimyung University, Daegu 704-701, Republic of Korea and 3School of Life Sciences and Biotechnology, College of Natural Sciences, Kyungpook National University, Taegu 702-701, Republic of Korea
Correspondence and offprint requests to: Kwon Moo Park; E-mail: [email protected]
A B S T R AC T Background. Progression of acute kidney injury to chronic kidney disease (CKD) is associated with inadequate recovery of damaged kidney. Hydrogen sulfide (H2S) regulates a variety of cellular signals involved in cell death, differentiation and proliferation. This study aimed to identify the role of H2S and its producing enzymes in the recovery of kidney following ischemia/reperfusion (I/R) injury. Methods. Mice were subjected to 30 min of bilateral renal ischemia. Some mice were administered daily NaHS, an H2S donor, and propargylglycine (PAG), an inhibitor of the H2Sproducing enzyme cystathionine gamma-lyase (CSE), during the recovery phase. Cell proliferation was assessed via 50 -bromo20 -deoxyuridine (BrdU) incorporation assay. Results. Ischemia resulted in decreases in CSE and cystathionine beta-synthase (CBS) expression and activity, and H2S level in the kidney. These decreases did not return to sham level until 8 days after ischemia when kidney had fibrotic lesions. NaHS administration to I/R-injured mice accelerated the recovery of renal function and tubule morphology, whereas PAG delayed that. Furthermore, PAG increased mortality after ischemia. NaHS administration to I/R-injured mice accelerated tubular cell proliferation, whereas it inhibited interstitial cell proliferation. In addition, NaHS treatment reduced post-I/R superoxide formation, lipid peroxidation, level of GSSG/GSH and Nox4 expression, whereas it increased catalase and MnSOD expression. Conclusions. Our findings demonstrate that H2S accelerates the recovery of I/R-induced kidney damage, suggesting that the H2S-producing transsulfuration pathway plays an important role in kidney repair after acute injury. © The Author 2015. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved.
Keywords: CBS, CSE, hydrogen sulfide, ischemia, reactive oxygen species, regeneration
INTRODUCTION Hydrogen sulfide (H2S) plays many important roles as a signaling molecule and antioxidant in mammalian cells, including kidney tubular epithelial cells [1, 2]. H2S is synthesized together with cysteine, a component of glutathione, by transsulfuration pathways; two pyridoxal-50 -phosphate-dependent enzymes, cystathionine β-synthase (CBS) and cystathionine γ-lyase (CSE), and a pyridoxal-50 -phosphate-independent enzyme, 3-mercaptopyruvate sulfurtransferase (3-MST) [3]. CBS and CSE are enriched in the kidney [4, 5]. Defects in transsulfuration pathways result in reduced glutathione, which is the most abundant antioxidant molecule in mammalian cells [6]. It has been reported that CSE gene deficiency in mice increases kidney susceptibility to ischemia and reperfusion (I/R) injury [7]. Ischemia/reperfusion in the kidney induces tubular epithelial cell disruption, resulting in renal functional impairment. Kidney tubular epithelial cells injured by I/R can be restored through proliferation within several days after renal blood flow recovery. If recovery from kidney damage is delayed or insufficient, the kidney progresses to chronic kidney disease (CKD), which is characterized by fibrotic lesions, so it is very important to find recovery accelerating-factor from ischemic injury. The inadequate recovery after injury is associated with various factors, including reactive oxygen species (ROS). Recently, we found that tissue ROS generated by I/R are sustained for at least 16 days after I/R in the mouse kidney, whereas plasma creatinine (PCr) and blood urea nitrogen levels returned to 1
Downloaded from http://ndt.oxfordjournals.org/ at Univ. of Massachusetts/Amherst Library on July 5, 2015
Sang Jun Han1, Jee In Kim2, Jeen-Woo Park3 and Kwon Moo Park1
ORIGINAL ARTICLE
M AT E R I A L S A N D M E T H O D S Animal preparation Eight-week-old C57BL/6 male mice (Koatech, Gyounggido, Korea) were used in experiments. All studies were conducted in accordance with the guidelines provided by the Animal Care and Use Committee of Kyungpook National University. For the induction of ischemia, kidneys were exposed through flank incisions, and the renal pedicles were clamped completely for 30 min using microaneurysm clamps while the mice were anesthetized with pentobarbital sodium (50 mg/kg BW). The same procedure, except for the clamping of the renal pedicle, was used during the sham operation. Mice received daily intraperitoneal administration of sodium hydrosulfide hydrate (NaHS; 500 µg/kg BW: Sigma, St. Louis, MO), an H2S donor, DL-Propargylglycine (PAG; 50 mg/kg BW: Sigma), an inhibitor of CSE, or 0.9% NaCl (vehicle), beginning 2 days after ischemia until the end of the experiment. Chosen NaHS and PAG dosages were based on our and other previous studies [1, 10, 11]. There were no significant changes of body weight, behavior and fur condition after NaHS and PAG administrations compared with vehicle-treated animals. NaHS and PAG were prepared freshly before use. Each experimental animal group included more than five mice. For use in biochemical and histological studies, kidneys were frozen in liquid nitrogen immediately after extraction or perfusion fixing, respectively. Plasma creatinine concentration Blood was taken from the retrobulbar vein plexus at the times indicated in the figures. Plasma creatinine concentrations were measured using a Vitros 250 (Johnson & Johnson, Rochester, NY, USA). BrdU-incorporation experiment To detect proliferating cells, 5-bromo-20 -deoxyuridine (BrdU; 50 mg/kg body weight; Sigma) was intraperitoneally
2
administered daily into mice beginning 2 days after either ischemia or sham operation until mice were sacrificed. The BrdU-incorporation experiment is commonly utilized to detect proliferating cells in vivo, as it is incorporated into the newly synthesized DNA of replicating cells (during the S phase of the cell cycle) instead of thymidine [12]. Histology Kidneys were perfusion-fixed with PLP (4% paraformaldehyde, 75 mM L-lysine, 10 mM sodium periodate; Sigma). Kidney paraffin sections were stained with PAS stain according to a standard protocol. To determine morphological tubular damage, kidney damage in a PAS-stained kidney section was scored at 10 fields in the outer medulla per kidney, as previously described [13]. Western blot analysis Western blot was performed as previously described [14]. Western blot was performed using proliferating cell nuclear antigen (PCNA; 1:10 000; DAKO, Glostrup, Denmark), catalase (1:10 000; Fitzgerald, Concord, MA, USA), manganese superoxide dismutase (MnSOD; 1:2000; Calbiochem, La Jolla, CA, USA), cystathionine β-synthase (CBS; 1:2000; Santa Cruz Biotechnology, Santa Cruz, CA, USA), cystathionine γ-lyase (CSE; 1:1000; Santa Cruz Biotechnology), 4-hydroxynonenal (HNE; 1:10 000; Abcam, Cambridge, UK), phospho-ERK ( p-ERK; 1:5000; Cell Signaling, Danvers, MA, USA), total-ERK (t-ERK; 1:5000; Cell Signaling), NADPH oxidase 4 (Nox4; 1:4000; Santa Cruz Biotechnology) and GAPDH (1:20 000; NOVUS, Littleton, CO, USA) antibodies. Sirius red staining To evaluate collagen deposition in the kidney, kidney paraffin sections were Sirius red-stained according to a standard protocol. Briefly, de-waxing paraffin kidney sections were incubated with picrosirius red for 1 h, washed by acidified water (0.5% glacial acetic acid) two times and rehydrated in alcohol. Sirius-red-positive area was measured using an image analysis program (i-solution, IMT, Korea). Immunohistochemical staining Immunohistochemical staining was performed using antiBrdU (1:100; Abcam) antibody as described previously [8]. Sections were observed under a Leica microscope (DM2500; Wetzlar, Germany). Measurement of H2S levels and H2S-producing capacity in the kidney H2S levels in kidney tissue were determined as previously described with slight modifications [1]. Briefly, kidney was 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 r.p.m. at 4°C. The supernatant was taken, and
S. J. Han et al.
Downloaded from http://ndt.oxfordjournals.org/ at Univ. of Massachusetts/Amherst Library on July 5, 2015
almost normal within the same time frame [8]. In addition, we found that the removal of ROS in recovering I/R-injured kidney retarded the progression of fibrosis and that ROS performed different roles in the restoration of damaged tubular epithelial cells and interstitial cells depending on cell type and ROS concentration [9]. Recently, we found that I/R injury in the kidney resulted in the dysfunction of H2S-producing transsulfuration pathways [2] and fibrotic changes of kidney after unilateral ureteral obstruction [1] was improved by supplementation with an H2S donor. However, the role of the transsulfuration system in the recovery process of the kidney after I/R injury remains to be defined. Therefore, in this present study, we investigated the role of H2S and its synthetic pathway, the transsulfuration pathway, in the repair of kidney tubular epithelial cells after I/R injury. Here, we report that H2S accelerates the recovery of I/R-injured kidney by accelerating the regeneration of tubular epithelial cells with an increased antioxidant effect. These findings indicate that H2S plays a critical role in the recovery from acute kidney injury (AKI), suggesting that H2S pathway is highly associated with the progression of AKI to CKD.
Measurement of the oxidized glutathione (GSSG) to total glutathione ratio in the kidney The ratio of oxidized glutathione (GSSG) to total glutathione (GSH; reduced glutathione + GSSG) was measured using an
Statistical analysis Results were expressed as means ± SEM. Statistical differences among groups were calculated using Students t-tests and one-way analysis of variance for comparison between two groups. A P-value of
