J Vet Clin 26(5) : 441-444 (2009)

441

1Corresponding author.E-mail : woohm@kangwon.ac.kr

A model of Isolated Renal Hemoperfusion

Hyun-Suk Nam and Heung-Myong Woo1

School of Veterinary Medicine and Institute of Veterinary Science, Kangwon National University, Chuncheon 200-701, Korea

(Accepted: April 06, 2009)

Abstract : Ischemia-reperfusion (I/R) injury is associated with an increased risk of acute rejection, delayed graft function

and long-term changes after kidney transplantation. The reperfusion models remain unsolved complications such asvascular obstruction and blood leakage. We developed an alternative model of isolated hemoperfusion in porcinekidneys. In the present study we introduced a newly developed reperfusion method. A connector was used insteadof surgical suture for the vascular anastomosis on the inguinal region in which main femoral vessels are parallel andbig enough to perfuse the kidney. To assess renal perfusion quality of the modified hemoreperfusion model, we analyzedboth hemodynamic values and patterns of I/R injury following a renal reperfusion. Following unilateral nephrectomy,the kidneys were preserved for 0, 24 and 48 hours at 4oC with histidine-tryptophan ketogluatarate (HTK) solutionand reperfused for 3 hours by vascular anastomosis connected to the femoral artery and vein in inguinal region.Histolopathological examinations were assessed on kidney biopsy specimens, taken after each cold storage andreperfusion. No differences of hemodynamic values were observed between aorta and femoral artery. The average warmischemia time before reperfusion start was 7.0 ± 1.1 minutes. There were no complications including vascular obstructionand blood leakage during the reperfusion. I/R injury of the perfused kidneys in this model was dependent upon thecold ischemia time. The results support that the modified perfusion model is simple and appropriate for the studyof early renal I/R injury and transplant immunology.

Key words : kidney, reperfusion model, Ischemia-reperfusion injury, porcine, cold storage.

Introduction

Ischemia-reperfusion (I/R) injury is associated with an

increased risk of acute rejection, delayed graft function and

long-term changes after kidney transplantation (2,9). As the

donor kidneys are subject to warm and cold ischemia due to

the surgical process of explantation, the investigation of the

ischemia- and reperfusion-related injury and consecutive devel-

opment of reperfusion injury-minimizing techniques could

improve graft survival (10). Numerous models of isolated

perfused kidneys have been established in the past years but

limitations such as organ size, perfusate and ethical standards

have restricted a widespread research in this area (3). Fur-

thermore the reperfusion models remain unsolved complica-

tions such as vascular obstruction and blood leakage. Additional

warm ischemia during and after the reperfusion is due to the

time-consumed vascular anastomosis, which is for the connec-

tion of the donor organ to the recipient (2-3,6).

In the present study we introduced a newly developed rep-

erfusion method using femoral site, in which a parallel vas-

culature, femoral artery and vein, are big enough to perfuse

the kidney. To evaluate whether a modified renal reperfusion

model is effective in the study of the early I/R injury in pigs,

we investigated with regard to hemodynamic between renal

artery and femoral artery in the miniature pigs. In addition, to

evaluate the efficacy of the reperfusion model the degree of

I/R injury on the cold ischemia time was evaluated.

Materials and Methods

Ten miniature-pigs (PWG Genetics Korea, Korea) weigh-

ing 28.3 to 37.5 kg were used in this study. All animals were

treated according to the regulations for the Care and Use of

Laboratory Animals in Kangwon National University.

Animals were premedicated with ketamine (Ketamine®,

Yuhan Pharmaceutical Co., korea, 15 mg/kg, IM) and atropine

sulfate (Atropine®, Daehan Pharmaceutical Co., korea, 0.05 mg/

kg, IM). At the beginning of general anesthesia they were

administrated thiopental sodium (Thionyl®, Daehan Pharma-

ceutical Co., korea, 10 mg/kg, IV). An endotracheal tube was

placed and anesthesia maintained with 1.5-2% of isoflurane

(Forane®, Choongwae Pharmaceutical Co., korea) and 100% of

oxygen. During the surgical procedure animals received 0.9%

saline intravenously (10 ml/kg/hour).

The left renal and femoral vessels were atraumatically iso-

lated and intra-arterial catheters (MIKRO-TIP® pressure cathe-

ter; Millar instruments) were inserted into the vessels. Invasive

blood pressure (IBP) was measured by pressure volume sys-

tem (MPVS-300®; Millar instruments) in the aorta and femoral

artery.

The kidneys were harvested, immediately flushed with the

442 Hyun-Suk Nam and Heung-Myong Woo

iced preservation solutions and stored for 0, 24 and 48 hours

at 4oC with histidine-hryptophan ketogluatarate (HTK) solution

(Custodiol®, Odyssey Pharmaceutical Co., East Hanover). The

cold stored kidney vessels were connected to the femoral

artery and the vein respectively, using a connector set con-

sisted of polyethylene connector and silicone tube (Fig 1 and

2). The kidney in inguinal area was reperfused for 3 hours.

After the animal was euthanized, tissue samples from the

cortex-outer medullar region of kidney were fixed in 10%

buffered formalin, embedded in paraffin, and stained with

either hematoxylin and eosin or Periodic Acid Schiff (PAS).

Histolopathological examinations were assessed on kidney

biopsy specimens, taken after each cold storage and reperfu-

sion. The histopathological lesions were semi quantitatively

graded on the basis of severity of cellular injury.

Statistical analyses were performed using SAS® 9.0 statis-

tical software (Cary, NC, USA). The paired t test was used to

investigate whether blood pressure differed between aorta at

the renal artery level and femoral artery. Differences at a P

value of less than 0.05 were considered to be significant.

Results

To prove the efficacy of a modified auto-perfusion model

using femoral vessels, blood pressure was measured in the

aorta and femoral artery. Blood pressure was no significant

differences between aorta and femoral artery (P > 0.05). The

values of blood pressures in the aorta and femoral artery

were systolic (122.99 ± 4.51 and 120.63 ± 3.83 mmHg), dias-

tolic (83.37 ± 9.48 and 87.26 ± 3.16 mmHg) and mean blood

pressures (96.58 ± 4.27 and 98.39 ± 3.31 mmHg) respectively

(Table 1).

In general, warm ischemia injury occurs during organ har-

vest and anastomosis. The average warm ischemia time until

hemo-reperfusion start was evaluated, it was 7.0 ± 1.1 min-

utes. The preserved organs displayed a visible urine excre-

tion immediately when reperfusion was started.

After the kidney reperfusion through the femoral artery

and vein, histopathological changes of the kidneys were char-

acterized by pyknosis, degeneration and exfoliation of distal

tubular epithelial cells, brush border loss and loss of glomer-

ular tufts (Fig 3). Morphological changes observed depen-

dent on cold ischemia time. The damage of renal tubule cells

was increased after 48 hours more than 24 hours cold-pre-

served kidney. In particular, exfoliation of distal tubular epi-

thelial cells was observed in the kidney stored for 24 hours

followed by reperfusion, markedly became severe in the 48

hours (Fig 3).

Fig 2. Schematic diagram of the femoral auto-hemoperfusion technique. A. The renal artery and vein were connected to the femoral

artery and vein using polyethylene connector respectively in an end-to-end fashion. B. Photograph of reperfusion.

Fig 1. Schematic overview of a newly developed reperfusion

model. The kidneys were harvested (A), immediately flushed and

preserved with the iced preservation solutions (B). After cold

preservation, the stored kidney was connected to the femoral

artery and the vein in inguinal region for hemo-reperfusion (C).

Table 1. Comparison of blood pressures and diameter betweenthe aorta and the femoral artery in miniature pig

Aorta Femoral artery P value

Systolic(mmHg) 122.99 ± 4.51 120.63 ± 3.83 0.3262

Diastolic(mmHg) 83.37 ± 9.48 87.26 ± 3.16 0.0677

MAP(mmHg) 96.58 ± 4.27 98.39 ± 3.31 0.2483

Abbreviation: MAP = mean arterial pressure

A model of Isolated Renal Hemoperfusion 443

Discussion

Renal I/R injury is encountered in many clinical situations

such as kidney transplantation, partial nephrectomy, cardiop-

ulmonary bypass, trauma and hydronephrosis (11). Numer-

ous models of I/R injury-related studies have been established

but limitation such as surgery techniques and perfusion sys-

tem have restricted. For the experimental studies of I/R injury,

autotransplant and warm-ischemia (occlusion of renal pedi-

cle) reperfusion model have been used to investigate the

effect of drug in I/R-induced renal injury (7). The isolated

and perfused kidney (IPK) has been used to study the effects

of drugs and toxic substances on renal functions (4). How-

ever, IPK model is inadequate for the study of I/R injury in

vivo. In the present study we introduced a newly developed

reperfusion method using femoral site, which has a parallel

vasculature enough to perfuse the kidney. Kidney mainly has

been transplanted in the iliac fossa. In general, blood pressure

between iliac and renal arteries was similar, and diameters of

iliac arteries were larger than the renal arteries (8). There were

no significant differences in blood pressure between renal arter-

ies and femoral arteries in pigs. Consequently, our data dem-

onstrated that experimental I/R model may be a suitable to

the study of the I/R injury in pigs.

Following I/R injury, the reperfused kidney may have his-

topathologic characteristics, such as extensive tubular dam-

age, tubular cell necrosis, glomerular injury, and signs of tubular

obstruction with cell debris (1). As expected (2,5), this model

shows that cold I/R produced tubulo-interstitial damage dur-

ing kidney preservation. The result of the present study was that

cold ischemia, 48 hours induced further tissue damage com-

pared to 24 hours of preservation.

The average warm ischemia time was decreased in this model

other than intra-abdominal transplantation. The organs dis-

played a visible urine excretion immediately after reperfusion.

Definitely, the newly modified model has the advantages

including simple and easy anastomosis of renal vessels for I/

R injury study. The site of the graft is easy to approach. In

addition, this procedure was able to reduce surgical time, cost

and complication such as blood leakage at anastomosis site.

However, a major limitation of the present model is repre-

sented by the limited duration of the reperfusion. Although

the reperfusion time was limited to 3 hour, our reperfusion

model may be effective in study of early renal I/R injury. This

model may provide a useful means for studying renal I/R inju-

ries as well as the effects of preservation solutions and storage

conditions.

Acknowledgments

“This work was supported by the Korea Research Founda-

tion Grant funded by the Korean Government (MOEHRD,

Basic Research Promotion Fund) (KRF--2006-311-E00528)”,

and also supported by “the Seoul R&BD program (#10548)”

and a grant (20070401034006) from BioGreen 21 Program,

Rural Development Administration, Republic of Korea.

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Fig 3. Histology after reperfusion. The kidneys respectively stored in the cold HTK preservation solutions at 4oC for 0 hour (A, D),

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edly became severe in the 48 hours (C, arrow). H&E staining (A, B and C), PAS staining (D, E and F). Magnification = ×200.

444 Hyun-Suk Nam and Heung-Myong Woo

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허혈/재관류 손상연구를 위한 체외 신장 재관류 모델

남현숙·우흥명1

강원대학교 수의학부대학 외과학교실

요 약 :허혈/재관류 손상은 장기이식 분야에서 해결해야 할 주요 문제점으로 알려져 있다. 본 연구에서는 대퇴 동,정

맥 부위에서 기존의 혈관 문합법 대신 맥관 connector를 이용하여 간단하면서 효과적인 체외 재관류 모델을 개발하였

기에 소개하고자 한다. 개발된 모델이 허혈/재관류 손상연구에 효과적인지 알아보기 위해 혈액 동력학적 평가와 신장

의 재관류 후 손상 양상을 분석하였다. 기존의 재관류 모델에서 사용되는 문합 부위인 복강 대동맥의 혈압과 본 연구

에서 재관류 부위로 활용된 대퇴동맥의 혈압은 유의적 차이가 없었다. 허혈 손상 후 재관류 효과를 알아보기 위해 미

니돼지에서 적출한 신장을 HTK용액에 24, 48시간 동안 각각 저온보관 후 대퇴부에 이식하여 재관류 한 결과, 신장의

재관류까지 수술시간은 평균 7.0 ± 1.1분 소요되었으며, 3시간 재관류 후 재관류 손상 정도는 저온보관시간에 따라 증

가되는 것이 확인되었다. 이는 개발된 모델이 맥관 문합 없이 간단한 관류방법이면서도 기존의 복잡한 수술에 의한 재

관류 방법과 유사한 손상 모델을 만들 수 있는 효과적인 허혈/재관류 동물모델이라는 것을 의미한다. 따라서 본 연구

에서 개발한 신장 재관류 모델은 초기 허혈/재관류 손상 연구와 장기이식에서 이식면역연구에 효과적인 모델이라 사

료된다.

주요어 :허혈/재관류 손상, 동물모델, 신장, 돼지, 혈압, 저온보관.