Int J Clin Exp Pathol 2015;8(3):2680-2689 www.ijcep.com /ISSN:1936-2625/IJCEP0005501
Original Article Inhalation of hydrogen gas ameliorates glyoxylate-induced calcium oxalate deposition and renal oxidative stress in mice Zhongjiang Peng1*, Wei Chen1*, Li Wang1, Zhouheng Ye2, Songyan Gao3, Xuejun Sun2, Zhiyong Guo1 Department of Nephrology, Changhai Hospital, Second Military Medical University, Shanghai, China; 2Department of Aviation Medicine, Faculty of Naval Medicine, Second Military Medical University, Shanghai, China; 3 School of Pharmacy, Second Military Medical University, Shanghai, China. *Equal contributors. 1
Received January 4, 2015; Accepted February 26, 2015; Epub March 1, 2015; Published March 15, 2015 Abstract: The aim of this study is to evaluate the protective effect and underlying mechanism of hydrogen gas (H2) to glyoxylate induced renal calcium oxalate (CaOx) crystal deposition in mice. In present work, rodent renal CaOx crystal deposition model was introduced by intra-abdominal injection of glyoxylate (100 mg/kg/d) for 5 days. Two days before administration of glyoxylate, inhalation of H2 for 30 min per day was initiated and continued for 7 days. By the end of the study, the samples of 24 hours urine, serum and renal tissue were collected for biochemical and pathological assay. According to levels of urine calcium excretion, renal calcium deposition, a serum excretion of kidney injury molecule-1 (KIM-1) assay and a TUNEL assay, inhalation of H2 could successfully decrease the CaOx crystallizations and protect against renal injury. Crystal deposition in the kidneys is associated with oxidative stress, which was indicated by increased levels of renal malondialdehyde (MDA) and 8-hydroxydeoxyguanosine (8-OHdG) and decreased activities of superoxide dismutase (SOD), glutathione (GSH) and catalase (CAT). These effects were reversed by a high-dose H2 pretreatment. The renal expressions of osteopontin (OPN), CD44, monocyte chemoattractant protein-1 (MCP-1) and interleukin-10 (IL-10) were markedly increased in glyoxylate-treated mice, and H2 significantly attenuated the increase of OPN, CD44 and MCP-1 but upregulated the expression of IL-10. Our findings demonstrate that inhalation of H2 reduces renal crystallization, renal oxidative injury and inflammation and it may be a candidate agent with few adverse effects for prevention of nephrolithiasis. Keywords: Hydrogen gas, calcium oxalate, kidney injury molecule-1, glyoxylate, oxidative stress
Introduction Stone disease, also known as urolithiasis, is becoming a major public health burden worldwide [1, 2]. Nephrolithiasis, deposition of crystal in the kidneys and the formation of kidney stones, is a common urological disorder. Calcium oxalate (CaOx) is the most common type of nephrolithiasis; hyperoxaluria is the major risk factor [3]. Nephrolithiasis formation consists of several stages including supersaturation, nucleation, growth, aggregation, and retention within renal tubules. Although the specific mechanism is not clear, there is accumulating evidence that crystal deposition in the kidneys is associated with reactive oxygen species (ROS) production, oxidative stress development, inflammation and renal tubule injury [4, 5]. Anti-oxidative treatment has become an
experimental strategy of anti-nephrolithiasis, such as vitamin E [6], vitamin C [7], and some traditional medicinal herbs [8, 9]. Nevertheless, no such substance could be translated into clinical practice because some side-effects were present [7]. Thus, developing an effective alternative with few adverse effects is an urgent need in this field. Hydrogen is considered an inert gas and has been used in medical applications to prevent decompression sickness in deep divers [10]. In 2007, it was reported that an inhalation of H2 has antioxidant and anti-apoptotic properties that can protect the brain against ischemia/ reperfusion injury by selectively neutralizing hydroxyl radicals [11]. This report aroused considerable interest worldwide. Thus far, the therapeutic effects of molecular hydrogen on vari-
Hydrogen ameliorates glyoxylate induced kidney injury ous diseases, including kidney diseases [12, 13], have been tested mostly regarding its antioxidation capability [14], but also its antiinflammation [15] and anti-apoptosis [16] capabilities. Compared with traditional antioxidants, hydrogen is a small molecule that can easily dissipate throughout the body and cells, and it is sufficiently mild that it does not disturb metabolic oxidation-reduction reactions or disrupt ROS mediated cell signaling. Thus, it may be a safe and effective antioxidant for nephrolithiasis. To date, most inhaled hydrogen gas research has focused on a low dose (2%-4%), but the effect of inhaling a high dose has rarely been tested. In theory, high-dose hydrogen might have good effects and can be easily used to explore the dose-dependent response of hydrogen with increasing dosage. In this study, we hypothesized that pretreatment with a high dose of hydrogen gas will attenuate renal crystallization by anti-oxidation, anti-inflammation, and protection from renal tubule injury.
intra-abdominal injections with glyoxylate (100 mg/kg/d, TCI, Tokyo, Japan) from day 3. 4) Hydrogen-oxygen gas pretreatment (HO plus CaOx) group: The animals inhaled mixed gas consisting of 67% H2 and 33% O2 (vol/vol) for 30 min daily for 7 days in addition to daily intraabdominal injections with glyoxylate from day 3. All mice were placed in individual metabolic cages with free access to water after the glyoxylate injection for 24 hours of urine collection. After urine and blood collection, the kidneys were harvested. The left kidney was frozen at -80°C for future use, and the right kidney was fixed in 4% paraformaldehyde for histologic examination. Gas administration
Male C57BL/6 mice (8 weeks old) were given water and food ad libitum and were kept for one week under a controlled 12-hour light/dark cycle at 20-25°C with the relative humidity set at 55-65%. This study was approved by the Animal Care and Use Committee of Second Military Medical University.
The mixed gas consisting of 67% H2 and 33% O2 was produced by the AMS-H-01 hydrogen oxygen nebulizer (Asclepius, Shanghai, China), which was specifically designed to extract the hydrogen and oxygen from water. The mice were placed into a transparent closed box (20 × 18 × 15 cm, length x width x height), into which the mixed gas was introduced at a rate of 200 ml/min throughout the experiments. The box was flushed with mixed gases for 30 min to replace the air in the box. Mixed gas consisting of 67% N2 and 33% O2 was administered at the same rate. During each experiment, the concentration of hydrogen gas in the box was monitored by Thermal trace GC ultra gas chromatography (Thermo Fisher, MA, USA).
Study groups
Urine biochemistry
Twenty-four male C57BL/6 mice were randomly divided into the following four groups of 6 mice each: 1) Nitrogen-oxygen gas (NO) group: The animals inhaled mixed gas consisting of 67% N2 and 33% O2 (vol/vol) for 30 min daily for 7 consecutive days in addition to daily intra-abdominal injections with a normal volume of saline (20 ml/kg/day) from day 3. 2) Hydrogen-oxygen gas (HO) group: The animals inhaled mixed gas consisting of 67% H2 and 33% O2 (vol/vol) for 30 min daily for 7 consecutive days in addition to daily intra-abdominal injections with a normal volume of saline (20 ml/kg/day) from day 3. 3) CaOx group: The animals inhaled mixed gas consisting of 67% N2 and 33% O2 (vol/vol) for 30 min daily for 7 days in addition to daily
The ratios of calcium, phosphate, magnesium and creatinine in urine were measured using a BC-2800 Vet animal auto biochemistry analyzer (Shihai, Guangdong, China).
Materials and methods Animals
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Serum excretion of Kidney injury molecule-1 (KIM-1) We measured the level of serum excretion of KIM-1 using an enzyme-linked immunosorbent (ELISA) kit (Uscn, Wuhan, China), according to the manufacturer’s instructions. Histological analysis Paraffin-embedded sections (3 μm) of mice kidneys were stained with a Von Kossa staining
Int J Clin Exp Pathol 2015;8(3):2680-2689
Hydrogen ameliorates glyoxylate induced kidney injury Table 1. The ratios of the urine concentration of calcium (Ca), phosphate (P) and magnesium (Mg) to creatinine in NO, HO, CaOx and HO plus CaOx groups Ca/Creatinine 0.33 ± 0.05 0.35 ± 0.09 0.63 ± 0.15* HO + CaOx 0.43 ± 0.10# NO HO CaOx *
P/Creatinine 3.72 ± 0.47 4.18 ± 0.56 8.12 ± 1.17* 5.33 ± 1.20#
Mg/Creatinine 0.27 ± 0.08 0.32 ± 0.12 0.23 ± 0.06 0.29 ± 0.07
P < 0.05 versus NO group, #P < 0.05 versus CaOx group.
commercial kit (Showbio, Shanghai, China) to quantify crystallization. The area of crystal deposits in kidney tissue was counted in 20 randomly selected fields and photographed under an Eclipse 50i light microscope (Nikon, Japan) at ×400 magnification. The level of calcium in kidney tissue We measured the level of calcium in kidney tissue using a colorimetric assay kit (Nanjing Jiancheng, Nanjing, China), according to the manufacturer’s instructions.
benzidine kits (Maixin, Fuzhou, China) and counterstained with hematoxylin solution (Beyotime, Haimen, China). TUNEL staining was performed using a FragELTM DNA Fragmentation Detection Kit and Colorimetric-TdT Enzyme (Merck & Co Inc, NJ, USA). All stained sections were counted in 10 randomly selected fields and photographed under an Eclipse 50i light microscope (Nikon, Japan) at ×400 magnification. Statistical analysis Results were presented as mean ± SD. We used one-way factorial ANOVA to test for overall differences among the groups, followed by Student-Newman-Keuls test to compare separate groups. Nonparametric data were analyzed by the Kruskal-Wallis H-test. P < 0.05 denoted the presence of a statistically significant difference. Results
Oxidative stress index in kidney tissue
Urine biochemistry
A portion of the tissue preparation was made from kidney tissue that was homogenized with normal saline, and the preparation was centrifuged at 2500 rpm at 4°C for 10 min. The resultant supernatant was equilibrated with saline and used to determine the levels of proteins in the tissue homogenates. The levels of superoxide dismutase (SOD), malondialdehyde (MDA), glutathione (GSH), catalase (CAT), myeloperoxidase (MPO), and 8-hydroxydeoxyguanosine (8OHdG) were determined using commercial kits (Nanjing Jiancheng, Nanjing, China). The determinations were performed strictly according to the manufacturer’s instructions.
As shown in Table 1, the ratio of calcium and creatinine significantly increased in the CaOx group compared with the NO group (P < 0.05) and decreased in the HO plus CaOx group (P < 0.05). Additionally, the ratio of phosphate and creatinine increased in the CaOx group (P < 0.05) and decreased in the HO plus CaOx group (P < 0.05). Additionally, H2 pretreatment alone showed no effect on the ratio of calcium, phosphate and creatinine.
Immunohistochemistry Kidney tissues sections (3 µm) were subjected to immunohistochemistry. The sections were incubated with primary antibodies of osteopontin (OPN) (1:100) (Santa Cruz, CA, USA), CD44 (1:100) (Proteintech, IL, USA), monocyte chemoattractant protein-1 (MCP-1) (1:100) (Santa Cruz, CA, USA) and interleukin-10 (IL-10) (1:100) (Santa Cruz, CA, USA) at 4°C overnight. After rinsing with PBST, secondary antibodies (1:500) (Proteintech, IL, USA) were added to the sections, conjugated to horseradish peroxidase and incubated at 37°C for 30 min. The resultant sections were visualized using di-amino2682
The level of serum excretion of KIM-1 The level of serum excretion of KIM-1 increased significantly after the administration of glyoxylate, and the effect was reversed by H2 pretreatment. Meanwhile, H2 pretreatment alone showed no effect on the level of serum excretion of KIM-1 (Figure 1A). TUNEL assay To further evaluate the potential protective effect of H2, renal cell apoptosis was assessed using TUNEL assay. The number of TUNELpositive cells was dramatically increased by glyoxylate treatment, to 8.7%, but this increase was dramatically blunted by pretreatment with H2, to 3.1% (Figure 1B, 1C). Int J Clin Exp Pathol 2015;8(3):2680-2689
Hydrogen ameliorates glyoxylate induced kidney injury
Figure 1. Changes in the level of serum excretion of KIM-1 and the cell death index. A. Serum excretion of KIM-1 as determined by enzyme-linked immunosorbent assay was significantly increased in CaOx group and was significantly reduced by H2 pretreatment. B and C. Represent the average number of TUNEL positive stained cells every 10 high power fields. *P < 0.05 versus NO group, #P < 0.05 versus CaOx group.
Von Kossa-stained kidney sections To examine the presence of CaOx crystal deposition in the kidney, Von Kossa-stained sections of kidney were examined under light microscopy. As shown in Figure 2, in histological examination of kidneys in the CaOx and HO plus CaOx groups, crystals were observed in the lumen and were primarily located at the border between the renal cortex and medulla (×40 magnification). Compared with the CaOx 2683
group, histological examination of kidneys in the HO plus CaOx group showed fewer crystal depositions in the kidney, and no crystals were observed in the NO or HO groups. The concentration of calcium in kidney tissue The concentration of calcium in renal tissue increased significantly (P < 0.05) after the mice were administered glyoxylate and decreased significantly (P < 0.05) in the HO plus CaOx Int J Clin Exp Pathol 2015;8(3):2680-2689
Hydrogen ameliorates glyoxylate induced kidney injury
Figure 2. Detection of renal calcium oxalate crystal deposition by Von Kossa staining and the concentration of renal calcium. (A Magnification x40) and (B Magnification x400) represent calcium oxalate crystal deposition, (C) Semiquantitative analysis of calcium deposition by the area of positive staining from every 20 random views, (D) The concentration of renal calcium. *P < 0.05 versus NO group, #P < 0.05 versus CaOx group.
group compared with the CaOx group (Figure 2D). H2 pretreatment alone showed no effect on the renal calcium level. ROS production and antioxidative enzyme The levels or activities of SOD, MDA, GSH, CAT, MPO, and 8-OHdG in renal tissue were determined to assist the H2 in protecting renal tissue from oxidative damage (Figure 3). The levels of MDA and 8-OHdG increased significantly (P < 0.05), and the activity of SOD and CAT and the GSH level decreased significantly (P < 0.05) in the CaOx group. In the HO plus CaOx group, a significant reduction of MDA and 8-OHdG levels (P < 0.05) was observed, and a significant increase in SOD and CAT activities and GSH levels (P < 0.05) was observed. However, the level of MPO did not change significantly. Immunohistochemical analysis of the kidneys Immunostaining was used to determine the expression of OPN, CD44, MCP-1 and IL-10 in 2684
the kidneys. OPN and CD44 expression was almost nonexistent in the NO group but was highly expressed in the renal tubules of the CaOx group (Figure 4). MCP-1 was also highly expressed in the adjacent interstitium of renal tubules in the CaOx group (Figure 4). Staining for OPN, CD44, and MCP-1 demonstrated a marked reduction in the kidneys of glyoxylateinduced mice pretreated with H2. Compared with the NO group, the expression of IL-10 increased in the CaOx group, and a markedly increased expression was clearly observed in the HO plus CaOx group. Discussion Hyperoxaluria is considered a major risk factor of CaOx stone disease. According to a previous study [17], we used glyoxylate, an oxalate precursor, to induce intratubular CaOx deposition. Based on the changes in urine biochemistry, the renal calcium concentration and the area of calcium deposition (Von Kossa staining), we Int J Clin Exp Pathol 2015;8(3):2680-2689
Hydrogen ameliorates glyoxylate induced kidney injury
Figure 3. The levels or activities of SOD, MDA, GSH, CAT, MPO, 8-OHdG, *P < 0.05 versus NO group, #P < 0.05 versus CaOx group.
found that H2 pretreatment could reduce renal crystallization. Hyperoxaluria and CaOx crystal deposition are associated with renal tubular epithelial cell injury. KIM-1 is a sensitive marker of the presence of renal tubular damage [18]. Urinary excretion and the renal expression of KIM-1 have been reported in association with renal CaOx crystal deposition [9]. In this study, we found that the level of serum excretion of
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KIM-1 was markedly upregulated after the administration of glyoxylate and downregulated by pretreatment with H2. Additionally, we examined renal apoptosis using TUNEL staining and found that kidneys in the CaOx group had more TUNEL positive cells compared with the NO group. H2 pretreatment significantly decreased TUNEL positive cells in the HO plus CaOx group. These results indicated that H2 could attenuate Int J Clin Exp Pathol 2015;8(3):2680-2689
Hydrogen ameliorates glyoxylate induced kidney injury
Figure 4. Changes in the expression of OPN, CD44, MCP-1, IL-10. The expression of OPN (A and B), CD44 (D and E), MCP-1 (G and H), IL-10 (J and K) increased significantly in CaOx group, and the expression of OPN (B and C), CD44 (E and F), MCP-1 (H and I) decreased significantly as well as IL-10 (K and L) increased significantly after H2 pretreatment.
the renal tubular injury. Simultaneously, H2 pretreatment alone showed no effect on the ratios of calcium, phosphate and creatinine, the concentration of renal calcium or the level of serum excretion of KIM-1. It seems that H2 has no side effects on crystal deposition and renal function.
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Many studies have revealed that ROS overproduction and decreased antioxidants lead to oxidative stress and are involved with kidney stone formation [4, 5]. ROS are generally considered cytotoxic and can damage a variety of macromolecules, including lipids, proteins and DNA [19]. MDA is an end product of lipid peroxida-
Int J Clin Exp Pathol 2015;8(3):2680-2689
Hydrogen ameliorates glyoxylate induced kidney injury tion that is usually used as a measure of the lipid peroxidation status of the body. MPO is a catalyst for oxidants. 8-OHdG is a marker of oxidative damage of DNA. SOD, GSH and CAT are common antioxidant substances in the body. H2 is considered a specific scavenger of hydroxyl radical and peroxynitrite that could decrease the levels of MDA, MPO and 8-OHdG and increase antioxidant enzyme activity [20]. In agreement with a previous study, the pretreatment with H2 in this study was associated with the reduced production of MDA and 8-OHdG and with enhanced activities of antioxidant enzymes such as SOD, GSH and CAT in the renal tissue of mice; however, the MPO level did not change significantly. Inflammation is accompanied with oxidative stress in crystal nephropathy, and crystallization modulators, such as OPN and CD44, play a significant role in inflammation and tissue repair [5]. OPN is implicated in crystal retention within the kidneys by affecting the adherence of CaOx crystals to renal epithelial cells and may be a chemoattractant for renal interstitium [21, 22]. CD44 serves as a cell surface receptor for both hyaluronan and OPN and as a crystal attachment promoter [5, 23]. The interaction with OPN and CD44, as expressed by injured/regenerating tubular cells, is a prerequisite for the retention of crystals in the kidney [23]. MCP-1, a chemokine with potent chemotactic activity for monocytes/macrophages, is a mediator of local inflammatory responses to interstitial crystals [24]. The interaction of renal epithelial cells with CaOx monohydrate crystals has been shown to stimulate both the expression of OPN and MCP-1 [25] and an increase in the generation of ROS from nicotinamide adenine dinucleotide phosphate as a second messenger system [26]. In the present study, we found that the expression of OPN, CD44 and MCP-1 increased significantly in the CaOx group and that this effect was reversed by H2 pretreatment. IL-10, initially denoted cytokine synthesis inhibitory factor plays a renoprotective role in several kidney models [27-29]. In lipopolysaccharide induced neuroinflammation mice, the expression of IL-10 was upregulated by the administration of hydrogen-enriched electrochemically reduced water, suggesting a strong anti-inflammatory response aiming at restoring the redox homeostasis and functional recovery [30]. Consistent with previous studies, the expression of IL-10 increased significantly
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in the CaOx group and was markedly upregulated in the HO plus CaOx group. These results indicated that H2 pretreatment could upregulate the expression of IL-10 to protect renal tubular epithelial cell injury from inflammation. It seems that H2 pretreatment could regulate the expression of crystallization modulators, suppress the pro-inflammatory cytokine and upregulate the anti-inflammatory factor in response to inflammation involved in CaOx crystal deposition. In summary, our results support that H2 reduced renal crystallization and protected renal tubular epithelial cells against glyoxylate-induced renal injury by suppressing oxidative stress and inhibiting inflammation. This study investigates the anti-nephrolithiasis effect of H2. It may provide a novel clinical treatment of kidney stones. Future studies should provide more information concerning the effects of different times and doses of inhaled H2 on kidney stones. Acknowledgements This work was supported by grants from the National Scientific Foundation of China (81270773, 81403261), the Basic Science Key Program (13401900105) from the Science and Technology Commission of Shanghai Municipality and the Innovation program of scientific research (14ZZ080) from the Shanghai Education Commission. Scientific program from Changhai Hospital (CH125520301). Dr. Changcheng Liu provided technical assistance. Disclosure of conflict of interest None. Address correspondence to: Xuejun Sun, Department of Aviation Medicine, Faculty of Naval Medicine, Second Military Medical University, 800 Xiangyin Road, Shanghai, China. Tel: +86-2181871143; Fax: +86-21-65492382; E-mail: sunxjk@ hotmail.com; Zhiyong Guo, Department of Nephrology, Changhai Hospital, Second Military Medical University, 168 Changhai Road, China. Tel: +86-2131161406; Fax: +86-21-31161418; E-mail: [email protected]
References [1]
Scales CD Jr, Smith AC, Hanley JM, Saigal CS. Prevalence of kidney stones in the United States. Eur Urol 2012; 62: 160-5.
Int J Clin Exp Pathol 2015;8(3):2680-2689
Hydrogen ameliorates glyoxylate induced kidney injury [2]
[3] [4] [5]
[6]
[7]
[8]
[9]
[10]
[11]
[12]
[13]
[14]
Edvardsson VO, Indridason OS, Haraldsson G, Kjartansson O, Palsson R. Temporal trends in the incidence of kidney stone disease. Kidney Int 2013; 83: 146-52. Asplin JR. Hyperoxaluric calcium nephrolithiasis. Endocrinol Metab Clin North Am 2002; 31: 927-49. Khan SR. Hyperoxaluria-induced oxidative stress and antioxidants for renal protection. Urol Res 2005; 33: 349-57. Khan SR. Reactive oxygen species as the molecular modulators of calcium oxalate kidney stone formation: evidence from clinical and experimental investigations. J Urol 2013; 189: 803-11. Santhosh KM, Selvam R. Supplementation of vitamin E and selenium prevents hyperoxaluria in experimental urolithic rats. J Nutr Biochem 2003; 14: 306-13. Baxmann AC, De O G Mendonca C, Heilberg IP. Effect of vitamin C supplements on urinary oxalate and pH in calcium stone-forming patients. Kidney Int 2003; 63: 1066-71. Lee HJ, Jeong SJ, Lee HJ, Lee EO, Bae H, Lieske JC, Kim SH. 1,2,3,4,6-Penta-O-galloyl-beta-Dglucose reduces renal crystallization and oxidative stress in a hyperoxaluric rat model. Kidney Int 2011; 79: 538-45. Zuo J, Khan A, Glenton PA, Khan SR. Effect of NADPH oxidase inhibition on the expression of kidney injury molecule and calcium oxalate crystal deposition in hydroxy-L-proline-induced hyperoxaluria in the male Sprague-Dawley rats. Nephrol Dial Transplant 2011; 26: 178596. Lafay V, Barthelemy P, Comet B, Frances Y, Jammes Y. ECG changes during the experimental human dive HYDRA 10 (71 atm/7,200 kPa). Undersea Hyperb Med 1995; 22: 51-60. Ohsawa I, Ishikawa M, Takahashi K, Watanabe M, Nishimaki K, Yamagata K, Katsura K, Katayama Y, Asoh S, Ohta S. Hydrogen acts as a therapeutic antioxidant by selectively reducing cytotoxic oxygen radicals. Nat Med 2007; 13: 688-94. Nakashima-Kamimura N, Mori T, Ohsawa I, Asoh S, Ohta S. Molecular hydrogen alleviates nephrotoxicity induced by an anti-cancer drug cisplatin without compromising anti-tumor activity in mice. Cancer Chemother Pharmacol 2009; 64: 753-61. Nakayama M, Nakano H, Hamada H, Itami N, Nakazawa R, Ito S. A novel bioactive haemodialysis system using dissolved dihydrogen (H2) produced by water electrolysis: a clinical trial. Nephrol Dial Transplant 2010; 25: 3026-33. Fukuda K, Asoh S, Ishikawa M, Yamamoto Y, Ohsawa I, Ohta S. Inhalation of hydrogen gas suppresses hepatic injury caused by isch-
2688
[15]
[16]
[17]
[18]
[19]
[20]
[21] [22] [23]
[24]
[25]
[26]
emia/reperfusion through reducing oxidative stress. Biochem Biophys Res Commun 2007; 361: 670-4. Zhang Y, Sun Q, He B, Xiao J, Wang Z, Sun X. Anti-inflammatory effect of hydrogen-rich saline in a rat model of regional myocardial ischemia and reperfusion. Int J Cardiol 2011; 148: 91-5. Cai J, Kang Z, Liu WW, Luo X, Qiang S, Zhang JH, Ohta S, Sun X, Xu W, Tao H, Li R. Hydrogen therapy reduces apoptosis in neonatal hypoxia-ischemia rat model. Neurosci Lett 2008; 441: 167-72. Okada A, Nomura S, Higashibata Y, Hirose M, Gao B, Yoshimura M, Itoh Y, Yasui T, Tozawa K, Kohri K. Successful formation of calcium oxalate crystal deposition in mouse kidney by intraabdominal glyoxylate injection. Urol Res 2007; 35: 89-99. Han WK, Bailly V, Abichandani R, Thadhani R, Bonventre JV. Kidney Injury Molecule-1 (KIM1): a novel biomarker for human renal proximal tubule injury. Kidney Int 2002; 62: 237-44. Linnane AW, Kios M, Vitetta L. Healthy aging: regulation of the metabolome by cellular redox modulation and prooxidant signaling systems: the essential roles of superoxide anion and hydrogen peroxide. Biogerontology 2007; 8: 44567. Ohno K, Ito M, Ichihara M, Ito M. Molecular hydrogen as an emerging therapeutic medical gas for neurodegenerative and other diseases. Oxid Med Cell Longev 2012; 2012: 353152. Wesson JA, Ward MD. Role of crystal surface adhesion in kidney stone disease. Curr Opin Nephrol Hypertens 2006; 15: 386-93. Khan SR. Role of renal epithelial cells in the initiation of calcium oxalate stones. Nephron Exp Nephrol 2004; 98: e55-60. Asselman M, Verhulst A, De Broe ME, Verkoelen CF. Calcium oxalate crystal adherence to hyaluronan-, osteopontin-, and CD44-expressing injured/regenerating tubular epithelial cells in rat kidneys. J Am Soc Nephrol 2003; 14: 3155-66. Umekawa T, Chegini N, Khan SR. Oxalate ions and calcium oxalate crystals stimulate MCP-1 expression by renal epithelial cells. Kidney Int 2002; 61: 105-12. Khan SR. Crystal-induced inflammation of the kidneys: results from human studies, animal models, and tissue-culture studies. Clin Exp Nephrol 2004; 8: 75-88. Umekawa T, Tsuji H, Uemura H, Khan SR. Superoxide from NADPH oxidase as second messenger for the expression of osteopontin and monocyte chemoattractant protein-1 in renal epithelial cells exposed to calcium oxalate crystals. BJU Int 2009; 104: 115-20.
Int J Clin Exp Pathol 2015;8(3):2680-2689
Hydrogen ameliorates glyoxylate induced kidney injury [27] Jin Y, Liu R, Xie J, Xiong H, He JC, Chen N. Interleukin-10 deficiency aggravates kidney inflammation and fibrosis in the unilateral ureteral obstruction mouse model. Lab Invest 2013; 93: 801-11. [28] Li YW, Zhang Y, Zhang L, Li X, Yu JB, Zhang HT, Tan BB, Jiang LH, Wang YX, Liang Y, Zhang XS, Wang WS, Liu HG. Protective effect of tea polyphenols on renal ischemia/reperfusion injury via suppressing the activation of TLR4/NFkappaB p65 signal pathway. Gene 2014; 542: 46-51.
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[29] Deng J, Kohda Y, Chiao H, Wang Y, Hu X, Hewitt SM, Miyaji T, McLeroy P, Nibhanupudy B, Li S, Star RA. Interleukin-10 inhibits ischemic and cisplatin-induced acute renal injury. Kidney Int 2001; 60: 2118-28. [30] Spulber S, Edoff K, Hong L, Morisawa S, Shirahata S, Ceccatelli S. Molecular hydrogen reduces LPS-induced neuroinflammation and promotes recovery from sickness behaviour in mice. PLoS One 2012; 7: e42078.
Int J Clin Exp Pathol 2015;8(3):2680-2689
Original Article Inhalation of hydrogen gas ameliorates glyoxylate-induced calcium oxalate deposition and renal oxidative stress in mice Zhongjiang Peng1*, Wei Chen1*, Li Wang1, Zhouheng Ye2, Songyan Gao3, Xuejun Sun2, Zhiyong Guo1 Department of Nephrology, Changhai Hospital, Second Military Medical University, Shanghai, China; 2Department of Aviation Medicine, Faculty of Naval Medicine, Second Military Medical University, Shanghai, China; 3 School of Pharmacy, Second Military Medical University, Shanghai, China. *Equal contributors. 1
Received January 4, 2015; Accepted February 26, 2015; Epub March 1, 2015; Published March 15, 2015 Abstract: The aim of this study is to evaluate the protective effect and underlying mechanism of hydrogen gas (H2) to glyoxylate induced renal calcium oxalate (CaOx) crystal deposition in mice. In present work, rodent renal CaOx crystal deposition model was introduced by intra-abdominal injection of glyoxylate (100 mg/kg/d) for 5 days. Two days before administration of glyoxylate, inhalation of H2 for 30 min per day was initiated and continued for 7 days. By the end of the study, the samples of 24 hours urine, serum and renal tissue were collected for biochemical and pathological assay. According to levels of urine calcium excretion, renal calcium deposition, a serum excretion of kidney injury molecule-1 (KIM-1) assay and a TUNEL assay, inhalation of H2 could successfully decrease the CaOx crystallizations and protect against renal injury. Crystal deposition in the kidneys is associated with oxidative stress, which was indicated by increased levels of renal malondialdehyde (MDA) and 8-hydroxydeoxyguanosine (8-OHdG) and decreased activities of superoxide dismutase (SOD), glutathione (GSH) and catalase (CAT). These effects were reversed by a high-dose H2 pretreatment. The renal expressions of osteopontin (OPN), CD44, monocyte chemoattractant protein-1 (MCP-1) and interleukin-10 (IL-10) were markedly increased in glyoxylate-treated mice, and H2 significantly attenuated the increase of OPN, CD44 and MCP-1 but upregulated the expression of IL-10. Our findings demonstrate that inhalation of H2 reduces renal crystallization, renal oxidative injury and inflammation and it may be a candidate agent with few adverse effects for prevention of nephrolithiasis. Keywords: Hydrogen gas, calcium oxalate, kidney injury molecule-1, glyoxylate, oxidative stress
Introduction Stone disease, also known as urolithiasis, is becoming a major public health burden worldwide [1, 2]. Nephrolithiasis, deposition of crystal in the kidneys and the formation of kidney stones, is a common urological disorder. Calcium oxalate (CaOx) is the most common type of nephrolithiasis; hyperoxaluria is the major risk factor [3]. Nephrolithiasis formation consists of several stages including supersaturation, nucleation, growth, aggregation, and retention within renal tubules. Although the specific mechanism is not clear, there is accumulating evidence that crystal deposition in the kidneys is associated with reactive oxygen species (ROS) production, oxidative stress development, inflammation and renal tubule injury [4, 5]. Anti-oxidative treatment has become an
experimental strategy of anti-nephrolithiasis, such as vitamin E [6], vitamin C [7], and some traditional medicinal herbs [8, 9]. Nevertheless, no such substance could be translated into clinical practice because some side-effects were present [7]. Thus, developing an effective alternative with few adverse effects is an urgent need in this field. Hydrogen is considered an inert gas and has been used in medical applications to prevent decompression sickness in deep divers [10]. In 2007, it was reported that an inhalation of H2 has antioxidant and anti-apoptotic properties that can protect the brain against ischemia/ reperfusion injury by selectively neutralizing hydroxyl radicals [11]. This report aroused considerable interest worldwide. Thus far, the therapeutic effects of molecular hydrogen on vari-
Hydrogen ameliorates glyoxylate induced kidney injury ous diseases, including kidney diseases [12, 13], have been tested mostly regarding its antioxidation capability [14], but also its antiinflammation [15] and anti-apoptosis [16] capabilities. Compared with traditional antioxidants, hydrogen is a small molecule that can easily dissipate throughout the body and cells, and it is sufficiently mild that it does not disturb metabolic oxidation-reduction reactions or disrupt ROS mediated cell signaling. Thus, it may be a safe and effective antioxidant for nephrolithiasis. To date, most inhaled hydrogen gas research has focused on a low dose (2%-4%), but the effect of inhaling a high dose has rarely been tested. In theory, high-dose hydrogen might have good effects and can be easily used to explore the dose-dependent response of hydrogen with increasing dosage. In this study, we hypothesized that pretreatment with a high dose of hydrogen gas will attenuate renal crystallization by anti-oxidation, anti-inflammation, and protection from renal tubule injury.
intra-abdominal injections with glyoxylate (100 mg/kg/d, TCI, Tokyo, Japan) from day 3. 4) Hydrogen-oxygen gas pretreatment (HO plus CaOx) group: The animals inhaled mixed gas consisting of 67% H2 and 33% O2 (vol/vol) for 30 min daily for 7 days in addition to daily intraabdominal injections with glyoxylate from day 3. All mice were placed in individual metabolic cages with free access to water after the glyoxylate injection for 24 hours of urine collection. After urine and blood collection, the kidneys were harvested. The left kidney was frozen at -80°C for future use, and the right kidney was fixed in 4% paraformaldehyde for histologic examination. Gas administration
Male C57BL/6 mice (8 weeks old) were given water and food ad libitum and were kept for one week under a controlled 12-hour light/dark cycle at 20-25°C with the relative humidity set at 55-65%. This study was approved by the Animal Care and Use Committee of Second Military Medical University.
The mixed gas consisting of 67% H2 and 33% O2 was produced by the AMS-H-01 hydrogen oxygen nebulizer (Asclepius, Shanghai, China), which was specifically designed to extract the hydrogen and oxygen from water. The mice were placed into a transparent closed box (20 × 18 × 15 cm, length x width x height), into which the mixed gas was introduced at a rate of 200 ml/min throughout the experiments. The box was flushed with mixed gases for 30 min to replace the air in the box. Mixed gas consisting of 67% N2 and 33% O2 was administered at the same rate. During each experiment, the concentration of hydrogen gas in the box was monitored by Thermal trace GC ultra gas chromatography (Thermo Fisher, MA, USA).
Study groups
Urine biochemistry
Twenty-four male C57BL/6 mice were randomly divided into the following four groups of 6 mice each: 1) Nitrogen-oxygen gas (NO) group: The animals inhaled mixed gas consisting of 67% N2 and 33% O2 (vol/vol) for 30 min daily for 7 consecutive days in addition to daily intra-abdominal injections with a normal volume of saline (20 ml/kg/day) from day 3. 2) Hydrogen-oxygen gas (HO) group: The animals inhaled mixed gas consisting of 67% H2 and 33% O2 (vol/vol) for 30 min daily for 7 consecutive days in addition to daily intra-abdominal injections with a normal volume of saline (20 ml/kg/day) from day 3. 3) CaOx group: The animals inhaled mixed gas consisting of 67% N2 and 33% O2 (vol/vol) for 30 min daily for 7 days in addition to daily
The ratios of calcium, phosphate, magnesium and creatinine in urine were measured using a BC-2800 Vet animal auto biochemistry analyzer (Shihai, Guangdong, China).
Materials and methods Animals
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Serum excretion of Kidney injury molecule-1 (KIM-1) We measured the level of serum excretion of KIM-1 using an enzyme-linked immunosorbent (ELISA) kit (Uscn, Wuhan, China), according to the manufacturer’s instructions. Histological analysis Paraffin-embedded sections (3 μm) of mice kidneys were stained with a Von Kossa staining
Int J Clin Exp Pathol 2015;8(3):2680-2689
Hydrogen ameliorates glyoxylate induced kidney injury Table 1. The ratios of the urine concentration of calcium (Ca), phosphate (P) and magnesium (Mg) to creatinine in NO, HO, CaOx and HO plus CaOx groups Ca/Creatinine 0.33 ± 0.05 0.35 ± 0.09 0.63 ± 0.15* HO + CaOx 0.43 ± 0.10# NO HO CaOx *
P/Creatinine 3.72 ± 0.47 4.18 ± 0.56 8.12 ± 1.17* 5.33 ± 1.20#
Mg/Creatinine 0.27 ± 0.08 0.32 ± 0.12 0.23 ± 0.06 0.29 ± 0.07
P < 0.05 versus NO group, #P < 0.05 versus CaOx group.
commercial kit (Showbio, Shanghai, China) to quantify crystallization. The area of crystal deposits in kidney tissue was counted in 20 randomly selected fields and photographed under an Eclipse 50i light microscope (Nikon, Japan) at ×400 magnification. The level of calcium in kidney tissue We measured the level of calcium in kidney tissue using a colorimetric assay kit (Nanjing Jiancheng, Nanjing, China), according to the manufacturer’s instructions.
benzidine kits (Maixin, Fuzhou, China) and counterstained with hematoxylin solution (Beyotime, Haimen, China). TUNEL staining was performed using a FragELTM DNA Fragmentation Detection Kit and Colorimetric-TdT Enzyme (Merck & Co Inc, NJ, USA). All stained sections were counted in 10 randomly selected fields and photographed under an Eclipse 50i light microscope (Nikon, Japan) at ×400 magnification. Statistical analysis Results were presented as mean ± SD. We used one-way factorial ANOVA to test for overall differences among the groups, followed by Student-Newman-Keuls test to compare separate groups. Nonparametric data were analyzed by the Kruskal-Wallis H-test. P < 0.05 denoted the presence of a statistically significant difference. Results
Oxidative stress index in kidney tissue
Urine biochemistry
A portion of the tissue preparation was made from kidney tissue that was homogenized with normal saline, and the preparation was centrifuged at 2500 rpm at 4°C for 10 min. The resultant supernatant was equilibrated with saline and used to determine the levels of proteins in the tissue homogenates. The levels of superoxide dismutase (SOD), malondialdehyde (MDA), glutathione (GSH), catalase (CAT), myeloperoxidase (MPO), and 8-hydroxydeoxyguanosine (8OHdG) were determined using commercial kits (Nanjing Jiancheng, Nanjing, China). The determinations were performed strictly according to the manufacturer’s instructions.
As shown in Table 1, the ratio of calcium and creatinine significantly increased in the CaOx group compared with the NO group (P < 0.05) and decreased in the HO plus CaOx group (P < 0.05). Additionally, the ratio of phosphate and creatinine increased in the CaOx group (P < 0.05) and decreased in the HO plus CaOx group (P < 0.05). Additionally, H2 pretreatment alone showed no effect on the ratio of calcium, phosphate and creatinine.
Immunohistochemistry Kidney tissues sections (3 µm) were subjected to immunohistochemistry. The sections were incubated with primary antibodies of osteopontin (OPN) (1:100) (Santa Cruz, CA, USA), CD44 (1:100) (Proteintech, IL, USA), monocyte chemoattractant protein-1 (MCP-1) (1:100) (Santa Cruz, CA, USA) and interleukin-10 (IL-10) (1:100) (Santa Cruz, CA, USA) at 4°C overnight. After rinsing with PBST, secondary antibodies (1:500) (Proteintech, IL, USA) were added to the sections, conjugated to horseradish peroxidase and incubated at 37°C for 30 min. The resultant sections were visualized using di-amino2682
The level of serum excretion of KIM-1 The level of serum excretion of KIM-1 increased significantly after the administration of glyoxylate, and the effect was reversed by H2 pretreatment. Meanwhile, H2 pretreatment alone showed no effect on the level of serum excretion of KIM-1 (Figure 1A). TUNEL assay To further evaluate the potential protective effect of H2, renal cell apoptosis was assessed using TUNEL assay. The number of TUNELpositive cells was dramatically increased by glyoxylate treatment, to 8.7%, but this increase was dramatically blunted by pretreatment with H2, to 3.1% (Figure 1B, 1C). Int J Clin Exp Pathol 2015;8(3):2680-2689
Hydrogen ameliorates glyoxylate induced kidney injury
Figure 1. Changes in the level of serum excretion of KIM-1 and the cell death index. A. Serum excretion of KIM-1 as determined by enzyme-linked immunosorbent assay was significantly increased in CaOx group and was significantly reduced by H2 pretreatment. B and C. Represent the average number of TUNEL positive stained cells every 10 high power fields. *P < 0.05 versus NO group, #P < 0.05 versus CaOx group.
Von Kossa-stained kidney sections To examine the presence of CaOx crystal deposition in the kidney, Von Kossa-stained sections of kidney were examined under light microscopy. As shown in Figure 2, in histological examination of kidneys in the CaOx and HO plus CaOx groups, crystals were observed in the lumen and were primarily located at the border between the renal cortex and medulla (×40 magnification). Compared with the CaOx 2683
group, histological examination of kidneys in the HO plus CaOx group showed fewer crystal depositions in the kidney, and no crystals were observed in the NO or HO groups. The concentration of calcium in kidney tissue The concentration of calcium in renal tissue increased significantly (P < 0.05) after the mice were administered glyoxylate and decreased significantly (P < 0.05) in the HO plus CaOx Int J Clin Exp Pathol 2015;8(3):2680-2689
Hydrogen ameliorates glyoxylate induced kidney injury
Figure 2. Detection of renal calcium oxalate crystal deposition by Von Kossa staining and the concentration of renal calcium. (A Magnification x40) and (B Magnification x400) represent calcium oxalate crystal deposition, (C) Semiquantitative analysis of calcium deposition by the area of positive staining from every 20 random views, (D) The concentration of renal calcium. *P < 0.05 versus NO group, #P < 0.05 versus CaOx group.
group compared with the CaOx group (Figure 2D). H2 pretreatment alone showed no effect on the renal calcium level. ROS production and antioxidative enzyme The levels or activities of SOD, MDA, GSH, CAT, MPO, and 8-OHdG in renal tissue were determined to assist the H2 in protecting renal tissue from oxidative damage (Figure 3). The levels of MDA and 8-OHdG increased significantly (P < 0.05), and the activity of SOD and CAT and the GSH level decreased significantly (P < 0.05) in the CaOx group. In the HO plus CaOx group, a significant reduction of MDA and 8-OHdG levels (P < 0.05) was observed, and a significant increase in SOD and CAT activities and GSH levels (P < 0.05) was observed. However, the level of MPO did not change significantly. Immunohistochemical analysis of the kidneys Immunostaining was used to determine the expression of OPN, CD44, MCP-1 and IL-10 in 2684
the kidneys. OPN and CD44 expression was almost nonexistent in the NO group but was highly expressed in the renal tubules of the CaOx group (Figure 4). MCP-1 was also highly expressed in the adjacent interstitium of renal tubules in the CaOx group (Figure 4). Staining for OPN, CD44, and MCP-1 demonstrated a marked reduction in the kidneys of glyoxylateinduced mice pretreated with H2. Compared with the NO group, the expression of IL-10 increased in the CaOx group, and a markedly increased expression was clearly observed in the HO plus CaOx group. Discussion Hyperoxaluria is considered a major risk factor of CaOx stone disease. According to a previous study [17], we used glyoxylate, an oxalate precursor, to induce intratubular CaOx deposition. Based on the changes in urine biochemistry, the renal calcium concentration and the area of calcium deposition (Von Kossa staining), we Int J Clin Exp Pathol 2015;8(3):2680-2689
Hydrogen ameliorates glyoxylate induced kidney injury
Figure 3. The levels or activities of SOD, MDA, GSH, CAT, MPO, 8-OHdG, *P < 0.05 versus NO group, #P < 0.05 versus CaOx group.
found that H2 pretreatment could reduce renal crystallization. Hyperoxaluria and CaOx crystal deposition are associated with renal tubular epithelial cell injury. KIM-1 is a sensitive marker of the presence of renal tubular damage [18]. Urinary excretion and the renal expression of KIM-1 have been reported in association with renal CaOx crystal deposition [9]. In this study, we found that the level of serum excretion of
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KIM-1 was markedly upregulated after the administration of glyoxylate and downregulated by pretreatment with H2. Additionally, we examined renal apoptosis using TUNEL staining and found that kidneys in the CaOx group had more TUNEL positive cells compared with the NO group. H2 pretreatment significantly decreased TUNEL positive cells in the HO plus CaOx group. These results indicated that H2 could attenuate Int J Clin Exp Pathol 2015;8(3):2680-2689
Hydrogen ameliorates glyoxylate induced kidney injury
Figure 4. Changes in the expression of OPN, CD44, MCP-1, IL-10. The expression of OPN (A and B), CD44 (D and E), MCP-1 (G and H), IL-10 (J and K) increased significantly in CaOx group, and the expression of OPN (B and C), CD44 (E and F), MCP-1 (H and I) decreased significantly as well as IL-10 (K and L) increased significantly after H2 pretreatment.
the renal tubular injury. Simultaneously, H2 pretreatment alone showed no effect on the ratios of calcium, phosphate and creatinine, the concentration of renal calcium or the level of serum excretion of KIM-1. It seems that H2 has no side effects on crystal deposition and renal function.
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Many studies have revealed that ROS overproduction and decreased antioxidants lead to oxidative stress and are involved with kidney stone formation [4, 5]. ROS are generally considered cytotoxic and can damage a variety of macromolecules, including lipids, proteins and DNA [19]. MDA is an end product of lipid peroxida-
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Hydrogen ameliorates glyoxylate induced kidney injury tion that is usually used as a measure of the lipid peroxidation status of the body. MPO is a catalyst for oxidants. 8-OHdG is a marker of oxidative damage of DNA. SOD, GSH and CAT are common antioxidant substances in the body. H2 is considered a specific scavenger of hydroxyl radical and peroxynitrite that could decrease the levels of MDA, MPO and 8-OHdG and increase antioxidant enzyme activity [20]. In agreement with a previous study, the pretreatment with H2 in this study was associated with the reduced production of MDA and 8-OHdG and with enhanced activities of antioxidant enzymes such as SOD, GSH and CAT in the renal tissue of mice; however, the MPO level did not change significantly. Inflammation is accompanied with oxidative stress in crystal nephropathy, and crystallization modulators, such as OPN and CD44, play a significant role in inflammation and tissue repair [5]. OPN is implicated in crystal retention within the kidneys by affecting the adherence of CaOx crystals to renal epithelial cells and may be a chemoattractant for renal interstitium [21, 22]. CD44 serves as a cell surface receptor for both hyaluronan and OPN and as a crystal attachment promoter [5, 23]. The interaction with OPN and CD44, as expressed by injured/regenerating tubular cells, is a prerequisite for the retention of crystals in the kidney [23]. MCP-1, a chemokine with potent chemotactic activity for monocytes/macrophages, is a mediator of local inflammatory responses to interstitial crystals [24]. The interaction of renal epithelial cells with CaOx monohydrate crystals has been shown to stimulate both the expression of OPN and MCP-1 [25] and an increase in the generation of ROS from nicotinamide adenine dinucleotide phosphate as a second messenger system [26]. In the present study, we found that the expression of OPN, CD44 and MCP-1 increased significantly in the CaOx group and that this effect was reversed by H2 pretreatment. IL-10, initially denoted cytokine synthesis inhibitory factor plays a renoprotective role in several kidney models [27-29]. In lipopolysaccharide induced neuroinflammation mice, the expression of IL-10 was upregulated by the administration of hydrogen-enriched electrochemically reduced water, suggesting a strong anti-inflammatory response aiming at restoring the redox homeostasis and functional recovery [30]. Consistent with previous studies, the expression of IL-10 increased significantly
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in the CaOx group and was markedly upregulated in the HO plus CaOx group. These results indicated that H2 pretreatment could upregulate the expression of IL-10 to protect renal tubular epithelial cell injury from inflammation. It seems that H2 pretreatment could regulate the expression of crystallization modulators, suppress the pro-inflammatory cytokine and upregulate the anti-inflammatory factor in response to inflammation involved in CaOx crystal deposition. In summary, our results support that H2 reduced renal crystallization and protected renal tubular epithelial cells against glyoxylate-induced renal injury by suppressing oxidative stress and inhibiting inflammation. This study investigates the anti-nephrolithiasis effect of H2. It may provide a novel clinical treatment of kidney stones. Future studies should provide more information concerning the effects of different times and doses of inhaled H2 on kidney stones. Acknowledgements This work was supported by grants from the National Scientific Foundation of China (81270773, 81403261), the Basic Science Key Program (13401900105) from the Science and Technology Commission of Shanghai Municipality and the Innovation program of scientific research (14ZZ080) from the Shanghai Education Commission. Scientific program from Changhai Hospital (CH125520301). Dr. Changcheng Liu provided technical assistance. Disclosure of conflict of interest None. Address correspondence to: Xuejun Sun, Department of Aviation Medicine, Faculty of Naval Medicine, Second Military Medical University, 800 Xiangyin Road, Shanghai, China. Tel: +86-2181871143; Fax: +86-21-65492382; E-mail: sunxjk@ hotmail.com; Zhiyong Guo, Department of Nephrology, Changhai Hospital, Second Military Medical University, 168 Changhai Road, China. Tel: +86-2131161406; Fax: +86-21-31161418; E-mail: [email protected]
References [1]
Scales CD Jr, Smith AC, Hanley JM, Saigal CS. Prevalence of kidney stones in the United States. Eur Urol 2012; 62: 160-5.
Int J Clin Exp Pathol 2015;8(3):2680-2689
Hydrogen ameliorates glyoxylate induced kidney injury [2]
[3] [4] [5]
[6]
[7]
[8]
[9]
[10]
[11]
[12]
[13]
[14]
Edvardsson VO, Indridason OS, Haraldsson G, Kjartansson O, Palsson R. Temporal trends in the incidence of kidney stone disease. Kidney Int 2013; 83: 146-52. Asplin JR. Hyperoxaluric calcium nephrolithiasis. Endocrinol Metab Clin North Am 2002; 31: 927-49. Khan SR. Hyperoxaluria-induced oxidative stress and antioxidants for renal protection. Urol Res 2005; 33: 349-57. Khan SR. Reactive oxygen species as the molecular modulators of calcium oxalate kidney stone formation: evidence from clinical and experimental investigations. J Urol 2013; 189: 803-11. Santhosh KM, Selvam R. Supplementation of vitamin E and selenium prevents hyperoxaluria in experimental urolithic rats. J Nutr Biochem 2003; 14: 306-13. Baxmann AC, De O G Mendonca C, Heilberg IP. Effect of vitamin C supplements on urinary oxalate and pH in calcium stone-forming patients. Kidney Int 2003; 63: 1066-71. Lee HJ, Jeong SJ, Lee HJ, Lee EO, Bae H, Lieske JC, Kim SH. 1,2,3,4,6-Penta-O-galloyl-beta-Dglucose reduces renal crystallization and oxidative stress in a hyperoxaluric rat model. Kidney Int 2011; 79: 538-45. Zuo J, Khan A, Glenton PA, Khan SR. Effect of NADPH oxidase inhibition on the expression of kidney injury molecule and calcium oxalate crystal deposition in hydroxy-L-proline-induced hyperoxaluria in the male Sprague-Dawley rats. Nephrol Dial Transplant 2011; 26: 178596. Lafay V, Barthelemy P, Comet B, Frances Y, Jammes Y. ECG changes during the experimental human dive HYDRA 10 (71 atm/7,200 kPa). Undersea Hyperb Med 1995; 22: 51-60. Ohsawa I, Ishikawa M, Takahashi K, Watanabe M, Nishimaki K, Yamagata K, Katsura K, Katayama Y, Asoh S, Ohta S. Hydrogen acts as a therapeutic antioxidant by selectively reducing cytotoxic oxygen radicals. Nat Med 2007; 13: 688-94. Nakashima-Kamimura N, Mori T, Ohsawa I, Asoh S, Ohta S. Molecular hydrogen alleviates nephrotoxicity induced by an anti-cancer drug cisplatin without compromising anti-tumor activity in mice. Cancer Chemother Pharmacol 2009; 64: 753-61. Nakayama M, Nakano H, Hamada H, Itami N, Nakazawa R, Ito S. A novel bioactive haemodialysis system using dissolved dihydrogen (H2) produced by water electrolysis: a clinical trial. Nephrol Dial Transplant 2010; 25: 3026-33. Fukuda K, Asoh S, Ishikawa M, Yamamoto Y, Ohsawa I, Ohta S. Inhalation of hydrogen gas suppresses hepatic injury caused by isch-
2688
[15]
[16]
[17]
[18]
[19]
[20]
[21] [22] [23]
[24]
[25]
[26]
emia/reperfusion through reducing oxidative stress. Biochem Biophys Res Commun 2007; 361: 670-4. Zhang Y, Sun Q, He B, Xiao J, Wang Z, Sun X. Anti-inflammatory effect of hydrogen-rich saline in a rat model of regional myocardial ischemia and reperfusion. Int J Cardiol 2011; 148: 91-5. Cai J, Kang Z, Liu WW, Luo X, Qiang S, Zhang JH, Ohta S, Sun X, Xu W, Tao H, Li R. Hydrogen therapy reduces apoptosis in neonatal hypoxia-ischemia rat model. Neurosci Lett 2008; 441: 167-72. Okada A, Nomura S, Higashibata Y, Hirose M, Gao B, Yoshimura M, Itoh Y, Yasui T, Tozawa K, Kohri K. Successful formation of calcium oxalate crystal deposition in mouse kidney by intraabdominal glyoxylate injection. Urol Res 2007; 35: 89-99. Han WK, Bailly V, Abichandani R, Thadhani R, Bonventre JV. Kidney Injury Molecule-1 (KIM1): a novel biomarker for human renal proximal tubule injury. Kidney Int 2002; 62: 237-44. Linnane AW, Kios M, Vitetta L. Healthy aging: regulation of the metabolome by cellular redox modulation and prooxidant signaling systems: the essential roles of superoxide anion and hydrogen peroxide. Biogerontology 2007; 8: 44567. Ohno K, Ito M, Ichihara M, Ito M. Molecular hydrogen as an emerging therapeutic medical gas for neurodegenerative and other diseases. Oxid Med Cell Longev 2012; 2012: 353152. Wesson JA, Ward MD. Role of crystal surface adhesion in kidney stone disease. Curr Opin Nephrol Hypertens 2006; 15: 386-93. Khan SR. Role of renal epithelial cells in the initiation of calcium oxalate stones. Nephron Exp Nephrol 2004; 98: e55-60. Asselman M, Verhulst A, De Broe ME, Verkoelen CF. Calcium oxalate crystal adherence to hyaluronan-, osteopontin-, and CD44-expressing injured/regenerating tubular epithelial cells in rat kidneys. J Am Soc Nephrol 2003; 14: 3155-66. Umekawa T, Chegini N, Khan SR. Oxalate ions and calcium oxalate crystals stimulate MCP-1 expression by renal epithelial cells. Kidney Int 2002; 61: 105-12. Khan SR. Crystal-induced inflammation of the kidneys: results from human studies, animal models, and tissue-culture studies. Clin Exp Nephrol 2004; 8: 75-88. Umekawa T, Tsuji H, Uemura H, Khan SR. Superoxide from NADPH oxidase as second messenger for the expression of osteopontin and monocyte chemoattractant protein-1 in renal epithelial cells exposed to calcium oxalate crystals. BJU Int 2009; 104: 115-20.
Int J Clin Exp Pathol 2015;8(3):2680-2689
Hydrogen ameliorates glyoxylate induced kidney injury [27] Jin Y, Liu R, Xie J, Xiong H, He JC, Chen N. Interleukin-10 deficiency aggravates kidney inflammation and fibrosis in the unilateral ureteral obstruction mouse model. Lab Invest 2013; 93: 801-11. [28] Li YW, Zhang Y, Zhang L, Li X, Yu JB, Zhang HT, Tan BB, Jiang LH, Wang YX, Liang Y, Zhang XS, Wang WS, Liu HG. Protective effect of tea polyphenols on renal ischemia/reperfusion injury via suppressing the activation of TLR4/NFkappaB p65 signal pathway. Gene 2014; 542: 46-51.
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[29] Deng J, Kohda Y, Chiao H, Wang Y, Hu X, Hewitt SM, Miyaji T, McLeroy P, Nibhanupudy B, Li S, Star RA. Interleukin-10 inhibits ischemic and cisplatin-induced acute renal injury. Kidney Int 2001; 60: 2118-28. [30] Spulber S, Edoff K, Hong L, Morisawa S, Shirahata S, Ceccatelli S. Molecular hydrogen reduces LPS-induced neuroinflammation and promotes recovery from sickness behaviour in mice. PLoS One 2012; 7: e42078.
Int J Clin Exp Pathol 2015;8(3):2680-2689
