ORIGINAL RESEARCH
published: 17 February 2021
doi: 10.3389/fimmu.2020.626295

Prevention of Chronic Rejectionof Marginal Kidney Graft by Usinga Hydrogen Gas-Containing
Preservation Solution and Adequate
Immunosuppression in a Miniature
Pig Model
Kotaro Nishi 1†, Satomi Iwai 1†, Kazuki Tajima 2,3, Shozo Okano 1, Motoaki Sano 4
and Eiji Kobayashi 5*
Edited by:
Hao Wang,
Tianjin Medical University General
Hospital, China
Reviewed by:Alexandra R. Lucas,
Arizona State University, United States
Zheng Jenny Zhang,Northwestern University,
United States*Correspondence:
Eiji Kobayashiorganfabri@a2.keio.jp†

These authors have contributed
equally to this work and shareﬁrst authorship

Specialty section:
This article was submitted to
Alloimmunity and Transplantation,a section of the journal
Frontiers in Immunology
Received: 05 November 2020
Accepted: 29 December 2020
Published: 17 February 2021
Citation:
Nishi K, Iwai S, Tajima K, Okano S,
Sano M and Kobayashi E (2021)
Prevention of Chronic Rejection of
Marginal Kidney Graft by Using a
Hydrogen Gas-Containing
Preservation Solution and
Adequate Immunosuppression
in a Miniature Pig Model.
Front. Immunol. 11:626295.
doi: 10.3389/fimmu.2020.626295

1 Laboratory of Small Animal Surgery 2, School of Veterinary Medicine, Kitasato University, Towada, Japan, 2 Laboratory of
Small Animal Internal Medicine 2, School of Veterinary Medicine, Kitasato University, Towada, Japan, 3 Department ofSurgery, Keio University School of Medicine, Tokyo, Japan, 4 Department of Cardiology, Keio University School of Medicine,Tokyo, Japan, 5 Department of Organ Fabrication, Keio University School of Medicine, Tokyo, Japan

In clinical kidney transplantation, the marginal kidney donors are known to develop chronic
allograft rejection more frequently than living kidney donors. In our previous study, we havereported that the hydrogen gas-containing organ preservation solution prevented thedevelopment of acute injuries in the kidney of the donor after cardiac death by using
preclinical miniature pig model. In the present study, we veriﬁed the impact of hydrogengas treatment in transplantation with the optimal immunosuppressive protocol based onhuman clinical setting by using the miniature pig model. Marginal kidney processed byhydrogen gas-containing preservation solution has been engrafted for long-term (longer
than 100 days). A few cases showed chronic rejection reaction; however, most were
found to be free of chronic rejection such as graft tissue ﬁbrosis or renal vasculitis. We
concluded that marginal kidney graft from donor after cardiac death is an acceptablemodel for chronic rejection and that if the transplantation is carried out using a strict
immunosuppressive protocol, chronic rejection may be alleviated even with themarginal kidney.
Keywords: kidney transplantation, hydrogen-containing organ preservation solution, chronic allograft
nephropathy, chronic rejection, marginal donor, mature minipig model, multi-immunosuppressant

INTRODUCTION
The number of patients with end-stage chronic kidney disease is high worldwide; this is a crucial
issue because of the substantial cost of medical treatment (1). A considerable number of patientswith end-stage chronic kidney disease are awaiting kidney transplantation because of a chronic
shortage of living donors. As a result, dialysis is required for these patients. Although the survival ofgrafts in living kidney transplantation has been greatly improved by the emergence of effective

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allograft tolerance occurred by administering a single
immunosuppressant for several weeks to several months after
surgery. From the viewpoint of chronic allograft nephropathy, thisalso makes it difﬁcult to extrapolate to human transplant recipients
who are maintained using several immunosuppressants.
In a previous study, we reported that hydrogen-containing
organ preservation solution for rinsing and preservation of grafts
reduced acute phase IRI in kidneys of adult minipigs (26). The
anti-inﬂammatory effects of hydrogen, such as inhibiting
inﬂammatory cytokines, inducing HO-1, and activating NFE2-related factor, have been reported in transplanted organs(27, 28). This solution has been shown to be effective insuppressing IRI in transplanted organs such as the heart, lung,and kidney (29–34). In the present study, we aimed to verify
the impact of hydrogen gas treatment in the prevention ofchronic rejection of marginal kidney graft with an optimal
immunosuppressive protocol based on human clinical settingby using the miniature pig model.

calcineurin inhibitors and antimetabolites (2, 3), it is critical tosecure organs from a wide range of donors to solve the problemof living donor shortage. Moreover, effective utilization of
marginal organs from donors after cardiac death (DCD)
is required.
Marginal kidneys from DCD carry a high risk of short-termor long-term graft loss, depending on the donor’s individual
circumstances (including age, sex, blood pressure, and medical
history) (4). In particular, ischemia-reperfusion injury (IRI) due
to prolongation of warm ischemic time in the transplanted
kidney causes strong expression of cytokines and oxidants suchas IL-1, IL-6, and TNF-a, resulting in primary nonfunction anddelayed graft function (4–7). In addition, T cell- and antibodymediated rejection of kidney tissues is factors that exacerbate
acute rejection (8, 9). The presence of acute rejection may trigger
and lead to chronic allograft rejection. Thus, IRI increases the riskof developing a chronic allograft rejection over the long-term posttransplantation period, and outcomes have not improved despite
appropriate immunosuppressant therapy after surgery (3, 10). It is
known not only in humans but also in animal models that ischemic
kidney transplantation from DCD is less likely to survive long-termand undergo chronic rejection than transplantation from living
donors (11, 12). It has been reported that chronic allograft rejection
involves immunological factors such as HLA histocompatibility
and acute rejection and non-immunological factors such as
nephrotoxicity by calcineurin inhibitor and ischemia-reperfusion(13, 14). However, only about 60% of chronic graft damage can be
explained by these risk factors (15). To date, several studies havebeen conducted on mechanical perfusion and the inclusion ofoxygen and carbon monoxide in organ preservation ﬂuids toreduce acute and chronic damage, some of which are usedclinically (16–18). However, despite the beneﬁts, chronic allograft
nephropathy remains a highlight. Therefore, it is thought that themechanism of chronic allograft rejection involves a combination ofseveral of these factors.
Currently, there have been several studies that attempted toalleviate acute and chronic allograft rejection by improving IRI
during transplantation at an early stage, using animal models suchas rodents, rabbits, dogs, and pigs (1, 19). In particular, the rat
model was used in several studies owing to its advantages of low
cost, easy maintenance, and established techniques for blood vessel
and ureteral anastomosis. Furthermore, in humans, chronicchanges such as thickening of the blood vessel wall and atrophyof the kidney tubules develop over several years to decades,
whereas in rat models, these results, as well as chronic tissuechanges, can be observed within several months (20).
Nevertheless, these rats are typically young and male, with mild
ischemic injury and an observation period of <3 months, and does
not show clinical manifestations of chronic allograft rejection.
Furthermore, most rat strains used have been allogeneictransplants when the rat MHC antigen subtypes such as F344
and LEW were the same or were a combination of strains with low
rejection (20–23). There have been few papers describing control
of ischemic kidney transplantation using a combination ofmultiple immunosuppressants in a system with strong rejection,
assuming actual clinical practice (13, 24, 25). In a rat study,

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MATERIALS AND METHODS
The pigs used in this study were micro-miniature pigs (FujimicraLtd., Shizuoka, Japan) with body weights not exceeding 30 kg
even when mature, as previously described (26, 35, 36). We usedfour males and eight females, with mean age of 25.8 ± 5.5 months(range, 14.8–37.7 months; median, 25.4 months) and mean
weight at 20.9 ± 2.4 kg (range, 17.4–26.8 kg; median, 20.3 kg).
The pigs were randomly selected donors and recipients and had
different swine leukocyte antigen (SLA) suitability. The pigs werehoused in cages under temperature- and light-controlled
conditions (12-h light/dark cycle) and were provided with foodand water ad libitum. The breeding room was clean andmaintained by washing twice daily. These experiments wereconducted with the approval of the Research Council andAnimal Care and Use Committee of Kitasato University(approval number, 18-133, 19-086). The animal experiments
were performed according to the ARRIVE (Animal Research:
Reporting of In Vivo Experiments) guidelines 2.0 as a guideline
for animal experiments, and all necessary checklist criteria
were met.

Pre-Operative Examination and Treatment
Blood and urine tests were performed at screening before surgery
to conﬁrm absence of health-related abnormalities, and bloodcompatibility testing was completed to determine the matchingdonor and recipient settings in all cases. The recipients began
receiving tacrolimus (0.15 mg/kg, PO, BID) and mycophenolate
mofetil (500 mg/head, PO, BID) for the induction of
immunosuppression 2 days before kidney transplantation.

Preparation for Hydrogenation
The organ preservation solution used for rinsing and preservationof organs was extracellular-type trehalose-containing Kyoto(ETK), which has been shown to have a renal-preservation2

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240 min in the same preservation solution as the rinse solution
and allowed to stand until transplantation.
The recipient underwent anesthesia as per the donor protocol.
A midline incision was made in the abdomen, and we incised theretroperitoneum to separate the kidney and renal artery and veinon the side to be transplanted. The vascular anastomosis method
was determined by the conﬁrmed shape of the blood vessel of thetransplanted kidney. The blood ﬂow of the abdominal aorta
or renal artery, posterior vena cava, or renal vein was blockedwith a vascular clamp, after which the recipient-kidney wasremoved, and then, the anastomosed blood vessel was formed.
Heparin (1,000 units/head) was administered intravenously
and was administered again 2 h after the ﬁrst administration.
The donor-kidneys were taken out from the preservation
solution and placed in the abdominal cavity of the recipient
after the preservation time of the donor-kidney was ﬁnished.
Subsequently, the kidney artery was sutured for end-to-side or
end-to-end anastomosis with 5–0 monoﬁlament non-absorbablethread and the kidney vein with 6-0 monoﬁlament nonabsorbable thread. In the case of anastomosis to the abdominal
aorta, the ischemic time of the recipient was set to within 30 min
from clamping of the abdominal aorta. After the arterial
anastomosis was competed, the blood vessel clamp was moved
to the renal artery, and the blood ﬂow in the abdominal aorta wasrestarted. Following kidney arteriovenous anastomosis, the blood
ﬂow block was released, and blood ﬂow into the kidney andnormal pulsation of the kidney artery were conﬁrmed (Figures
1B, C). The ureter was anastomosed with 6-0 monoﬁlament
non-absorbable thread and the retroperitoneum was sutured.
The abdominal wall was sutured using 1-0 and 3-0 synthetic
multiﬁlament absorbent threads. Furthermore, the skin was
routinely sutured using 2-0 nylon thread. An access port for
sampling blood and drug administration was installed in thejugular vein using a central vein (CV) catheter kit. The CV
catheter in the surviving pig was removed 1 month after surgery.

effect in previous studies (37). The hydrogen addition method to
ETK was performed using a hydrogen storage alloy canister as
described (26). The hydrogen was fed from a rubber stopper into
a bag of ETK cooled to 4°C and mixed when the internal pressureof the container reached 0.06 ppm. The hydrogen concentration
decreased slowly when stored at 4°C under normal pressure, anddissolved hydrogen concentration of 1 ppm or more could besecured for 4 h. Therefore, the pressure reduction was similar tothat reported previously and the product was stored at 4°C (26).
These operations were performed immediately before ischemic
nephrectomy of the donor.

Anesthesia Methods
The donor and the recipient at the time of transplantation weresedated by intramuscular administration of a mixed solution ofmedetomidine (20 µg/kg), midazolam (0.2 mg/kg), andbutorphanol (0.2 mg/kg) to the gluteal muscle. After sufﬁcient
sedation was induced, mask inhalation was performed with
isoﬂurane of 3.0%, and anesthesia was induced until thepharyngeal reﬂex disappeared. The 24-G catheter indwelling
needle was inserted into the central ear vein, and lactated
Ringer’s solution was started at 5 ml/kg/h. The infusion
volume was changed appropriately by monitoring anesthesia
during surgery. Then, a 5.5-Fr tracheal tube was introduced, andanesthesia was maintained with isoﬂurane 2.0%. An 8-Fr balloon
catheter was placed in the bladder of the recipient to secure
visuals of the surgical ﬁeld and conﬁrm urination during surgery.
The catheter was removed after surgery. All pigs wereadministered with enroﬂoxacin [5 mg/kg, SC (subcutaneously),
BID (bis in die/twice daily)] and cefazolin sodium hydrate (30mg/kg, IV, BID) as antibiotics and buprenorphine (20 µg/kg, IV,
BID) as an analgesic from the preoperative period.

Kidney Transplantation Procedure
Two kidneys obtained from one donor were transplanted to
two recipients, and both kidneys of the recipients were excised.
The group using hydrogenated ETK was classiﬁed as H-DCD20(n = 4) and the normal ETK group as NH-DCD20 (n = 3).
The living kidney-transplanted individual (DCD 0, n = 1) was
treated with the same protocol as the other groups except forischemic manipulation.
The donor was anesthetized and held in a supine position.
The chest and abdomen were shaved and disinfected. A midline
incision was made in the abdomen with an electric scalpel. Bloodﬂow to the aorta and posterior vena cava was blocked on the
cranial and caudal sides of the kidney arteries and veins to induce
kidney ischemic time for 20 min. An anticoagulant was not usedin blocking the blood ﬂow of the donor. Twenty minutes later,
the abdominal aorta and posterior vena cava, left and rightkidneys, and ureter were separated and excised. The excised
kidney was immediately cannulated from the kidney artery andrinsed with cooling hydrogenated ETK or normal cooled ETKfrom a height of 1 m by natural dropping for 10 min (Figure 1A).
Further, it was immersed in a cooled storage solution similar to
the rinse liquid. Excessive soft tissue around the kidney hilum
was trimmed in the preservation solution, and anastomosis of theblood vessel was formed. The kidneys were stored for 60 orFrontiers in Immunology | www.frontiersin.org

Perioperative Managementand Long-Term Observation
The pigs receiving kidney transplantation were provided with foodand water ad libitum from the ﬁrst day after surgery. Antibiotics
and analgesics were administered postoperatively, and tacrolimus[0.1 mg/kg/day, IV (intravenously)] was intravenously
administered continuously until ingestion was observed if theanimal had no appetite. Tacrolimus (0.15–0.6 mg/kg, p.o., BID),
mycophenolate mofetil (250–500 mg/head, po, BID), andmethylprednisolone (0.5–2 mg/kg, IV or PO, BID) wereadministered during the follow-up period. Tacrolimus andmycophenolate mofetil doses were set based on previous reports(38). Tacrolimus trough levels were measured routinely and thedose adjusted to maintain trough levels of 5–15 ng/ml. When adecrease in the trough level of tacrolimus was observed, the dose of
tacrolimus was gradually increased to adjust the dose. In situations
considered to represent acute rejection, the dose of prednisolone
was increased for 3 days (2 mg/kg, BID) and gradually decreased
over 1 week. As an analgesic, buprenorphine (20 µg/kg, IV, BID)
was administered. As appropriate, famotidine (0.5 mg/kg, PO or
IV BID) was orally or intravenously administered, as well as

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A

B

C

FIGURE 1 | The kidneys during rinse and after reperfusion of blood. (A) A typical image showing the kidney being rinsed by the organ preservation solution. The
kidney of ① is rinsed with a hydrogenated organ preservation solution. ② is rinsed with a non-hydrogenated organ preservation solution. The whole area of ① isﬂushed compared to ②. (B, C) The transplanted kidney immediately after reperfusion after rinsing and storage with an organ preservation solution. B, hydrogenatedkidney; C, non-hydrogenated kidney.

darbepoetin (30 mg/head, SC, once a week) and iron which wasadministered orally.
Blood sampling from the CV catheter was performed until thekidney function stabilized. Blood was collected every day until
the ﬁrst week after the operation and at 2 weeks, 1 month, 2months, and 100 days postoperatively. We recorded thecomplete blood count (CBC), blood urea nitrogen (BUN), andcreatinine levels. In addition, syndecan-1 levels were measured
using plasma before surgery and 4 h after transplantation using
an ELISA kit (MBS101321, MyBioSource, CA, USA), because theincrease in syndecan-1 levels due to angiopathy occurs at
approximately 2–4 h postoperatively, according to a previous
report (39). Urine was collected at the same time as blood
sampling, and the urine was evaluated for speciﬁc gravity, pH,
and sediment.
The observation period was 100 days after the operation, andindividuals surviving 100 days or more were sacriﬁced to remove
the transplanted kidney. In addition, individuals withpostoperatively elevated creatinine levels, anuria for 6 days or
more, and exacerbated clinical symptoms (frequent vomiting,diarrhea, inability to stand, loss of appetite) were sacriﬁced. The
method of sacriﬁce was the same method as for anesthesia
during surgery, followed by rapid administration of pentobarbital(100 mg/kg, IV) and potassium chloride (25 ml/head, IV).

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Ten minutes later, we conﬁrmed cardiopulmonary arrest. At thetime of sacriﬁce, blood was collected and ultrasonography andcontrast-enhanced computed tomography (CT) were performed
to evaluate the kidneys.

Ultrasonography and Contrast-Enhanced
CT Examination
Ultrasonography was performed under sedation to conﬁrm
blood ﬂow and for morphological evaluation of thetransplanted kidney. The pig was placed in the supine position.
The vascular resistance coefﬁcient (RI) was measured by thecalculated systolic and diastolic blood ﬂow rates using the pulsed
Doppler method. RI is known to be high in rejection andkidney artery stenosis and has been shown to be associatedwith antibody-related rejection and acute tubular necrosis(40). In order to avoid variations in results due to differencesin ﬂow velocity depending on the measurement position, RI
measurements were performed uniformly on the interlobar arteryin all individuals. The RI was measured three times consecutively,
and the average value was calculated. In contrast-enhanced CT
examination, blood ﬂow to the renal arteries and veins under
sedation was observed. The pig was in the supine position, andiohexol (1 ml/kg) was rapidly administered intravenously
for imaging.

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TABLE 2 | Concentration of Syndecan-1.

Histopathological Examination
The removed kidney, renal artery and vein, and ureter were
immersed in 4% paraformaldehyde-phosphate buffer (4% PFA)
and ﬁxed at 4°C for 24–48 h. The kidneys were sectioned
longitudinally to include the papilla centering on the cortex.
The artery, vein, and ureter were divided along the lumen to alongitudinal section and were embedded. After embedding, thinlayer slices (3 µm) were prepared using a conventional method.
Thin sections were stained with hematoxylin and eosin,
Masson’s trichrome stain, and Elastica van Gieson stain.
Histological examination was performed by blindedpathologists, and kidney graft pathology was assessed
according to the Banff classiﬁcation (41, 42).

No.

Pre (mg/ml)
Post (mg/ml)
Rate of variability (%)

H-DCD 20(n = 2)

NH-DCD 20(n = 2)DCD 0(n = 1)

3

4

5

6

8

5.08
9.06
40.0

2.48
5.46
78.3

10.32
16.46
120.3

10.99
15.13
59.4

5.24
7.34
37.7

ND, under than the limited value of measurement.

the individuals in NH-DCD20 continued to increase and did not
improve (Figure 2B). Syndecan-1 levels are displayed in Table 2.
The concentration in NH-DCD20 was higher than in DCD0 and
H-DCD20 before and after surgery. Only the H-DCD20 (3/4)
and DCD0 were able to provide urine after surgery. One
individual in H-DCD20 and all the individuals in NH-DCD20
did show urine in the bladder by ultrasonography; however,urine could not be collected. The result of urinary testing 2 weeks
after surgery showed that H-DCD20 (1.016 ± 0.004) had a lower
speciﬁc gravity than DCD0 (1.029). In summary, the renal
function in H-DCD20 could be earlier recovered than that of
NH-DCD20 and remain stable.

RESULTS
Recipient Information and Survival Days
Table 1 shows the survival days, preservation time, and warm
ischemic time in all groups. The warm ischemic time was thetime from the placement of the transplanted kidney in therecipient to the reperfusion. Ischemia was slightly prolonged in
the three individuals of H-DCD20 due to blood vessel site injury
and bleeding. DCD0 was able to survive for 100 days. The day
after the operation, appetite improved and urination wasobserved. Three H-DCD20 survivors (3/4 heads) survived for
100 days, whereas the remaining survivor had a nephrostomy
tube under anesthesia 28 days after surgery due to severe ureteralobstruction. The general condition improved and wasmaintained. The surviving animals gradually recovered theirappetite from postoperative days 2–3, and urinary excretionand normal feces were observed. NH-DCD20 was sacriﬁced 3–
7 days after surgery due to exacerbation of clinical symptoms
including anuria, vomiting, and deterioration of kidney function.
Taken together, these ﬁndings suggest H-DCD20 enabled longterm survival despite of the more severe ischemic conditions
than NH-DCD20.

Imaging Findings
Ultrasonography showed clear blood ﬂow in the kidneys of thesegmental arteries in the surviving DCD0 and H-DCD20
individuals from postoperative day 1 to follow-up (Figure 3).
One individual in the NH-DCD20 and H-DCD20 who died lessthan 7 days after transplantation had little or no kidney blood
ﬂow from the early postoperative period. In addition, the entire
kidney was hypoechoic, making anatomical evaluation of thekidney difﬁcult (Figure 3). The measured RI was higher for HDCD20 than for DCD0 after surgery; however, it gradually
decreased and became stable (Table 3). Only No. 5 could bemeasured in the NH-DCD20, and RI was higher than that of theother groups from the ﬁrst day after surgery. All surviving
individuals had clear contrast-enhanced images within therenal parenchyma using contrast-enhanced CT examination on
day 100 (Figure 3). The renal arteries and veins were clearlyvisualized. The individuals who died within 7 days after
operation did not show contrast medium inﬂux into the renalparenchyma. Collectively, data indicate that clear blood ﬂow inthe surviving H-DCD20 individuals could be observed byultrasonography and contrast-enhanced CT examination frompostoperative day 1 to follow-up.

Blood and Urinary Tests
The BUN and creatinine of Nos. 2, 3, and 8 peaked on
postoperative days 2–4 and gradually decreased; following that,the value remained stable (Figure 2A). No. 4 showed increased
BUN and creatinine due to ureteral obstruction; however, BUN
and creatinine improved with percutaneous nephrostomy tube
placement and medical management under anesthesia. The BUN
and creatinine of non-surviving individuals in H-DCD20 and all

TABLE 1 | Information of groups.
H-DCD 20 (n = 4)

Survival days
Warm ischemic time (min)Preservation time (min)NH-DCD 20 (n = 3)

DCD 0 (n = 1)

No. 1

No. 2

No. 3

No. 4

No. 5

No. 6

No. 7

No. 8

1
60
240

100
79
60

100
64
240

100
69
240

3
60
60

6
60
60

7
60
60

100
60
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A

B

FIGURE 2 | BUN and creatinine in the survival span. (A) The transition of BUN and creatinine in H-DCD20 individuals who were able to survive up to 100 days after
surgery. The solid line shows DCD0. No. 4 has a temporary increase in BUN and creatinine due to ureteral obstruction. In other individuals, BUN and creatinine
peaks within 5 days after surgery, and kidney function is stable until 100 days. (B) The transition of BUN and creatinine in individuals who died early after surgery.
These continue to rise after the operation, and no improvement is observed. BUN, blood urea nitrogen. *H-DCD20: n=4, NH-DCD20: n=3, DCD0: n=1.

there was no cell inﬁltration into the stroma. Additionally, thetubular atrophy and tubular inﬂammation were mild (Figures
4D, E). Therefore, No. 3 was identiﬁed to have no chronic
allograft nephropathy. In No. 4, mild interstitial ﬁbrosis andatrophy of renal tubules were observed, and cellular inﬁltration,arterial intima, and venous infarction were noted in some areas(Figures 4F, G). Thus, we concluded that this was a type I
chronic rejection, according to the Banff classiﬁcation (Table 5).
Three of the four individuals who died within 7 days showed
severe tubular necrosis over the entire area, and cellular
proliferation and hemorrhage from the intima to the adventitiaof the renal arteries and veins were detected (Figure 4H). In Nos.
1 and 7, there were infarcts due to thrombus in the renal arteries(Figure 4I).
The supplementary table shows the number of drops in theinfusion line when rinsing with ETK. As in previous reports,
H-ETK (n = 3) has a higher number of drops per minutethan NH-ETK (n = 3). The supplementary ﬁgure is the resultof histopathological examination. This result was examined after
the graft was treated by the same procedure as in this study and
immediately ﬁxed. As a result, the hydrogen-treated graft(hydrogen; n = 1) tended to have a mild tubular degeneration
to necrosis with dilatation score compared to the nonhydrogenized graft (control; n = 1).
Collectively, data indicate that the surviving individuals in
H-DCD20 showed no to mild chronic allograft nephropathy
since there was a possibility that the hydrogen improved an acute

Pathology Anatomy
No renal arteriovenous thrombus was observed in any individual
who survived for 100 days (Table 4). In addition, individuals
who survived for 100 days with H-DCD20 had ureteralanastomosis stenosis or adhesion to surrounding tissues, aswell as dilated renal pelvis and ureters. The individuals whodied within 1 week after surgery had thrombus in the kidneyarteries (Table 4). In individuals whose blood ﬂow could not be
conﬁrmed before dissection using ultrasound images, thrombus
was conﬁrmed in the renal arteries and in some of the segmental
arteries and veins. In summary, individuals in H-DCD20 had nothrombus formation in the renal artery and vein; however, theindividuals in NH-DCD20 had thrombus in the kidney arteries.

Histopathological Findings
The histopathological images are shown in Figure 4. DCD0
(No. 8) and the surviving individuals (Nos. 2, 3, and 4) showedchronic tissue changes. The kidney of No. 2 showed moderate
tubular inﬂammation and glomerulitis and mild mononuclear
cell inﬁltration into the stroma. Histological images showed mild
chronic allograft nephropathy such as tubular atrophy andinterstitial ﬁbrosis. Stenosis of the arterial lumen, necrosis of
the intima, edema of the media, and cell inﬁltration into theadventitia were observed (Figures 4A–C). These werecomprehensively evaluated and found to be type I chronic
allograft nephropathy, according to the Banff classiﬁcation(Table 5). In No. 3, the glomerulitis was moderate; however,

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FIGURE 3 | Ultrasonographic and contrast CT images. Upper row: Ultrasound images on the ﬁrst day after the operation are shown. Renal blood ﬂow in H-DCD20
individuals is clearly conﬁrmed; however, some areas are difﬁcult to see with a Doppler. Almost no renal blood ﬂow is observed in NH-DCD20 individuals, and therenal parenchyma shows a hypoechoic image similar to that of surrounding tissues. Kidney blood ﬂow in DCD0 is clearly conﬁrmed, and branching of interlobar
arteries and veins is observed from segmental arteries and veins. Middle/lower row: contrast-enhanced CT is used to demonstrate the vascular and excretory
phases, and 3D stereoscopic images are shown. In H-DCD20 and DCD0, inﬂow of contrast medium into the kidney is observed, and the ureter and bladder are
imaged. However, in individuals of NH-DCD20 and H-DCD20 without blood ﬂow, the renal arteries are not imaged, and the ureter and bladder are not imaged during
the excretory phase (yellow circle, transplanted kidney). CT, computed tomography; 3D, three-dimensional. *H-DCD20, n = 4; NH-DCD20, n = 3; DCD0, n = 1.

TABLE 3 | RI value by ultrasonography.
Day after operation

H-DCD20(n = 4)NH-DCD20(n = 3)DCD0(n = 1)

No. 1
No. 2
No. 3
No. 4
No. 5
No. 6
No. 7
No. 8

Day 1

Day 3

Day 5

1 week2 weeks

1 month

2 months

Day 100

NT
0.61
0.63
NT
0.71
NT
NT
0.50

Dead
0.68
0.59
NT
0.78
NT
NT
0.51

–
0.72
0.64
0.64
0.63
Dead
NT
0.53

–
0.53
0.59
0.64
0.82
–
Dead
NT

–
0.54
0.51
NT
Dead
–
–
0.47

–
0.51
0.72
0.63
–
–
–
NT

–
0.55
0.60
0.75
–
–
–
0.51

–
0.55
0.46
0.71
–
–
–
0.66

NT, not tested (no blood ﬂow or difﬁcult to measure).

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Hydrogen Effects for Chronic Rejection

TABLE 4 | Ultrasonographic and anatomical ﬁndings in the last day.

potassium concentration and cell edema, reduce reperfusion
injury, and inhibit further ischemia. Chronic tissue changes canbe expected to suppress inﬂammatory cytokines and reactive
oxygen species and improve ischemia. Therefore, thecombination of H and ETK may be one of the leading protocols
for ischemic kidney transplantation.
The glycocalyx present in vascular endothelial cells is anextremely fragile structure, consisting of proteoglycan,
glycoprotein, and glycan; it is responsible for maintaining
microcirculation and barrier function of blood vessels andeasily disintegrates upon stimuli such as IRI and cytokines (7,34, 47, 48). The glycocalyx is present on the surface of glomerular
capillaries and podocytes in the kidney and has been shown to be
exfoliated by exposure to lipopolysaccharides. Hydrogen isthought to protect the glycocalyx (49). There were decreased
expression levels of syndecan-1 in the glycocalyx when hydrogen
was inhaled in a non-traumatic hemorrhagic shock rat model,
showing that hydrogen contributed to the prolongation ofsurvival (49). One study showed that an increase in syndecan-1
levels in tubular epithelial cells was associated with improved
graft function and prolonged engraftment (50). Furthermore,
syndecan-1 knockout mice were often affected by renal IRI andwere prone to ﬁbrosis. Although measured syndecan-1 levels
increased postoperatively in all groups, the ﬂuctuating rate waslower in NH-DCD 20 than in H-DCD 20. The reason may be as
follows: NH-DCD 20 did not effectively rinse out cytokines andoxidants, thereby inducing thrombus formation immediately
after reperfusion. Systemic circulation of syndecan-1 did notoccur during reperfusion. This can further be determined by theappearance of the kidney after reperfusion (Figures 2B, C). Thissuggests that the increasing serum levels of syndecan-1 may havebeen underestimated. In contrast, in H-DCD 20, the rate ofchange of syndecan-1 expression was relatively high becausehydrogen removed microthrombi and blood entered thesystemic circulation from the graft.
No chronic rejection was observed in the transplanted kidneywith long-term engraftment of H-DCD 20, and there wereindividuals with the same histopathological image as DCD 0. In
previous studies, it has been shown that ischemic kidneytransplantation is likely to induce chronic rejection. The ischemic
state of the transplanted kidney contributes to non-immune
chronic rejection, and the induction of acute inﬂammation andrejection by IRI results in chronic organic changes in tissue,
including thickening of the arterial wall. We evaluated the rate ofdecrease during ETK ﬂow within the kidney and conﬁrmed that
rapid ﬂow of ETK decreased in the line, similar to previous reports(Supplementary Table). Furthermore, we conﬁrmed that thehistopathological ﬁndings of the kidney immediately after rinsing
with ETK showed no difference; however, tubule degeneration
associated with dilation was mild in the kidney using ETK
containing hydrogen (Supplemental Figure). Currently, moststudies on chronic rejection have been using rats with lowresponder combinations, and immunosuppressants have often
been used alone for short periods of time. In the present study,
we did not conﬁrm the haplotypes of SLA. Most MHC haplotypesof the microminiature pigs used have been identiﬁed; however,

H-DCD20 NH-DCD20 DCD 0(n = 4)(n = 3)(n = 1)
Ultrasonographic ﬁndings Renal artery ﬂow
Renal venous ﬂow
Ureteral dilation
Pelvis dilation
Anatomical ﬁndings
Arterial thrombus
Venous thrombus
Ureteral dilation
Pelvis dilation

3/4
3/4
3/4
3/4
1/4
1/4
3/4
3/4

3/3
3/3
0/3
0/3
1/3
0/3
0/3
0/3

1/1
1/1
0/1
0/1
0/1
0/1
0/1
0/1

tubular degeneration and necrosis compared to the nonhydrogen-treated kidney.

DISCUSSION
To the best of our knowledge, this is the ﬁrst study to observe longterm prognosis after transplantation using hydrogen in the ischemic
kidney of mature minipigs and to inhibit chronic rejection responseusing a strict multi-immunosuppressant protocol.
NH-DCD20 showed severe kidney damage in the acute phaseand was sacriﬁced, whereas H-DCD20 quickly recovered from
IRI in the acute phase and all survived for long periods except for
one individual. Kidney transplant dysfunction can lead to 6-day
survival. Therefore, we believe that the cause of an early death of
the individual in H-DCD20 was not transplanted kidney
dysfunction; rather, there were other causative factors. We
speculate that hydrogen improved microcirculation and that
this was one of the factors that caused the difference in thelong-term survival between the two groups.
In human studies, kidney allograft thrombosis accounts for
2%–7% of graft loss (43). The causes include technical factors,
vascular endothelial damage, decreased cardiac output, acutetubular necrosis, and acute rejection. In the case of ischemicorgan transplantation, rejection is strongly associated withmicrocirculatory changes induced by inﬂammatory cytokines
due to IRI (44, 45). Hydrogen has been shown to suppress the
production of the major inﬂammatory cytokines IL-1b, IL-6, andTNF-a, and it has anti-inﬂammatory effects that reduce tissue
damage (29, 34). Saito et al. reported that using hydrogenatedpreservatives for lung transplantation reduced the expressionof IL-1b and 8-hydroxyguanosine and reduced the number ofneutrophils attached to the vascular wall and hydrogen-inhibited
vascular endothelial damage (29). In addition, ETK potentiallycontributes to improving microcirculation. In actual humanclinical cases, its clinical application in lung transplantation has
been reported (46). Although its application in kidneytransplantation as a clinical example has not been reported, inexperimental animals, we has previously shown prolongation ofindividual survival by ETK preservation in ischemic kidneytransplant rat models compared to the University of Wisconsin
solution and lactated Ringer’s solution (37). The high-sodium/
low-potassium composition of ETK and the trehalose containedin it are thought to suppress vasoconstriction due to highFrontiers in Immunology | www.frontiersin.org

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Hydrogen Effects for Chronic Rejection

A

B

C

D

E

F

G

H

I

FIGURE 4 | Histopathological examinations. (A–C) The histopathological images in No. 2 are shown. (A) Localized cell inﬁltration is observed, and cell inﬁltration isobserved around the epithelial edema. (B) Mild ﬁbrosis is observed (Masson’s trichrome staining). (C) Stenosis of the lumen in the kidney artery, necrosis of theintima and edema of the media, and cell inﬁltration in the adventitia are observed. (D, E) The histopathological images in No. 3 are shown. (D) Borderline change isobserved to the extent that localized cell inﬁltration is observed. (E) Chronic glomerulosis due to thickening of the vascular loop (blue arrow) is shown with anincrease in mesangial substrate. (F, G) The histopathological images in No. 4 are shown. (F) Arterial infarction (arrow) and venous infarction (arrowhead) are
observed. (G) Fibrosis was mild but more extensive than No. 2 (Elastica van Gieson staining). (H, I) Representative histopathological images of individuals who diedearly after operation are shown. (H) Tubular necrosis is observed throughout the whole kidney. Inﬁltration of mononucleosis is observed near the capsule.
(I) Thrombus formation is observed in the renal artery.

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TABLE 5 | Histopathological ﬁndings for kidney allograft.
No.

Glomerulitis
Interstitial mononuclear cell inﬁltration
Tubulitis
Intimal arteritis
Chronic allograft nephropathy type/grade
Chronic transplant glomerulopathy
Interstitial ﬁbrosis
Tubular atrophy
Mesangial matrix
Vascular ﬁbrous intimal thickening

H-DCD 20 (n = 4)

NH-DCD 20 (n = 3)

DCD 0 (n = 1)

1

2

3

4

5

6

7

8

/
0
0
0
0
/
0
0
/
0

2
1
2
1
I
3
1
1
3
1

2
0
1
0
0
1
0
1
3
1

2
1
3
1
I
3
1
1
3
2

3
3
3
1
I
2
2
3
3
0

/
/
1
0
0
/
0
0
/
0

/
/
2
0
0
/
0
0
/
0

2
0
0
0
0
0
0
1
1
0

Grade 0, no change; 1, mild; 2, moderate; 3, severe;/, no examination.
Chronic allograft nephropathy type/grade.
I. Mild interstitial ﬁbrosis and tubular atrophy (<25% of cortical area).
II. Moderate interstitial ﬁbrosis and tubular atrophy (26%–50% of cortical area).
III. Severe interstitial ﬁbrosis and tubular atrophy/loss (>50% of cortical area).

is 30–60 min (54, 55). Therefore, it was speculated that thematurity of microminiature pigs was one of the factors giving riseto the difference in viability despite using the same animal
species. As mentioned previously, pigs generally used intransplantation experiments are young, typically less than 6months old. It cannot be denied that this acts as a kidney
protective effect because many factors such as immune
response and growth hormone can be confounded during the
growth period (56), and this precludes extrapolation to humans.
The ischemic kidney transplantation model of mature miniaturepigs created in this study is expected to contribute signiﬁcantly topreclinical studies in humans.
The limitations of this study were the small number ofindividuals included and the limited data obtained, unknown
detail SLA types, and the lack of complete elucidation of themechanism by which the improvement tendency of chronic
rejection occurred. Regarding this point, we are consideringexperiments considering the combination of SLAs in the future.
The purpose of this study was to conﬁrm long-term survival and theaccompanying changes in chronic tissue response; IHC, PCR,ELISA, and ﬂow cytometry were not performed on tissue sections.
However, Abe et al. reported that dissolving hydrogen in the UWorgan preservation solution during renal transplantation in rats
suppressed tubular apoptosis and reduced interstitial macrophageinﬁltration (57). We think this can be observed in miniature pigs aswell. In addition, we plan to analyze the inﬂammatory cytokine
cascade and its associated cell-mediated immune response.
Nevertheless, it is clear that hydrogen improves the blood ﬂow
environment in the ischemic kidney in ischemic kidneytransplantation in mature pigs. Hydrogen enables rapid recoveryof kidney function and long-term survival in the context ofadministration of multidrug immunosuppressive therapy.
Furthermore, the fact that chronic rejection was not observed or
was mild in ischemic kidney transplantation of pigs, we can infer that
this was mediated by hydrogen. This factor can contributesigniﬁcantly in explaining the chronic rejection mechanism.
In conclusion, hydrogen induced a positive effect on IRI,
possibly suppressing thrombus formation and microcirculation

this is currently under study (36). SLA has multiple haplotypes, as
in humans. This suggests the requirement for multiimmunosuppressant protocols similar to actual clinical practice.
Furthermore, recently, a mechanism called IL-6 ampliﬁer has been
considered for MHC class II-related inﬂammatory reactions. This
indicates that CD4+ T cell-derived cytokines such as IL-17A may
lead to sustained activation of IL-6 due to T-cell activation andlocal accumulation that are not related to antigen speciﬁcity (51).
To support this, it is shown that synaptotagmin-17, a type ofurinary exosome, is increased in association with IL-6 ampliﬁers inpatients with chronic active antibody-mediated rejection in kidneytransplantation (52). These were considered that may be relating
the suppression of cytokines by hydrogen and may affect the resultsof mild ﬁbrosis and tubular atrophy in the H-DCD20 (29, 34).
Hydroxyl radical (OH−) and reactive oxygen species cause
oxidative damage to kidney tissue, producing cytokines such as
TGF-b1 and tubular cells, causing epithelial-mesenchymaltransition (EMT). EMT causes ﬁbrosis by ﬁlling the tubularinterstitial with ﬁbrotic substances such as vimentin and asmooth muscle actin, causing arterial ischemia and atrophy ofglomeruli and kidney tubules. Therefore, it was suggested thatthe hydrogen-dissolved organ preservation solution suppresses
the upper cascade of ﬁbrosis by removing hydroxyl radicals,
reactive oxygen species, and inﬂammatory cytokines, and may
contribute to the reduction of chronic rejection.
The ischemic time affected long-term survival in the ischemic
kidney transplant model of miniature pigs used in this study. In
humans, it has been shown that the retention and survival rate oftransplanted organs decreased when the ischemic time was
30 min or more (53), and the ischemic time compatible withsurvival after transplantation differs depending on the animal
species. Previous studies in miniature pigs showed ischemic timesof 30 min and observation periods of less than 1 week (26). In
addition, we noted that survival after kidney transplantation inminiature pigs with an ischemia time of 30 min was difﬁcult forall (n = 9, unpublished data). Autologous transplantation using
general domestic pigs and miniature pigs involve young pigs andthere is no use of immunosuppressants; hence, the ischemic time

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Hydrogen Effects for Chronic Rejection

for lethal ischemic kidney transplantation in miniature pigs.
Furthermore, ﬁndings suggested suppression of chronic
rejection in long-term engrafted kidney tissue and showed arelationship between inﬂammation and rejection in the acutephase and chronic tissue changes. This is a signiﬁcant preclinicalmodel, and we believe that this model will provide further insight
into the mechanism of chronic rejection.

opinions of all the co-authors. All co-authors reviewed theﬁnal version of this paper and agreed to submit it. The authors
agreed to be accountable for all aspects of the work in ensuringthat questions related to the accuracy or integrity of any part of
the work are appropriately investigated and resolved. All authors
contributed to the article and approved the submitted version.

DATA AVAILABILITY STATEMENTFUNDINGThe original contributions presented in the study are included in
the article/Supplementary Material. Further inquiries can bedirected to the corresponding author.

This work was supported by grants from Doctors Man Co., who
had no role in study design, data collection and analysis, decision
to publish, or preparation of the manuscript (Grant number:
2225-7257, 2225-7265).

ETHICS STATEMENTACKNOWLEDGMENTS

The animal study was reviewed and approved by The ResearchCouncil and Animal Care and Use Committee of Kitasato
University (approval number: 18-133, 19-086).

This work was supported by grants from Doctors Man Co., Ltd. Sou
Hashimoto from this company assisted with devising the system
for infusing hydrogen gas into organs as preservation solution.

AUTHOR CONTRIBUTIONSKN, SI, and EK contributed substantially to the conception anddesign of the work. SI made the same contribution as KN in thisresearch. KN, SI, KT, SO, MS, and EK contributed to theacquisition, statistics, analysis, and interpretation of data. EK
supervised and conﬁrmed all the data and supervised the

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Nishi et al.

Hydrogen Effects for Chronic Rejection

Conﬂict of Interest: Co-authors of this manuscript, EK and MS, are medical
advisors to Doctors Man Co., Ltd.

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The remaining authors declare that the research was conducted in the absence ofany commercial or ﬁnancial relationships that could be construed as a potential
conﬂict of interest.
Copyright © 2021 Nishi, Iwai, Tajima, Okano, Sano and Kobayashi. This is an
open-access article distributed under the terms of the Creative Commons Attribution
License (CC BY). The use, distribution or reproduction in other forums is permitted,
provided the original author(s) and the copyright owner(s) are credited and that theoriginal publication in this journal is cited, in accordance with accepted academic practice.
No use, distribution or reproduction is permitted which does not comply with these terms.

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