Oral hydrogen water prevents chronic allograft nephropathy in rats& 2009 International Society of Nephrology Oral hydrogen water prevents chronic allograftnephropathy in rats Jon S. Cardinal1, Jianghua Zhan1, Yinna Wang1, Ryujiro Sugimoto1,2,3, Allan Tsung1,Kenneth R. McCurry1,3, Timothy R. Billiar1 and Atsunori Nakao1,2,3 1Department of Surgery, University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania, USA; 2Department of Surgery,Thomas E. Starzl Transplantation Institute, University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania, USA and 3Department ofSurgery, Heart, Lung and Esophageal Surgery Institute, University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania, USA Reactive oxygen species (ROS) contribute to the Chronic kidney disease is the ninth leading cause of death in development of interstitial fibrosis and tubular atrophy the United States accounting for over 40,000 deaths seen in chronic allograft nephropathy (CAN). As molecular annuallyDespite advances in renal replacement therapy, hydrogen gas can act as a scavenger of ROS, we tested the transplantation remains the preferred treatment for suitable effect of treatment with hydrogen water (HW) in a model candidates.However, in spite of improved postoperative of kidney transplantation, in which allografts from Lewis immunosuppression regimens, the 10-year graft survival rats were orthotopically transplanted into Brown Norway rates are 55 and 75% for cadaveric and live donor kidney recipients that had undergone bilateral nephrectomy.
allografts, respectively. The vast majority of late failures Molecular hydrogen was dissolved in water and recipients are attributable to chronic allograft nephropathy (CAN), were given HW from day 0 until day 150. Rats that were recently reclassified as interstitial fibrosis and tubular atrophy treated with regular water (RW) gradually developed with unknown etiology.The clinical course of CAN is proteinuria and their creatinine clearance declined, ultimately characterized by a progressive deterioration in renal function, leading to graft failure secondary to CAN. In contrast, manifested by increasing renal hypertension and proteinuria.
treatment with HW improved allograft function, slowed the Presently, no specific treatment is available for chronic progression of CAN, reduced oxidant injury and rejection in clinical transplantation despite a number of inflammatory mediator production, and improved overall successful approaches in animal models, including the use survival. Inflammatory signaling pathways, such as mitogen- of macrophage inhibitors, angiotensin converting enzyme activated protein kinases, were less activated in renal inhibitors, and endothelin A receptor antagonists.
allografts from HW-treated rats as compared with RW-treated A number of factors contribute to the development of rats. Hence, oral HW is an effective antioxidant and antiinflammatory agent that prevented CAN, improved rejection) and nonimmunological (for example, ischemia- survival of rat renal allografts, and may be of therapeutic reperfusion injury) factors.Oxidative stress is believed value in the setting of transplantation.
to be a common pathway that leads to both immunological Kidney International advance online publication, 11 November 2009; and nonimmunological stress in the setting of kidney transplantation and, ultimately, to the development of KEYWORDS: chronic rejection; hydrogen; inflammation; kidney transplanta- CAN.Markers of oxidative stress, such as plasma lipid tion; oxidative stress; rats peroxidases, are increased, whereas antioxidant markers,including glutathione, superoxide dismutase, and glutathioneperoxidase, are decreased in the setting of CAN.Despitethis association, few studies have attempted to examine theeffect of antioxidants on kidney allograft outcomes and thosethat have yielded mixed results. Vitamin E (a-tocopherol)supplementation did not prevent allograft injury in a modelof however, in the same model, L-arginine didattenuate proteinuria and glomerulosclerosis.Furthermore,a clinical trial using recombinant human superoxide Correspondence: Atsunori Nakao, Department of Surgery, Thomas E. Starzl dismutase resulted in significantly decreased acute and Transplantation Institute, Heart, Lung and Esophageal Surgery Institute, chronic rejection.Therefore, based on the seemingly University of Pittsburgh, E1551 BST, 200 Lothrop Street, Pittsburgh,Pennsylvania 15213, USA. E-mail: conflicting results of these few studies, there is a need foradditional investigations into the applicability of antioxidants Received 5 May 2009; revised 16 September 2009; accepted29 September 2009 for the prevention of CAN.
Kidney International JS Cardinal et al.: Hydrogen water prevented CAN Molecular hydrogen has recently been shown to have therapeutic value as an antioxidant through its ability to selectively reduce cytotoxic reactive oxygen species (ROS).
Inhaled hydrogen gas (B4% H2 in air) can reduce infarct size in rat models of focal cerebral and myocardial ischemia- reperfusion injuryMore recently, our group reported that perioperative hydrogen inhalation (2%) significantly amelio-rates intestinal transplant injury and prevents remote organ inflammation through its antioxidant effectDrinking Time after intake (min) Time after intake (min) water containing a therapeutic dose of hydrogen (hydrogenwater; HW) represents an alternative mode of delivery of molecular hydrogen. The primary advantages of HW are that it is a portable, easily administered, and safe means of delivering molecular hydrogen. Therefore, it may be of potential therapeutic value in the treatment of oxidativestress-induced pathologies. Interestingly, drinking HW, as wellas inhaling hydrogen gas, can alleviate cisplatin-induced nephrotoxicity, which is known to be mediated, in part, by the Time after intake (min) Time after intake (min) accumulation of ROS that occurs secondary to the ability ofcisplatin to inhibit the reducing form of glutathioneConsumption of HW ad libitum prevents the development of atherosclerosis in apolipoprotein E knockout mice, in part, through its ability to limit the amount and deleterious effects of oxidative stress in the blood vessels of these mice.
Furthermore, a clinical trial in type II diabetic patients given supplemental HW led to improved lipid and glucose metabolism compared with contrThe aim of the present study was to determine the efficacy of HW in preventingCAN after allogeneic kidney transplantation in rats.
Iso Iso Allo Allo Iso Iso Allo Allo Iso Iso Allo Allo Figure 1 Hydrogen water (HW) administration improves Oral administration of HW increased local and systemic levels kidney allograft function. (a) Hydrogen-rich water (HW) (3 ml) was orally administered to naı¨ve Lewis (LEW) rats by gavage.
To determine whether oral administration of HW results in Arterial blood and kidney tissue were taken at 15, 30, and 60 min increased local or systemic levels of molecular hydrogen, after oral administration of HW. Hydrogen concentration in blood unoperated naı¨ve Lewis (LEW) rats were given HW to drink and homogenized kidney tissue increased within 15 min and thenreturned to the basal levels (n ¼ 3). (*Po0.05 vs 0 min).
and, subsequently, the concentration of molecular hydrogen (b) Kinetic analysis of the blood hydrogen levels after HW (3 ml) was measured in the kidney and in the serum. Both local and administration in the transplanted recipients that had been systemic concentrations of molecular hydrogen peaked given HW for 60 days were performed. Both isograft recipients (n ¼ 3) and allograft recipients (n ¼ 3) showed similar changes 15 min after ingestion (), proving that HW is an in circulating hydrogen concentrations. (c) Male LEW rats were effective mode of delivery of molecular hydrogen. We also used as donors for either syngeneic (LEW recipient) or allogeneic tested whether transplant recipients given long-term, daily HW (Brown Norway recipient) renal transplantation. Recipients had increased concentrations of circulating hydrogen. Similar were given either regular water (RW) or HW ad libitum aftertransplantation, resulting in a total of four experimental groups: kinetic changes in hydrogen concentration were observed in isograft given RW (Iso/RW, n ¼ 8) or HW (Iso/HW, n ¼ 8); or allograft both LEW recipients with LEW grafts (control rats; syngeneic given RW (Allo/RW, n ¼ 18) or HW (Allo/HW, n ¼ 17). The recipients transplantation prevents CAN) and Brown Norway (BN) receiving daily HW had improved allograft function, as measured recipients with LEW grafts treated with HW for 60 days by blood urea nitrogen (BUN), creatinine clearance (CCr), and 24 hurinary protein excretion, as compared with those receiving RW at . The baseline levels of the hydrogen detected in 60 days posttransplantation. (*Po0.05 vs Allo/RW).
circulation after 60 days of HW treatment was comparable tothat of naı¨ve animals, suggesting that there was no hydrogenaccumulation during long-term HW administration.
recipients (LEW donor and LEW recipient) were included ascontrols, as these rats do not develop CAN. Using a model of Oral administration of HW improves kidney function after kidney transplantation followed by bilateral nephrectomy, we found that rats receiving daily HW ad libitum had improved We then sought to determine the effect of HW adminis- allograft function, as measured by blood urea nitrogen, tration on kidney function after allotransplantation. Isograft creatinine clearance, and proteinuria, compared with those Kidney International JS Cardinal et al.: Hydrogen water prevented CAN receiving regular water (RW) at 60 days posttransplantation harbinger of allograft failure and, ultimately, of death.
These results suggest that HW can improve HW-treated recipients exhibited a significant increase in kidney function after allotransplantation.
survival (median survival 4 150 days) compared withRW-treated controls (median survival 78 days) Oral administration of HW improves weight gain and overall These results show that the improved allograft function that survival after allotransplantation was observed at 60 days posttransplantation in HW-treated To determine whether the improved allograft function recipients, as compared with RW-treated controls, also leads that was observed in HW-treated recipients correlated with to improved survival outcomes.
global parameters of well being, we measured the weights oftransplant recipients and found that the majority of animals Oral administration of HW prevents the progression of CAN that had undergone allotransplantation followed by admin- To determine whether the improved allograft function and istration of RW began to lose body weight B40 days after overall survival observed in HW-treated animals were transplantation. This process was significantly diminished attributable to decreased chronic rejection, histological in the allograft recipients that received HW .
analysis was performed on allografts obtained 60 days Furthermore, we found that weight loss proved to be a posttransplantation from both RW- and HW-treated reci-pients. Hematoxylin and eosin staining of the allografts obtained from HW-treated recipients exhibited decreased evidence of the hallmarks of CAN, including less glomerulo-sclerosis and inflammatory cell infiltration, as compared with hematoxylin and eosin staining of allografts obtained from RW-treated recipients Furthermore, Masson's trichrome staining and a-smooth muscle actin (aSMA) staining on allografts obtained from HW-treated recipients showed less interstitial fibrosis and smooth muscle prolifera- tion, respectively, compared with allografts obtained from RW-treated controls The expression of Body weight gain (%) aSMA, indicating myofibroblast accumulation in the grafts, was mostly seen in the interstitial areas. There was nodefinitive, aSMA-positive staining in the tubular epithelial cells. Histopathology of the control isografts from the same time point was similar to that of normal naı¨ve animals and Days after transplantation was not affected by HW administration (data not shown). Inaddition, immunohistochemistry for both CD3 and CD68 revealed fewer graft-infiltrating T cells and macrophages,respectively, in allografts obtained from HW-treated recipi- ents compared with those obtained from RW-treated controlsThese results proved to be statistically significant when the number of positive-staining cells per high-power field was counted for each sample Taken in total, these histological results suggest that allografts from HW-treated recipients experienced less CAN than did Recipient survival (%) those from RW-treated controls.
Oral administration of HW is an effective antioxidant strategyin the setting of kidney allotransplantation Days after transplantation As mentioned previously, molecular hydrogen possesses potent Figure 2 Hydrogen water (HW) administration improves antioxidant properties. Furthermore, oxidant stress-induced long-term survival after kidney allotransplantation.
(a) Although isograft recipients showed fair body weight gains tissue damage is believed to be a common pathway in many of regardless of intake of HW, the recipients with allografts given the pathophysiological mechanisms involved in the develop- regular water (RW) gradually lost body weight by 60–80 days after ment of CAN. Therefore, owing to the fact that HW adminis- transplantation. The recipients given HW showed better body tration resulted in increased local and systemic concentrations weight gain compared with those without HW. (b) In correlationwith improved graft function, survival of allograft recipients of molecular hydrogen, as well as decreased histological was significantly prolonged with oral administration evidence of CAN in kidney allograft recipients compared with of HW. (Isograft given RW (Iso/RW), (n ¼ 5); isograft given HW RW-treated controls, we next determined whether the protec- (Iso/HW), (n ¼ 5); allograft given RW (Allo/RW) (n ¼ 13); allograft tion from CAN seen with HW administration was accom- given HW (Allo/HW) (n ¼ 12); Kaplan-Meier, log-rank test,Po0.05 Allo/RW vs Allo/HW).
panied by a decrease in markers of oxidative tissue injury.
JS Cardinal et al.: Hydrogen water prevented CAN Number of positive Number of positive Figure 4 Hydrogen water (HW) administration decreasesintragraft inflammatory cell infiltration after kidneyallotransplantation. CD3-positive (a) and CD68-positive (b)infiltrating cells were assessed by immunohistochemistryand quantitated as number of positive-staining cells per high-power field (HPF) (original magnification 400) (c). Images are Figure 3 Hydrogen water (HW) administration decreases representative of five individual animals for each group; (n ¼ 5 chronic allograft nephropathy after kidney allotransplantation.
for each group, original magnification 200, *Po0.05 versus Hematoxylin/eosin (a), Masson's trichome (b), or a-smooth muscle Allo/RW). Allo/HW, allograft given HW; Allo/RW, allograft given actin (c) staining was performed 60 days posttransplantation to RW; Iso HW, isograft given HW; Iso RW, isograft given RW; RW, assess histological and/or immunohistochemical evidence of regular water.
chronic allograft nephropathy. Images are representative of fourseparate, individual grafts from each experimental group. Originalmagnification 200. Allo/HW, allograft given HW; Allo/RW, of inflammatory cytokineTherefore, because HW allograft given RW; RW, regular water.
administration led to decreased oxidative stress and slowedthe progression toward CAN in kidney allografts, we next Tissue malondialdehyde (MDA) levels, which indicate lipid determined whether HW treatment was also associated with peroxidation in cells and tissues, were significantly decreased a decrease in local inflammatory cytokine production.
in allografts obtained 60 days posttransplantation from Quantitative reverse transcription PCR revealed significantly HW-treated recipients compared with those obtained from lower levels of interleukin-6, tumor necrosis factor-a, RW-treated controls . Furthermore, immunohisto- intracellular adhesion molecule-1, and interferon-g mRNA chemistry performed on allografts obtained from HW-treated in kidney allografts obtained from HW-treated recipients recipients exhibited less 4-hydroxy-2-nonenal (HNE) and 60 days posttransplantation as compared with those obtained peroxynitrite staining compared with that seen in allografts from RW-treated controls These results obtained from RW-treated controls . These indicate that HW administration can attenuate the local results show that the local and systemic levels of molecular production of inflammatory markers in the setting of kidney hydrogen that are achieved through the administration of HW are sufficient to effectively reduce oxidative stress-inducedtissue damage in the setting of kidney allotransplantation.
Oral administration of HW decreases the activationof inflammatory signaling cascades after kidney Oral administration of HW decreases the local production of inflammatory markers in the setting of kidney Inflammatory intracellular signaling pathway activation (most notably activation of mitogen-activated protein One mechanism by which oxidative stress leads to the deve- kinases; MAP kinases) is a well-described event that contri- lopment of chronic rejection is by increasing the production butes to the progression of kidney allografts toward chronic Kidney International
JS Cardinal et al.: Hydrogen water prevented CAN Figure 6 Hydrogen water (HW) administration decreasesthe intragraft production of inflammatory cytokines afterkidney allotransplantation. Interleukin-6 (IL-6) tumor necrosisfactor-a (TNFa) intracellular adhesion molecule-1 (ICAM-1), andinterferon-g (IFNg) mRNA levels in the kidney grafts taken60 days after transplant were measured by real-time quantitativereverse transcriptase PCR analysis. Although allograft rejectioncaused upregulation of the mRNAs for these markers, HWsignificantly reduced the mRNA levels as compared with regular Figure 5 Hydrogen water (HW) administration decreases water (RW). (n ¼ 5, *Po0.05 vs Allo/RW). Allo HW, allograft given intragraft markers of oxidative stress after kidney HW; Allo RW, allograft given RW; GAPDH, glyceraldehyde-3- allotransplantation. (a) Intragraft malondialdehyde (MDA) levels phosphate dehydrogenase; Iso HW, isograft given HW; Iso RW, were quantitated using a commercially available kit. MDA levels in isograft given RW.
the allografts treated with regular water (RW) increased 60 daysafter transplantation. HW significantly reduced tissue MDA levels.
(n ¼ 5, *Po0.05 vs Allo/RW). In addition, immunohisto-chemistry for 4-hydroxy-2-nonenal (HNE) (b) and peroxynitrite indicate that HW administration can inhibit intracellular (c) was performed on formalin-fixed sections obtained from the signaling pathways that are known to contribute to the allograft groups. Oxidative injuries were more prominent in development of CAN in the setting of kidney transplantation the allografts given RW (Allo/RW) compared with those given HW (Allo/HW). Arrows indicate HNE-positive cells. (Originalmagnification 400; images are representative of three separateexperiments). Allo/HW, allograft given HW; Allo/RW, allograft given RW; Iso HW, isograft given HW; Iso RW, isograft given RW.
In this study, we found that both allograft function andoverall survival were improved in rats that had been fed witha diet supplemented with HW. Allografts from HW-treated rejection.Oxidative stress can activate MAP kinase rats exhibited less infiltration of inflammatory cells and signaling, which ultimately contributes to the proliferation suppressed activation of intragraft inflammatory signaling of mesangial cells in the setting of diabetic nephropathy.
pathways. The allografts from the HW-treated rats mani- Mesangial cell proliferation is also involved in the develop- fested fewer markers of oxidative stress and, ultimately, fewer ment of CAN. Therefore, we next determined whether HW progressed toward CAN as compared with controls. These administration could suppress MAP kinase activation in the results indicate that HW represents a potentially novel kidney allografts. Western blot analysis showed that MAP therapeutic strategy in the prevention of CAN in kidney kinases, including c-Jun N-terminal kinase, p-38, extra- cellular signal-regulated protein kinase as well as upstream Molecular hydrogen is produced continuously under kinase cascades (MEK-1), were less activated in allografts normal physiological conditions, primarily during the obtained from HW-treated recipients than in allografts fermentation of nondigestible carbohydrates by intestinal obtained from RW-treated recipients These results bacteria in the large intestine. This physiological production Kidney International
JS Cardinal et al.: Hydrogen water prevented CAN the present study are that (i) HW improves allograft function and overall survival by preventing CAN in a rodent model ofkidney transplantation, doing so in part by (ii) reducing oxidative stress-induced damage and (iii) reducing theactivation of inflammatory signaling pathways and cytokine The basis for the present study was the fact that oxidative stress is believed to be a common pathway that leads to thedevelopment of chronic rejection in kidney transplantation.
As mentioned earlier, markers of oxidative stress are elevatedin kidney transplant recipients and, in contrast, markers ofantioxidant pathways are diminished. Mechanistically, ROS activate inflammatory intracellular signaling pathways invascular smooth muscle cells,induce the epithelial-to- mesenchymal transitionparticipate in extracellular matrixdeposition by mesangial cellsand contribute to renal tubular atrophy through apoptosiand inflammatillof which are key processes involved in the pathogenesisof CAN.
Given the association described above between oxidative Figure 7 Hydrogen water (HW) administration decreases stress and the development of CAN, the finding that HW intragraft inflammatory signaling cascade activation after administration effectively decreases the intragraft accumula- kidney allotransplantation. Western blot analysis showed that tion of markers of oxidative stress, such as MDA, 4-HNE, and mitogen-activated protein (MAP) kinases (JNK (c-Jun NH2-terminal kinase), p38, ERK1/2 (extracellular signal-regulated peroxynitrite, suggests that the antioxidant properties of kinase 1/2)) and upstream kinase cascades (MEK-1), were less molecular hydrogen are likely to be responsible for the activated in allografts obtained from recipients that were beneficial effects on the allograft function and the prevention HW-treated compared with those obtained from regular water of progression of CAN that were observed in this rat model (RW)-treated recipients. Images are representative of threeindependent experiments. Allo, allograft recipients; Iso, isograft of kidney allotransplantation. We examined the intragraft recipients; p, phosphorylated; t, total.
expression of tumor necrosis factor-a, interleukin-6, inter-feron-g, and intracellular adhesion molecule-1 at the mRNAlevel, as surrogate markers of the deleterious processes of hydrogen gas may be responsible for the baseline levels of downstream from oxidative damage, and found less expres- hydrogen detected in circulation. It is excreted as flatus, sion of these inflammatory cytokines in HW-treated further metabolized by gut flora, or exhaled as a natural recipients, which are well-described mediators of the component of abdominal gas. However, molecular hydrogen fibrogenesis phase of CAN.Furthermore, MAP kinase has known physiological roles during conditions of home- signaling, which is known to be induced by oxidative damage ostasis. Recent evidence indicates that inhaled hydrogen gas in the setting of kidney transplantation, was decreased in has antioxidant and anti-apoptotic properties that can HW-treated recipients. MAP kinase signaling contributes to protect organs from ischemia-reperfusion-induced injury by the development of CAN by mediating the action of growth selectively scavenging detrimental ROS. The mechanism of factors, such as transforming growth factor-bparticipating action of inhaled hydrogen gas in these models involves its in the proliferation of vascular smooth muscle cellsand the ability to prevent oxidative damage, as indicated by decreased extracellular matrix,and contributing to the intragraft nucleic acid oxidation and lipid peroxidation.Although infiltration of mononuclear inflammatory cells through the the concentration of gaseous molecular hydrogen used in the production of chemoattractan above studies (B4%) is lower than the threshold at which it Therefore, we conclude that HW, through its ability to act is known to be flammable (4.6%), flammability is still a as an effective method of delivery for molecular hydrogen, realistic concern which may limit the translational appli- can reduce the development of CAN in a rat model of kidney cability of inhaled molecular hydrogen. Therefore, HW allotransplantation. This improves allograft function and represents a novel and easily translatable method of delivery overall survival in HW-treated recipients. The mechanism of of molecular hydrogen. To our knowledge, this is the first protection afforded to HW-treated recipients likely involves report describing the preventative effects of molecular the ability of molecular hydrogen to reduce oxidative stress- hydrogen, delivered in water containing therapeutic doses, induced damage, which is believed to be an upstream on the development of chronic rejection in the setting mediator that contributes to the ultimate development of of kidney allotransplantation and, as such, represents a CAN. Consequently, HW may be an effective and novel tool potentially novel and easily applicable solution to a difficult in the clinical armamentarium against oxidative stress- clinical scenario (that is, CAN). The major novel findings of induced pathologies, in general, and CAN, in particular.
Kidney International JS Cardinal et al.: Hydrogen water prevented CAN MATERIALS AND METHODS were followed until recipient death due to graft failure or for 150 days after transplantation.
Inbred male Lewis (LEW, RT1l) and Brown Norway (BN, RT1n) ratsweighing 200–250 g were purchased from Harlan Sprague Dawley Evaluation of graft function (Indianapolis, IN, USA). Animals were maintained in cages in a Renal graft function was assessed by measuring plasma creatinine specific pathogen-free facility at the University of Pittsburgh and fed (P(Cr)) and blood urea nitrogen levels, as well as urinary protein a standard diet with free access to water. All procedures were excretion and urinary creatinine (U(Cr)) levels using an auto- performed according to the guidelines of the Institutional Animal analyzer (Beckman Instruments, Fullerton, CA, USA). Creatinine Care and Use Committee at the University of Pittsburgh and the clearance (CCr; ml/min) was calculated using the formula (CCr; National Research Council's Guide for the Humane Care and Use of ml/min) ¼ (U(Cr) urine volume)/(P(Cr) time). Urine samples Laboratory Animals.
were collected using metabolic cage systems.
Kidney transplantation transplantation was Formalin-fixed graft tissues were paraffin embedded, cut into 5 mm previously described technique.In short, after intravenous sections, and stained with hematoxylin/eosin, modified Masson's heparinization (300 U), the donor's left kidney was removed with trichrome, or Verhoeff 's elastic tissue stain (Rowley Biochemical the left renal artery in continuity with a short aortic segment and the Institute, Danvers, MA, USA). Sections were also immunohisto- left renal vein with a patch of vena cava. The excised graft was chemically analyzed using the avidin–biotin–peroxidase complex flushed with 3 ml of UW solution (Viaspan, Du Pont, Wilmington, method after antigen retrieval and incubation with mouse anti-SMA DE, USA). The left kidney graft was orthotopically transplanted into (a-SMA, DAKO, Carpinteria, CA, USA), monoclonal anti-rat the recipient by end-to-side microvascular anastomoses between CD68 (ED1, Serotec, Raleigh, NC, USA), or monoclonal anti-rat graft aorta and recipient infrarenal abdominal aorta, and between CD3 (Serotec), followed by incubation with LSAB þ horseradish graft renal vein and recipient infrarenal vena cava with 10-0 Micrin peroxidase (DAKO). Tissue oxidative injury was also assessed by suture. Both native kidneys of the recipient were removed, and end- immunostaining with rabbit polyclonal anti-nitrotyrosine (Santa to-end ureteral anastomosis was performed using 10-0 Micrin Cruz Biotechnology, Santa Cruz, CA, USA) and mouse monoclonal suture. Recipients received prophylactic antibiotics (Cefotetan anti-4-HNE antibody (JAICA, Shizuoka, Japan), followed by disodium, 100 mg/kg, intramuscular injection) for 3 days after the incubation with biotinylated anti-mouse IgG antibody (Vector Laboratories, Burlingame, CA, USA) and avidin–biotin–peroxidasecomplex (Vector Laboratories). Diaminobenzidine was used as the Oral administration of HW peroxidase substrate. In each study, a set of sections was stained Hydrogen water was produced by Blue Mercury (Tokyo, Japan) without the primary antibody as a negative control.
using a HW-producing apparatus, by which molecular hydrogen gaswas dissolved in water under a pressure of 0.4 MPa, as previously Assessment of tissue MDA Tissue MDA levels were assessed using the BIOXYTECH MDA-586 40.6 mmol/l) was stored in an aluminum bag and placed in a kit (OxisResearch, Portland, OR, USA), as previously described.
glass vessel twice a day. Control animals were treated with RW, Kidney samples were homogenized in buffer (pH 7.9) containing generated by degassing HW by gentle stirring for 24 h.
5 mmol/l butylated hydroxytoluene to prevent sample oxidation andstored at 80 1C. Once all samples were collected, they were thawed Determination of hydrogen levels in blood and tissue on ice and 10 mg of probucol was added to further minimize Hydrogen water (3 ml) was orally administered by gavage to naı¨ve oxidative reactions, then N-methyl-2-phenylindole (NMPI) in 25% LEW rats or transplant recipients that had already been treated with methanol and 75% acetonitrile were added to the supernatants, HW for 460 days. Arterial blood and kidney tissue were taken at followed by addition of 1 hydrogen chloride (HCL) and incubated 15, 30, and 60 min after oral administration of HW. Blood or at 45 1C for 60 min. A standard curve using 1,1,3,3-tetra-methoxy- homogenized kidney tissue was placed in a glass tube and air-phase propane (TMOP), which generates free MDA during the acid hydrogen levels were measured by gas-chromatography (Biogas hydrolysis step, was also prepared. All samples and standards were analyzer BAS-1000, Mitleben, Osaka, Japan).
centrifuged (10,000 g, 10 min) and absorbance at 586 nm wasmeasured using the Spectronic Biomate 3 (Thermo Scientific, Experimental groups Waltham, MA, USA) according to the manufacturer's protocol.
Four experimental groups were analyzed: LEW to LEW syngeneicgrafts treated with RW (Group 1), LEW to LEW syngeneic grafts SYBR green real-time reverse transcription PCR treated with HW (Group 2), LEW to BN allogeneic grafts treated The levels of mRNAs for interleukin-6, intracellular adhesion with RW (Group 3), and LEW to BN allogeneic grafts treated with molecule-1, tumor necrosis factor-a, interferon-g, and glyceral- HW (Group 4). Recipients of allografts received daily intramuscular dehyde-3-phosphate dehydrogenase were quantified in duplicate injections of tacrolimus (FK506, Astellas Pharmaceutical, Tokyo, using SYBR Green two-step, real-time reverse transcription PCR, as Japan) at a dose of 0.5 mg/kg for 7 days (days 0–6), whereas those of previously Briefly, 1 mg of RNA from each sample was isografts received no immunosuppressant. Recipients were killed at used for reverse transcription with oligo dT primers (Invitrogen, 60 or 150 days after transplantation, and blood and kidney graft Carlsbad, CA, USA) and Superscript II enzyme (Invitrogen) to samples were obtained after being cleared of blood by flushing with generate first-strand cDNA. The PCR reaction mixture was prepared Lactated Ringers solution. Kidney tissue was snap-frozen and stored using SYBR Green PCR Master Mix (PE Applied Biosystems, Foster at 80 1C until use or fixed in 10% buffered formalin for routine City, CA, USA). Each sample was analyzed in duplicate using the histopathology. For survival study experiments, recipient animals conditions recommended by the manufacturer. Gene expression was Kidney International JS Cardinal et al.: Hydrogen water prevented CAN normalized with glyceraldehyde-3-phosphate dehydrogenase mRNA Amuchastegui SC, Azzollini N, Mister M et al. Chronic allograft nephropathy in the rat is improved by angiotensin II receptor blockadebut not by calcium channel antagonism. J Am Soc Nephrol 1998; 9:1948–1955.
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