RESEARCH ARTICLE

Organ preservation solution containing
dissolved hydrogen gas from a hydrogenabsorbing alloy canister improves function oftransplanted ischemic kidneys in miniature
pigs
Eiji Kobayashi1,2,3, Motoaki Sano ID1,3*

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1 Department of Cardiology, Keio University School of Medicine, Tokyo, Japan, 2 Department of Organ
Fabrication, Keio University School of Medicine, Tokyo, Japan, 3 Center for Molecular Hydrogen Medicine,
Keio University, Tokyo, Japan* msano@a8.keio.jp

Abstract
OPEN ACCESS
Citation: Kobayashi E, Sano M (2019) Organpreservation solution containing dissolvedhydrogen gas from a hydrogen-absorbing alloy
canister improves function of transplanted
ischemic kidneys in miniature pigs. PLoS ONE 14(10): e0222863. https://doi.org/10.1371/journal.
pone.0222863
Editor: Tohru Minamino, Niigata Daigaku, JAPANReceived: May 29, 2019
Accepted: September 9, 2019
Published: October 1, 2019
Peer Review History: PLOS recognizes thebenefits of transparency in the peer review
process; therefore, we enable the publication of
all of the content of peer review and author
responses alongside final, published articles. The
editorial history of this article is available here:
https://doi.org/10.1371/journal.pone.0222863
Copyright: © 2019 Kobayashi, Sano. This is an
open access article distributed under the terms of
the Creative Commons Attribution License, which
permits unrestricted use, distribution, andreproduction in any medium, provided the original
author and source are credited.

Various methods have been devised to dissolve hydrogen gas in organ preservation solutions, including use of a hydrogen gas cylinder, electrolysis, or a hydrogen-generating
agent. However, these methods require considerable time and effort for preparation. We
investigated a practical technique for rapidly dissolving hydrogen gas in organ preservation
solutions by using a canister containing hydrogen-absorbing alloy. The efficacy of hydrogen-containing organ preservation solution created by this method was tested in a miniature
pig model of kidney transplantation from donors with circulatory arrest. The time required for
dissolution of hydrogen gas was only 2–3 minutes. When hydrogen gas was infused into a
bag containing cold ETK organ preservation solution at a pressure of 0.06 MPa and the bag
was subsequently opened to the air, the dissolved hydrogen concentration remained at 1.0
mg/L or more for 4 hours. After warm ischemic injury was induced by circulatory arrest for
30 minutes, donor kidneys were harvested and perfused for 5 minutes with hydrogen-containing cold ETK solution or hydrogen-free cold ETK solution. The perfusion rate was fasterfrom the initial stage with hydrogen-containing cold ETK solution than with hydrogen-free
ETK solution. After storage of the kidney in hydrogen-free preservation solution for 1 hour
before transplantation, no urine production was observed and blood flow was not detectedin the transplanted kidney at sacrifice on postoperative day 6. In contrast, after storage inhydrogen-containing preservation solution for either 1 or 4 hours, urine was detected in the
bladder and blood flow was confirmed in the transplanted kidney. This method of dissolvinghydrogen gas in organ preservation solution is a practical technique for potentially converting damaged organs to transplantable organs that can be used safely in any clinical setting
where organs are removed from donors.

Data Availability Statement: All relevant data arewithin the paper and its Supporting Information
files.

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Dissolving hydrogen gas in organ preservation solution with a hydrogen-absorbing alloy canister

Funding: This study was funded by Doctors Man
Co., Ltd. (to Eiji Kobayashi). This funder had no rolein study design, data collection and analysis,
decision to publish, or preparation of themanuscript.
Competing interests: Eiji Kobayashi received a
research grant from Doctors Man Co., Ltd.. Keio
University and Doctors Man Co., Ltd. have jointly
applied for a patent (patent no. 2019-081425). Eiji
Kobayashi and Motoaki Sano are the inventors of
this patent. This does not alter our adherence to
PLOS ONE policies on sharing data and materials.

Introduction
Transplantation of marginal organs from donors after circulatory arrest is an important option
to reduce the waiting period for recipients. Progress has been made in perfusion of donororgans with diluted donor blood [1], but there are few methods that can be easily used at manysites where donors may be found.
Hydrogen gas has been shown to have various biological effects, including suppression ofischemia-reperfusion injury in animal studies [2–5]. Ischemia-reperfusion injury is an inevitable complication of solid organ transplantation and limiting this type of injury can increasegraft survival. Use of hydrogen gas has been reported to be effective in transplantation models
of various organs, including the small intestine [6–7], lung [8–14], liver [15–18], heart [19, 20],
osteochondral tissue [21], and kidney [22]. It is possible to expose the excised organ to hydrogen gas ex vivo without the donor and/or recipient inhaling the gas, and various methods havebeen devised to dissolve hydrogen gas in organ preservation solutions, including use of ahydrogen gas cylinder [6], electrolysis [18, 20, 21, 22], or a hydrogen-generating agent [17].
However, these methods require bulky equipment and dangerous high-pressure cylinders withstrict regulations for handling, resulting in the need to expend considerable time and effort for
preparation. Therefore, it is probably unrealistic to attempt the introduction of such methods
into the clinical setting. Accordingly, a simple technique for rapidly dissolving hydrogen gas inorgan preservation solutions is required.
A hydrogen-absorbing alloy is a compound that absorbs hydrogen when it is cooled orpressurized and then releases hydrogen when it is heated or depressurized. A hydrogenabsorbing alloy canister is filled with such an alloy, and these canisters have been used to supply hydrogen for fuel cells. We have developed a method of using a hydrogen-absorbing alloy
canister to rapidly and conveniently dissolve hydrogen in organ preservation solutions at highconcentrations. The canister storing hydrogen can be safely transported anywhere and can beeasily connected to a bag containing conventional organ preservation solution, allowinghydrogen to be dissolved in organ preservation solution within a few minutes at the site ofdonor organ harvesting.
In the present study, the efficacy and safety of cold organ preservation solution containinghydrogen dissolved by this method were tested in a miniature pig model of kidney transplantation from donors with circulatory arrest. Previous studies were performed in juvenile domestic
pigs, but we used miniature pigs to more closely reflect the clinical setting [23]. After circulatory arrest for 30 minutes, kidneys were harvested from the donor, flushed out, and stored ineither hydrogen gas-containing organ preservation solution or conventional organ preservation solution. Then early kidney function after transplantation was compared between the two
methods of preservation.

Materials and methods
Miniature pigs
Conventionally, animal studies on kidney transplantation have been performed using juvenile
domestic pigs (about 4 months old). However, the donors and recipients were adult miniaturepigs in the present study because this was considered to more closely correspond to the clinicalsetting. Miniature pigs do not exceed 30 Kg in weight when fully grown after 2 years. We usedfemale pigs aged 25–40 months and weighing 20–26 Kg, purchased from Fujimicra Ltd., Shizuoka, Japan. Animals were treated in accordance with the Animal (Scientific Procedures)
Protection Act 1986 of the United Kingdom. The pigs were housed in cages under temperatureand light-controlled conditions (12-hour light/dark cycle) and were provided with food and

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Dissolving hydrogen gas in organ preservation solution with a hydrogen-absorbing alloy canister

water ad libitum. The pigs were fasted for 12 hours prior to surgery with free access to water.
Sedation with a mixture of midazolam/medetomidine/butorphanol was followed by endotracheal intubation and mechanical ventilation. Anesthesia was maintained with inhalational isoflurane. Midazolam and medetomidine were added according to the depth of anesthesia.
Buprenorphine was administered as an intraoperative analgesic management. After intravenous administration of pentobarbital, saturated potassium chloride was rapidly administeredintravenously to euthanize. Three animals were used to examine the flushing effect of thehydrogen-containing organ preservation solution and 9 animals were used for the organ transplantation experiment (one donor and two recipients were used for one set of experiments;n = 3). These experiments were conducted with the approval of the Research Council and Animal Care and Use Committee of Keio University [approval no: 16072-(1)]. Surgery was performed by a surgeon with over 200 clinical transplant operations, who is a steering memberof the transplantation society and a permanent director of the transplantation society of Japan(E.K.).

Hydrogen gas dissolution system based on a hydrogen-absorbing alloy
canister
The hydrogen-absorbing alloy canister (Japan Steel Works, Ltd.) was a cylinder containing ahydrogen-absorbing alloy that can reversibly absorb and release hydrogen (Fig 1A). Since theinternal pressure of the canister does not exceed 1 MPa, it does not correspond to high-pressure gas according to the Japan High-Pressure Gas Safety Act. The device for transferringhydrogen was assembled just before use and hydrogen was infused into the organ preservation
solution from the hydrogen-absorbing alloy canister via a pressure regulating valve (Fig 1Band 1C). When the gauge pressure reached 0.06 Mpa, the plastic bag containing the organ
preservation solution was disconnected from the pressure regulating valve. After vigorous
manual shaking for 30 seconds or more, the bag containing the organ preservation solution
was placed on ice and opened to the atmosphere. Then the concentration of hydrogen gas in
the solution was measured over time by gas chromatography (TRIlyzer mBA-3000; Taiyo Co.,
Ltd., Kochi, Japan).

Investigation of the effectiveness of hydrogen-containing preservation
solution for flushing kidneys from donor pigs with circulatory arrest
A midline upper abdominal incision was made under general anesthesia, and the left and rightkidneys were dissected. Then the chest was opened and the thoracic aorta was clamped toinduce ischemia of all abdominal organs. After 30 minutes of warm ischemia, the kidneys were
removed for use as donor organs. The donor animal was not treated with an anticoagulant
such as heparin. The excised kidneys were immediately placed on the back table, divided into
the right and left kidneys, and perfused for 5 minutes by gravity feed from a height of 1 m.
One kidney was perfused with hydrogen-containing cold extracellular-type trehalose-containing Kyoto (ETK) solution and the other was perfused with hydrogen-free cold ETK solution.
The number of drops of the solution per minute was counted for each kidney.

Evaluation in a kidney transplantation model
After flushing with either hydrogen-containing cold ETK solution or hydrogen-free cold ETK
solution for 5 minutes, the harvested kidneys were immersed and stored in the same ETK solution for 1 to 4 hours until transplantation into the recipient.

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Dissolving hydrogen gas in organ preservation solution with a hydrogen-absorbing alloy canisterFig 1. Novel method of dissolving hydrogen gas in organ preservation solution by using a hydrogen-absorbing
alloy canister. (A) Mechanism of the hydrogen absorbing alloy. (B) Process of dissolving hydrogen in the organ
preservation solution by using the hydrogen-absorbing alloy canister. (1) A hydrogen-absorbing alloy canister and apressure regulating valve are connected to a bag of organ preservation solution. (2) Hydrogen gas is injected throughthe rubber stopper using a bottle needle. When the pressure reaches 0.06 Mpa, the bag of organ preservation solution is
disconnected from the pressure regulating valve and hydrogen-absorbing alloy canister. (3) The organ preservation
solution in the plastic bag is shaken vigorously by hand for more than 30 seconds. (4) The bag is opened to theatmosphere. (C) Components of the device for infusing hydrogen gas into organ preservation solutions.
https://doi.org/10.1371/journal.pone.0222863.g001

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Dissolving hydrogen gas in organ preservation solution with a hydrogen-absorbing alloy canister

Under general anesthesia, a midline abdominal incision was made to expose the recipient’s
left renal artery and vein [24]. After intravenous administration of 1 ml of heparin, the abdominal aorta was clamped immediately above the renal artery bifurcation. Then the renal vein
was clamped with bulldog forceps, and the left kidney was resected from the renal artery leaving a Carrel patch configuration. Next, the stored donor kidney was anastomosed end-to-end
with 5–0 nylon to the renal artery at the Carrel patch site. After arterial anastomosis, theperipheral renal artery was clipped again before unclamping the abdominal aorta. The total
aortic clamp time was 30 minutes. Subsequently, the renal veins were joined by end-to-end
continuous anastomosis with 6–0 nylon, and finally the ureter was anastomosed with 6–0
nylon knotted sutures. The transplanted kidney was wrapped in a thermal bag, and the temperature was kept at 20 ˚C or less by appropriate infusion of cold ETK solution [25]. Warmischemic time was set at 1 hour. After reperfusion, blood flow was confirmed in the transplanted kidney. Then the right native kidney was excised and the abdomen was closed. Cefazolin sodium (25mg/kg) was given on the first postoperative day, and the animals were allowed
free access to both food and water. The animals were observed for up to 6 days after surgery,and then were sacrificed for laboratory tests, urinalysis, and histopathological examination of
the transplanted kidneys. Blood urea nitrogen (BUN) and creatinine (CRE) were measured inperipheral blood and urine samples by the urease-GLDH method and enzymatic method,respectively. Total urinary protein was measured by the pyrogallol red technique and urinary
electrolytes were measured by an ion-selective electrode method, while inorganic phosphate(IP) was determined by an enzymatic method and glucose by the hexokinase/glucose-6-phosphate dehydrogenase method. The transplanted kidneys were fixed in 10% neutral bufferedformaldehyde solution and cut in the longitudinal direction to include the papilla centering on
the cortex. After embedding by a conventional method, thin sections were prepared and were
stained with hematoxylin and eosin (H. E.) and Elastica van Gieson (EVG) stain. Histologicalexamination was done by blinded pathologists and renal graft pathology was assessed according to the Banff classification [26, 27].

Results
Hydrogen concentration profile in organ preservation solutions
The time required for infusion of hydrogen gas was 2–3 minutes. Changes of the hydrogenconcentration in three organ preservation solutions after infusion of hydrogen gas are shownin Fig 2. At the same pressure, UW solution and HTK solution had higher hydrogen concentrations than ETK solution. The hydrogen concentration declined very slowly at 4 ˚C undernormal pressure and was 1 mg/L or more after 4 hours in all of the solutions. As ETK solution
does not require strict temperature control for kidney preservation [28, 29], subsequent experiments on flushing and preservation of donor kidneys were performed using this solution.

Flushing effect of hydrogen-containing organ preservation solution
Transplantation of marginal donor organs was performed to assess the effectiveness of coldhydrogen-containing ETK solution generated by our hydrogen gas infusion method. We usedminiature pigs (25–40 months old) to more closely reflect the clinical transplantation setting.
After warm ischemic injury was created by circulatory arrest for 30 minutes, the donor kidneys
were harvested and perfused for 5 minutes with hydrogen-containing cold ETK solution orhydrogen-free cold ETK solution (Fig 3A). When hydrogen-containing cold ETK solution wasused, the perfusion rate was faster from the initial stage of perfusion than with hydrogen-free
ETK solution (Table 1). At the end of perfusion, visual inspection indicated that perfusion

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Dissolving hydrogen gas in organ preservation solution with a hydrogen-absorbing alloy canisterFig 2. Changes of the hydrogen concentration in organ preservation solutions (UW, HTK, and ETK) at atmospheric pressure after
infusion of hydrogen gas.
https://doi.org/10.1371/journal.pone.0222863.g002

Fig 3. Effectiveness of kidney flushing with hydrogen-containing ETK solution. A. The kidneys were perfused for 5 minutes by gravity feed
(1 m). One kidney each was perfused with hydrogen-containing ETK solution and hydrogen-free ETK solution. B. 1) Kidney perfused withhydrogen-containing ETK solution; 2) Kidney perfused with hydrogen-free ETK solution.
https://doi.org/10.1371/journal.pone.0222863.g003

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Dissolving hydrogen gas in organ preservation solution with a hydrogen-absorbing alloy canister

Table 1. ETK solution perfusion rate by gravity feed (1 m).
Experiment 1

Experiment 2

Time (min)

0–1

1–2

2–3

3–4

4–5

0–1

1–2

2–3

3–4

4–5

Hydrogen-containing ETK solution

71

71

81

88

89

137

139

146

150

168

Hydrogen-free ETK solution

60

63

68

68

73

78

78

83

85

88

The perfusion rate is shown as drops per minute.
https://doi.org/10.1371/journal.pone.0222863.t001

with hydrogen-containing ETK solution was more effective than use of hydrogen-free ETK
solution (Fig 3B).

Early post-transplant assessment of kidneys preserved with or withouthydrogen-containing ETK solution
All of the recipient miniature pigs survived until the end of the observation period. After kidney storage for 1 hour before transplantation in hydrogen gas-free preservation solution, nourine was observed in the bladder and no blood flow was detected in the transplanted kidneyat sacrifice on postoperative day 6 (Table 2). On the other hand, after kidney storage in hydrogen gas-containing preservation solution for either 1 hour or 4 hours, blood flow was detectedin the transplanted kidney and urine was found in the bladder on postoperative day 6. Aftertransplantation of kidneys stored for 1 hour in hydrogen gas-free ETK solution, the blood levels of BUN and creatinine in the recipients were 270 ± 29.5 mg/dL and 4.72 ± 2.72 mg/dL,
respectively (n = 3). After transplantation of a single kidney stored for 1 hour in hydrogencontaining ETK solution, blood levels of BUN and creatinine were 83 mg/dL and 4.4 mg/dL(n = 1), respectively. Therefore, the kidney storage time before transplantation was extended
to 4 hours for the remaining two pigs in the hydrogen-containing ETK solution group. Despite
longer storage, blood levels of BUN and creatinine were low at the time of sacrifice in 1 ofthese 2 animals, being 108 mg/dL and 8.2 mg/dL, respectively.
Histopathological findings are displayed in Fig 4. In the hydrogen-free ETK solution group,
all three kidneys showed extensive cortical necrosis (pan-necrosis). In the hydrogen gas-containing ETK solution group, there was evidence of acute tissue injury, but not cortical necrosis.
Tubular dilation and cellular infiltration were noted at a low magnification, while infiltrationof mononuclear cells and capillary occlusion were observed in the glomeruli at a highmagnification. Infiltration of mononuclear cells and lymphocytes was also observed in the
Table 2. Laboratory data on day 6 after kidney transplantation.
Blood
Group
Hydrogen-free ETK solution

Hydrogen-containing ETK
solution

Storage time (hr)

Urine

BUN
CRE
U-TP
U-UN U-CRE
U-Na
U-K
U-CI
U-IP
U-GLU
(mg/dL) (mg/dL) (mg/dL) (mg/dL) (mg/dL) (mEq/L) (mEq/L) (mEq/L) (mg/dL) (mg/dL)

No.1

1

261

22.8

N.T

No.2

1

224

20.0

N.T

No.3

1

325

29.2

No.1

1

83

4.4

327.6

405.5

99.07

15

35.5

12

0.8

8

No.2

4

108

8.2

41.0

682.5

81.28

23

30.6

13

44.2

1

No.3

4

239

33.6

2491.0

290.1

51.11

89

39.9

75

17.9

61

N.T

N.T; Not tested (no urine in the bladder)
https://doi.org/10.1371/journal.pone.0222863.t002

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Dissolving hydrogen gas in organ preservation solution with a hydrogen-absorbing alloy canisterFig 4. Histological findings of the kidneys on day 6 after transplantation. Specimen numbers correspond to those in
Table 2. A, C, E, G: low magnification (40x), B, D, F, H: high magnification (400x). (A, B) A kidney preserved by usinghydrogen-free ETK solution shows extensive necrosis in most of the microscopic field, indicating pan-necrosis of thecortex. (C-H) No cortical necrosis was observed in kidneys preserved by using hydrogen gas-containing ETK solution.
However, tubular dilation and interstitial mononuclear cell infiltration are evident.
https://doi.org/10.1371/journal.pone.0222863.g004

tubulointerstitial region. The pathological findings of transplanted kidneys preserved by usinghydrogen-containing ETK solution are summarized in Table 3.

Discussion
In this study, we developed a method of producing hydrogen-containing organ preservation
solution by rapidly infusing hydrogen gas into organ preservation solution from a canistercontaining hydrogen-absorbing alloy. Since the source of hydrogen gas is a hydrogen-absorbing alloy canister, it can be easily and safely brought to the site of donor organ extraction, andhydrogen gas can be quickly infused into the organ preservation solution even in an emergency. Then the hydrogen-containing organ preservation solution can be used to flush thedonor organ and preserve it during transport. When hydrogen gas was infused into a bag containing cold ETK organ preservation solution at a pressure of 0.06 MPa and the bag was subsequently opened to the air, we confirmed that the dissolved hydrogen concentration remained
at 1.0 mg/L or higher for 4 hours.
While it has been shown that hydrogen gas is effective for preventing ischemia reperfusion
injury [1–4], hydrogen gas is flammable at high concentrations and clinical use is limited bysafety considerations. Our previous investigator-initiated clinical trial demonstrated that inhalation of hydrogen gas combined with coronary intervention could inhibit left ventricularremodeling after myocardial infarction [30]. In that trial, the subjects inhaled a gas mixture ofhydrogen (1.3%), oxygen, and nitrogen. However, it is considered difficult to use a cylinder ofpure hydrogen gas to dissolve hydrogen in an organ preservation solution at the site of organ
donation.
Previous methods for dissolving hydrogen in organ preservation solutions have involved
generation of hydrogen by electrolysis [18, 20, 21, 22] or by use of a hydrogen-generating
agent [17], after which the hydrogen gas undergoes passive diffusion into the organ preservation solution. However, these methods are complicated and are unsuitable for clinical use

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Dissolving hydrogen gas in organ preservation solution with a hydrogen-absorbing alloy canister

Table 3. Detailed histopathological findings of transplanted kidneys preserved with hydrogen-containing ETK
solution.
Findings/ specimen no.

1

2

3

Cortical necrosis

0

0

0

Cell debris

0

0

0

Acute rejection

2

1

3

Glomerulitis

3

1

2

Glomerular thrombus

0

0

1

Thrombus002Interstitial mononuclear cell infiltration

3

2

3

Tubulitis

3

2

3

Intimal arteritis

2

0

3

Intimal arteritis in medulla

0

3F)

0

Chronic allograft nephropathy

1

1

1

Chronic transplant glomerulopathy, mesangial proliferation

2

1

2

Interstitial fibrosis

1

1

1

Tubular atrophy and loss

1

2

3

Tubular degeneration and necrosis

0

1

2

Tubular dilatation

2

2

3

Tubular regeneration

0

1

1

0

2

3

Hyaline casts

1

2

3

Cellular casts

1

1

2

Neutrophil/ eosinophil infiltration

1

1

1

Hemorrhage

1

1

1

Congestion, outer medulla

2

1

1

Fibrous intimal thickening with elastica fragmentation
Other

Grading system: 0; No change, 1: Mild, 2: Moderate, 3; Severe.
Fibrinoid change: nonspecific changes in arteries or veins

F)https://doi.org/10.1371/journal.pone.0222863.t003

since it requires 24–48 hours for the hydrogen gas concentration in the solution to reach 1
mg/L.
When hydrogen-containing ETK solution was compared with hydrogen-free ETK solution
for perfusion of donor kidneys, the former showed a higher flow rate from the start of perfusion. Examination of the perfused tissue revealed dilation of capillaries and washout of
microthromboses.
In this study, we performed short-term observation (up to the 6th postoperative day) after
transplantation of kidneys with warm ischemic injury from donor miniature pigs with circulatory arrest. When transplantation was done after storage of the kidney in hydrogen gas-freeorgan preservation solution for 1 hour, the outcome was always primary non-function. In contrast, when transplantation was performed after storage in hydrogen-containing solution for
up to 4 hours, blood flow was detected in the transplanted kidney and production of urine wasobserved. These results suggest that our method could potentially be used clinically for restoring damaged organs to transplantable organs.
Some limitations of our study should be considered. We focused on the acute postoperative
period, so it is also necessary to examine long-term renal function in recipients on immunosuppressant therapy in the future. In addition, kidneys treated with the hydrogen gas-containing organ preservation solution still developed severe acute tubulointerstitial injury. However,

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Dissolving hydrogen gas in organ preservation solution with a hydrogen-absorbing alloy canister

it is possible that the combination of hydrogen-containing organ preservation solution with an
intraoperative rinse [31], intraoperative or postoperative immunosuppressant therapy, or
adjuvant cell therapy using cells such as mesenchymal stromal cells [32, 33] could allow kidneys from marginal donors that have not been usable to be transplanted in the future.

Conclusion
In conclusion, we developed a device that can be used to rapidly infuse hydrogen gas into
organ preservation solution at the site of donor organ removal. It is possible that flushing andpreservation of organs with hydrogen gas-containing solution could increase the feasibility of
performing transplantation from marginal donors.

Supporting information
S1 File. NC3Rs ARRIVE guidelines checklist.
(PDF)

Acknowledgments
This work was supported by grants from Doctors Man Co., Ltd. (E.K.). Sou Hashimoto (Doctors Man Co., Ltd.) assisted with devising the system for infusing hydrogen gas into organ
preservation solution.

Author Contributions
Supervision: Motoaki Sano.
Writing – original draft: Eiji Kobayashi, Motoaki Sano.

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