犬における移植腎早期急性拒絶反応抑制を目的とした3剤併用免疫抑制療法の適用

誌名誌名 獣医麻酔外科学雑誌

ISSNISSN 09165908

著者著者

片山, 泰章深井, 和紘五十嵐, 宏之谷, 健二百田, 豊神志那, 弘明佐藤, かつ江高橋, 公正多川, 政弘

巻/号巻/号 41巻1号

掲載ページ掲載ページ p. 9-16

発行年月発行年月 2010年4月

農林水産省 農林水産技術会議事務局筑波産学連携支援センターTsukuba Business-Academia Cooperation Support Center, Agriculture, Forestry and Fisheries Research CouncilSecretariat

Triple drug Therapy for the Prevention of Early Acute Renal Allograft Rejection in Dogs

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Masaaki KATAYAMA !), Kazuhiro FUKAI! ), Hiroyuki IGARASHI! ), Kenji TANI2l, Yutaka MOMOTA3), Hiroaki KAMISHTNA4), Katsue SAT05),

Kimimasa T AKAHASHI5) and Masahiro T AGA W A 6)

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I)Division of Small Animal S/./Igel)·, Department ofVeterinaJY Medicine. Faculty (}f Agriculture, Iwate University, 3-/8-8 Ueda, Morioka, /wate 020-8550, 2)Division (}f VeterinGl), Surgel)', Department of Veterinal ), Medicine, Faculty o/Agriculture, Yamagu­chi University, /677-/ Yoshida, Yamaguchi, Yamaguchi 753-85 I 5, 3) Department of VeterinGl), Nursing and Technology, Nippon VeterinaJ)' and Li/e Science University, /-7-/ KyounclI7, Musashino, Tokyo /80-8602,4) Division ol'Small Animal Internal Medi­cine, Department of VeterinaJ), Medicine, Faculty of Agriculture, /wate University, 3-/8-8 Ueda, Morioka, lH'ate 020-8550, 5) Division of VeterinGl ;l' Pathology, Department of Veterinarv Medicine, Nippon Veterinary and Life Science Universit) ', 1-7-1 Kyollnan, Musashino, Tokyo /80-8602, and 6) Division of VeterinGl ;v Sill gel)" Department of VeterinGl), Medicine, Nippon Veter­inGl)' and Life Science Universi~v. 1-7-1 K),ounan, Musashino, Tokyo 180-8602, Japan

SUMMARY: A triple drug therapy with emulsified cyclosporine, mycophenolate mofetil and predonisolone was uscd

for 90 days for the prevention of acute rejection in six renal transplanted beagles . Five dogs survived for 90 days, but

one dog died suddenly from unknown reason with a functioning graft 9 days after transp lantation. Allografts of three

of the five survived dogs showed no evidence of acute rejection based on Banff 07 histopathological classification. The

other two dogs showed acute allograft rejection on histopathological evaluation and urinary tract infection at the end

of the study. None of the dogs showed adverse effects such as hepatotoxicity and myelosuppression. At necropsy,

abnormal cystic dil atation of the crypts with secretory accumu lation was found in the duodenum mucosa in all dogs

survived for 90 days. With further research, this triple drug immunosuppressive therapy could become a potential alter­

native of the classical immunosuppressive protocol (cyclosporine, azathiopurine and predonisolone) for the prevention

of acute allograft rejection in dogs.

Key words: cyclosporine, dog, mycophenolate mofetil , renal transplantation

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Jpl7. 1. Vet. Anesth. Slirg. 41(1): 9- 16, 2010.

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10 Masaaki KAT A Y AMA, Kazuhiro FUKAI, Hiroyuki IGARASHI , el al.

Table.1. Pairs of recipient and donor dogs

Donor & Recipient

Pairs

Dog No. B.w. (Kg) Sex Age (years)

Pair A

Pair B

Pair C

2

3

4

5

6

9.4

11.8

10.5

9.1

10.0

8.3

female

female

female

female

male

female

7

3 7

7

8

8

Five female and one male, non-sibling, healthy beagle dogs were used in the current study. Each pair of the donor

and recipient was determined based on the compatible results of the blood cross matching (major and minor).

Renal transplantation is considered as one of the

treatment options for the end-stage chronic renal failure

in humans and cats. In contrast, prevention of strong

acute rejection episode has been challenging in dogs

because of a lack of an effective immunosuppressive

regimen. Moreover, adverse effects of

immunosuppressive drugs are common in dogs. It is

thought that the combination of drugs with different

modes of action should be more effective to suppress

acute rejection episodes than a single use of each drug.

Bernsteen et al. repOlted the use of triple drug regimen

using emulsified form of cyclosporine (E-CsA),

antimetabolic drug azathiopurine (Aza) and

predonisolone (Pre) in canine renal transplant model !).

In their study, two of the four dogs survived for 100 day

study period and one of them showed acute rejection

(AR) at the study end. In addition, three dogs had

hepatotoxicity which was induced by Aza. Aza has been

known to induce hepatotoxicity and bone marrow

suppression in dogs II , 14). If these adverse effects

develop, Aza has to be withdrawn or used with a reduced

dosage. As a result, acute rejection episode may develop.

Therefore, it is important to search for more effective and

safer immunosuppressive protocol as an excellent

alternative for this triple drug regimen consisted of E­

CsA, Aza and Pre in canine renal transplantation.

Mycophenolate mofetil (MMF) is used as one of the

immunosuppressive drugs against acute rejection

episode in conjunction with Aza in human renal

transplantation. While Aza widely suppresses both de

novo and salvage pathways for purine synthesis for cell

proliferations, MMF exhibits immunosuppressive effects

through restraint of cytotoxic T lymphocyte production

and the antibody production ability by selective

suppression of only de novo pathway on which

lymphocytes depend. Therefore, MMF could target

lymphocytes intensively and may decrease the incidence

of the adverse effects associated with the antimetabolic

immunosuppressive drug compared with Aza. We

hypothesized that the triple agent therapy of E-CsA,

MMF and Pre is effective and safe for the prevention of

acute allograft rejection in renal transplanted dogs. The

aim of this study was to investigate the clinical efficacy

of this E-CsA based triple drug therapy in preventing

acute renal allograft rejection in red blood cells cross­

matched beagles.

In a canine renal transplant model, acute allograft

rejection occurs within 7- 13 days with inadequate

immunosuppression8). An early acute rejection episode

of transplants within 3 months post-transplantation has

been indicated as an unfavorable prognostic factor in

human patients5). We therefore chose the 90 day study

period to evaluate whether the triple drug

immunosuppressive protocol with E-CsA, MMF and Pre

is effective in preventing early acute allograft rejection in

dogs before assessing its efficacy in a clinical setting.

Materials & Methods

Five female and one male, non-sibling, healthy beagle

dogs were used in the current study. Body weights

ranged from 8.6 to 12.5 kg and the ages from 3 to 8 years

old. Three pairs of the donor and recipient were

determined based on the compatible results of the blood

Triple drug Therapy for the Prevention of Early Acute Renal Allograft Rejection in Dogs II

cross matching (major and minor)8) (Table I). This study

was approved by the Iwate University Animal Care and

Use Committee.

Each dog received a heterotopic renal transplantation

from a paired dog and the native kidney was removed,

leaving only the transplanted kidney to the recipient.

Two surgical teams performed the same procedure

simultaneously on each paired dog. Each dog served as

both a kidney donor and recipient. The donor and

recipients were premedicated with butorphanol (0.5 mgt

kg intravenously [IV]) and midazolam (0.3 mg/kg IV).

Anesthesia was induced with propofol (4 mg/kg IV to be

effect) and maintained with isoflurane in 100% oxygen.

All dogs were administered a balanced electrolyte

solution (lactated Ringer's solution; 10 mllkg/hr) and

dopamine (3 fig/kg/min) through a catheter placed in the

cephalic vein. Antibiotics (cefmetazole Na 25 mg/kg [V)

was administered at the anesthesia induction and 2 hours

later, then continued for 7 days twice a day. Mannitol

was administered 20 minutes prior to kidney harvest at

the dose of I g/kg [V and again just after completion of

renal vessel anastomosis at the dose of 0.5 g/kg IV.

The kidney was harvested, flushed with cold

phosphate buffered sucrose (PBS) solution, and placed in

4°C PBS solution before anastomosis. The average

warm ischemia time was 47 minutes (range: 42- 50

minites).

The renal vein was anastomosed in end-to-side fashion

to the external iliac vein with a simple continuous suture

pattern using 5-0 silk. The renal artery was anastomosed

in an end-to-end fashion to the external iliac artery with a

simple interrupted suture pattern using 6-0 nylon. The

ureter was anastomosed to the bladder mucosa with a

modified ureteroneocystostomy technique with 3-0

polypropylene in a simple continuous pattern. The

transplanted kidney was stabilized by sutured placement

in a retroperitoneal pocket with 6-0 nylon in a simple

interrupted pattern.

Dogs were monitored continuously for 24 hours and

buprenorphine (0.2 mg/head) was administered to

control pain. IV fluids were administered at a

maintenance rate for 24 hours after surgery.

Serum creatinine (Cre), blood urea nitrogen (BUN)

and CsA trough levels were measured every 3- 5 days

after transplantation. In this study, serum Cre and BUN

values were used as an index of the renal function

because it depended only on the transplanted kidney.

Complete blood count, serum biochemistry panel and

urinalysis were performed every 2 to 3 weeks to monitor

the adverse effects such as hepatotoxicity and

myelosuppression.

The day of renal transplantation was designated as day

O. Starting on day -2, all dogs were orally administered

with E-CsA (Cicporal capsules; Nichi-iko, Toyama,

Japan) which was adjusted to around 10 mg/kg twice

daily. Pre (0.5 mg/kg twice daily) was also orally

administered. The dosage of Pre was tapered every 2

weeks and discontinued on day 80. All dogs started 5

mg/kg of MMF (Cellcept capsules; Chugai, Tokyo,

Japan) twice daily on day 2 and the dosage was increased

to 10 mg/kg twice dai lyon day 10 and continued unti I the

end of the study. CsA trough levels were monitored

using a fluorescence polarization immunoassay (FPIA)

(TDx Cyclosporine A Dinapack kit; Abbott, Tokyo,

Japan). The dose of CsA was adjusted to keep the CsA

trough level within the therapeutic range of 400- 600 ng/

mI. [n this study, fluconazole (Fcz) was added to the

regimen in order to decrease the CsA dosage required to

keep CsA trough levels within the therapeutic range4).

Detail data of the CsA dosage after Fcz administration is

presented in our previous study3). Once CsA blood levels

were stabilized, oral Fcz was given daily at 5 mg/kg 2

hours before CsA administration. The dose of CsA was

then re-adjusted to keep the trough level within the

therapeutic range. Upon achieving the stable CsA blood

levels, the frequency of CsA dosing was changed from

twice to once daily. In this study, groups of no treatment,

CsA alone and MMF alone were not included for humane

reasons.

Dogs were euthanized 90 days after transplantation.

Necropsy was performed in all dogs immediately after

euthanasia, and tissue samples of the heart, lung, liver,

stomach, small intestine, large intestine, and allograft

were collected. These samples were fixed in 10 %

formalin and processed for histopathological evaluation.

Evaluation of acute allograft rejection was based on the

severity of the infiltration of mononuclear cells into the

interstitium and tubles with or without arteritis according

12 Masaaki KATAYAMA, Kazuhiro FUKAI, Hiroyuki IGARASHI, era!.

12

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Fig. J. Serum creatinine (a) and blood urea nitrogen (b) values in renal transplanted dogs treated with a combined emulsified cyclosporine I mycoph enolate mofetil I predonisolone immunosuppressive regimen .

500

~ 400 ,

~ 300

; lOO

", .. _. -II-

' . ' .

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3000

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a 2000

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o 1000 :§

~ 500

o 10 20

10 20

30 40 50 60 ; 0 so 90

D:.ysAfter TnDspbnt3rioD

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- . D022

--'-Do:3 --)(-'Dog4

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c

to Banff'07 classification which

for

IS the standard

histopathological

transplantation J 5).

criteria

Results

human renal

Five (Dog 2- 6) of the 6 dogs survived for the 90 days

study periods. One dog (Dog I) died suddenly with a

functioning graft with no evidence of acute rejection 9

days after transplantation. Necropsy could not reveal

specific reasons for the sudden death in this dog. The

serum Cre and BUN values of Dog 2, Dog 3, Dog 5 and

100

g 90

:; so ~ iO

'2 60

~ SO c 'c 40

~ ~o~..."..-....,r ~ 20

< 10

10 20 30 40 50 60 70

Days After TI";lRlp~nl:;ttioD

b

- ... Do,l

-. Do&2 ........... Do&3 --X- -Dog4 __ DogS

-"- ' Du&6

so 90

Fig. 2. Alanine aminotransferase (a), aspartate aminotransferase (b) and alkaline phosphatase (c) values in renal transplanted dogs treated with a combined em ulsified cyclosporinc I mycophenolate mofetil l predonisolone immunosuppressive regimen.

Dog 6 were maintained within the reference range

(Fig.l.a and b, respectively) , and clinical symptoms

indicative of acute rejection such as fever, inappetence,

vomiting, loss of activity or pain of the transplantation

kidney region were not observed throughout the study.

Dog 4 developed bladder atony and presented azotemia

throughout the study period. Changes of Liver enzymes

in each dog throughout the study period were shown in

Fig. 2 .a-c. Elevation of alanine aminotransferase (AL T)

and alkaline phosphatase (ALP) were observed in all

survived dogs, which tended to return to reference ranges

after discontinuation of oral administration of Pre. The

Triple drug Therapy for the Prevention of Early Aeute Renal Allograft Rejection in Dogs 13

1600

1400

'"3 1200

~ 1000 -~

soo :., 600

400 -200

-

120

~ 100

§ SO

:> ." 60

" 40

" 20

- ..... Dogl - __ Dog!

----.- Dog3 --)(-- Dog",

~Dog5

- .... -- Dog 6

.'~::-::~"?( ___ ~::::= ... =~::-------*-----__ ~ _________ ~ ____ -------x

10 30 40 50 60

DaysAfter T ranspJantation

a

10 20 30 40 50 60

D:tysAfter Tr:tnspbntation

c

10

70

80

- ... Dogl - .... Dog.:!

~Dog3

--X--Dog-'

--'-Dog5 - .... --Oog6

so

90

90

40000

35000

i 30000

25000 ."

~ 20000

_ 15000

~ 10000

5000

100

SO

" - 60 , -

40

~ 20

10 20

10 20

-~ Dogl - __ Dog2

--"-Dog3 --)(-- Dog ..

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30 40 50 60 i O so 90 D:a.ysAfter Transplantation

b

- ... Dogl - __ Dog2

-'-DogJ --X--Dog4

--"'Dog5 - .... --Dog 6

30 40 50 60 70 80 90

Days After Tr:msplaDtarioD

d

Fig. 3. Number of white blood cells (a), red blood cells (b)and platelets (c), and packed cell volume (d) in renal transplanted dogs treated

w ith a combined emulsified cyclosporine I mycophenolate mofetil / predonisolone immunosuppressive regimen.

adverse effects which were related to the

myelosuppression such as leucocytopenia,

thrombocytopenia, or anemIa were not observed (Fig.

3. a- d).

Urinary infection by E.coli was found in Dog 3 and

Dog 4 at day 66 and 35, respectively. Dog 3 and 4 were

treated with cefazolin Na (25 mg/kg IV or PO) twice

daily followed by fosfomycin (20 mg/kg PO) twice daily

based on the result of a urine bacterial sensitivity test.

Dog 4 developed acute renal failure after IV

administration of fosfomycin (20 mg/kg) on day 61, but

recovered by fluid therapy. The IV administration of

fosfomycin (20 mg/kg) was changed to oral

administration thereafter. Although treatments with

fosfomycin were continued in both Dog 3 and Dog 4

until the end of the study, a urinary bacterial infection

remained.

All 5 dogs who survived for 90 days showed temporal

weight reduction after transplantation. They most

decreased their weight 0.8- 1.3 kg (average; 1.0 kg)

between day 12- 17 (average; day 14) compared with

pre-transplant. Vomiting was observed in 2 of the 5 dogs

(Dog 2, Dog 4) w ithin 6 days after transplantation, but it

was not related to a raise ofCre value. In addition, a soft

stool was observed in Dog I on day I, but this was

improved without treatments within a few days. Mild

dehydration was observed in all dogs but improved by

discontinuation of Pre on day 80.

Dog 2, Dog 5 and Dog 6, had a mild infiltration of

lymphocytes and plasma cells in the cortex of their

allografts and classified as borderline changes (Fig. 4).

Dog 3 and Dog 4 had acute rejection I B (Fig. 5) and III

(Fig. 6), respectively. However, both dogs had urinary

tract infection with E.coli during the study. Renal

allograft injury is known to be associated with urinary

tract infection caused by E. coli in human renal transplant

patients I3 ). Therefore, these two dogs should be

excluded from histopathological evaluation of the

allografts.

In all 5 dogs who survived for the 90 days study

period, mild to moderate lymphocytic-plasmacytic

inflammation and the cystic dilatation of the crypts with

secretory accumulation in the duodenum mucosa were

found (Fig. 7). No abnormities were found in the heart,

14 Masaaki KATA Y AMA. Kazlihiro FUKAI, HirOYlIki IGARASHI. el al.

Fig. 4. I-listopathology orthe allograft in Dog 2 on 90 days post­

transplant. Mild Iymphocytic-plasmacytic interstitial infiltration

is seen in the cortex (arrow). This lesion was class i tied as

borderline changes according to Ban/T07 crite ri a. HE stain.

Bar=50 ,LIm.

Fig. 6. Histopathology of the allograft in Dog 4 on 90 days post­

transplan t. Severe IYll1phocytic-plasll1acytic interstitial inliltration

and artel'ior fibrin oid change (aITow) in the cortex. Thi s lesion

was classi ti ed as acute rejection III according to Ban 1'1'07 criteria.

J-J E slain. Bar= 10 )fill.

lungs, liver, stomach, small intestine, large intestine, and

the spleen at necropsy. The nati ve kidneys removed a t

the surgery also showed no abnormalities.

Discussion

Acute reject ion IS suspected based on clinical

symptoms such as fever, inappetence , vomiting, loss of

activity or pain on palpation of the area of the a llograft,

elevation of BUN and ere values , and histological

Fig. 5. Histopathology orthe allograft in Dog 3 on 90 days post­

tran splant. Moderate lymphcytic-p laSl11acytic interstiti al­

infiltration in the cortex. Thi s les ion was class ified as acute

rejectionlB accordi ng to Ban/l'07 criteria. HE stain. 8ar= 10 ifill.

Fig. 7. Hi stopathology oCthe duodenuill in Dog 3 on 90 days

post- transplant. Abnormal cystic dilatation oCthe crypts with

secretol'y accul11ulation (arrow) and mild lymphocytic infiltration

are see ll ill the mucosa. HE stain. B<II= IO p111.

evidence of acute rejection episodes according to Bantf'

07 criteria. Banff classification has been Llsed to evaluate

renal a llograft hi sto logy in dogs as well as hUll1ans7).

There are three types of acute rejection (I, II and Ill) with

subclassifications of A and B, which define the severity

of the lesions. Type I is tubulointerstitial rej ection

without arteritis. Type II IS vascular rejection

characterized by intimal arteritis. Type III tS severe

rejecti on with transmural arteritis. The cases with only

mild tubulitis with or without only mild focal interstitia l

Triple drug Therapy for the Prevention of Early Aeute Renal Allograti Rejection in Dogs 15

inflammation are classified as borderline changes l2, 15),

Some inflammatory changes like borderline changes are

expected in any allograft and do not necessarily mean

that acute rejection is occurringI2). In our study, three of

the five dogs survived for 90 days were classified as

borderline changes and were thought to bc inappropriate

to be included into the rejection categories.

In canine renal transplantation, the mean graft survival

time was reported to be 25 days with monotherapy of oil­

based cyclosporine (O-CsA: 25 mg/kg/day)'J). With

MMF alone at the dosage of 10, 20 and 40 mg/kg/day,

the mean graft survival time was 6, 9 and 36 days,

respectively9, IU). When MMF (20 mg/kg/day) was

combined with O-CsA (5 mg/kg/day) and

methylpredonisolone (0.1 mg/kg/day), the mean allograft

survival time was significantly extended to 122.4 days IU).

However Papalois et al. reported that MMF (20 mg/kg/

day) with O-CsA (25 mg/kg/day) had the mean allograft

survival time of only 13 days9) These variabilities of the

allograft survival time may be partially due to the drug

formulation of CsA. In these studies, O-CsA which has

unstable intestinal absorption properties was used and

monitoring of whole blood CsA trough concentrations

was not performed. Kyles et 01. reported that three ofthc

five dogs treated with E-CsA (20 mg/kg/day) alone

survived for 100 day study period, but exhibited

moderate to severe mononuclear cell infiltrates into the

allografts6), In the present study, we chose the

combination therapy of E-CsA, MMF and Pre and

adjusted the CsA trough levels between 400 and 600 ng/

ml using FPIA method throughout the study. Triple drug

therapy used in this study could prevent early acute

allograft rejection in three of five dogs and prolong graft

survival time in five of six dogs up to 90 days post­

transplant. A longer study period is needed to evaluate

the effects of our immunosuppressive protocol on a late

acute rejection.

In this study, lower urinary tract infection with E.coli.

was found in two dogs. Bacterial infections have been

known as one of the serious complications in canine renal

transplant patients because of the lifelong

ImmunosuppressIon. Although prophylactic use of

antibiotics may be effective for the prevention of the

bacterial infections after transplantation, it may induce

renal toxicity and the development of antibiotic resistant

bacteria. In this study, we chose cefmetazole Na (twice a

day) up to 7 days after surgery as post operative

antibiotic therapy. However, further study is required to

investigate the effectiveness and safety of long term

prophylactic antibiotic therapy for call1ne renal

transplantation,

MMF, a noncompetitive inhibitor of 1110Sl11e

monophosphate dehydrogenase and guanosl11e

monophosphate syntherase, regulates the growth-rate of

rapidly proliferating cells. In this study, cystic dilatation

of the crypts with secretory accumulation was found in

the duodenum mucosa in all 5 dogs survived for 90 days.

Mild to moderate lymphocytic-plasmacytic duodenitis

was also found in these dogs, Secretory accumulation in

the crypts was not due to the hyper-secretion or

dysfunction of secretary system. These findings may be

due to the down-regulation of turnover-rate of intestinal

mucosa by anti-metabolic effects ofMMF. Chanda et 01.

reported acute cryptitis and crypt abscesses without

glandular architectural abnormalities or other chronic

features in beagles treated with MMF2) Severe

vomiting, diarrhea, bleeding in intestinal tracts, and loss

of appetite were mentioned as clinical signs of MMF

toxicities, however these signs were not observed in our

dogs. It is also of importance to evaluate whether cryptal

dilatation in the gut progresses to acute cryptitis and

crypt abscesses in a long term study.

In conclusion, the triple immunosuppressive protocol

of E-CsA combined with MMF and Pre could become a

potential alternative regImen of the classical

immunosuppressive therapy (CsA, Aza and Pre) for the

prevention of acute allograft rejection in canine renal

transplantation. However, all dogs in this study showed

subclinical mild to moderate Iymphocytic-plasmacytic

interstitial infiltration and cystic dilatation of the crypts

with secretory accumulation in the duodenum. Further

investigation IS needed to see the long term

immunosuppressive effects and adverse effects

associated with this protocol.

References

I. Bernsteen, L, Gregory, C.R" Kyles, A.L Griffeyn, S.M. and

Patzn. J. (2003): Microel1111lsitied cyclosporine-based il11l11111l0-

16 Masaaki KATA Y AMA. Ka z uhiro FUKAI. I-liroy uki IGARASHI , el al.

suppression for the prevention of acute renal allografi rejection in

unrelatcd dogs: preliminary experimenta l study. Vet. Surg. 32:

213- 219.

2. Chanda. S.M" Sellin . .I .H" Torres, e.M. and Yec, J.P. (2002):

Comparative gastrointestinal effects of mycophenolatc mofctil

capsules and cnteric-coated tablet s of sodium-mycopheno lic acid

in beagle dogs. Transplant. Proc . 34: 3387- 3392.

3. Kata yama, M. , Iga rashi . 1-1" Fukai , K. , Tani , K. , Momota. Y. ,

Kamishina, H. and Taga\\'a. M. (20 I 0): Fluconazole decreases

cyclosporine dosagc in renal transplantcd dogs. Research in vete r­

inary science 89: 124-125.

-to Katayama, M" Igarashi. H" Tani. K" Nezu. Y., Harada , Y" Yogo ,

T" Hara. Y .. Aoki, S. and Tagawa, M. (2008): Effect of multiple

oral dosing of Iluconazo!c on the pharmacokinetics of cyclospo­

rine in healthy beagles. J. Vet. Mcd. Sci. 70: R5- R8.

5. Kinukawa. T" Ohshima, S. , Ono. Y. and Ilattori, R. (2002):

[Effect of acute rejection episode on occu rrence of chroni c rej ec­

tion afier kidney transplantation I. Hinyokika Ki yo. 48: 687- 69 1.

6. Kyles, A.E., Gregory, e.R. , Griffey, S.M. , Jackson, J. , Bernsteen.

L. and Morris, R.E. (2002 ): An el'aluation of combined immuno­

suppression with MNA 715 and microemulsitied cyclosporine on

renal allografi rejection in misJ11atched mongrel dogs. Vet. Surg.

31: 358- 366.

7. Kyles. A.E., Gregory, e.R. , Griffey. S.M., Bernsteen. L., Pierce ,

.I., Lilja, H.s. and Morris, R.E. (2003): ImJ11unosuppression lI'ith a

cOJ11bination of the lellunoJ11ide analog. FK 778. and microeJ11ulsi ­

licd cyclosporine for renal transplantation in mongrel dogs. Trans­

plantation 75: I 128- 1 133 .

8. MinaJ11i , T" Watanabc, T. , Muto, M. , Wakao. Y" Suzuki. T . and

Takahashi. M. (1993): Prcliminary results of short-term combina­

tion immunosuppressions of mi?oribine, azathioprine. and prcd­

nisolone with pretreatment to caninc kidney transplantation. J.

Vet. Med. Sci. 55: 409-414.

9. Papalois. B., Wahon: D., Leone, .I.. Aashcim, T .. Gilmore, T. and

Sutherland. D. E. (1996): The effect of a nonstcroid immunosup-

pressi ve regimen with RS-61443 and cyelosporine on kidney

allograll surv iva l in dogs. Transplant. Proc. 28: 937.

10. Platz. K.P., Sollinger, 1-I.W., Ilullen. D.A., Eckhoff. D.E., Eugui .

E.M. and Allison , A.e. (1991): RS-61443- a nell' , potent immun­

osuppressive agent. Transplantation 51: 27- 31.

II . Plumb. D.e. (1999) Veterinar. Drug Handbook, 3rd ed. Phanna

Vet, Whiet Bear Lake. MN.

12 . Racusen , L.e. , Solez, K .. Colv in. R.B .. Bonsib, S.M. , Castro,

M.C .. Cavallo, T .. Croker. B.P .. Dcmctris. A . .I. , Drachenberg,

e.B., Fogo. A.B., Furness, 1'. , Gaber. L.W .. Gibson. I.W. , Glotz,

D., Goldberg, .I.e. , Grande . ./., Halloran, P.F., Hansen, H.E., lIan­

ley, B., Hayry, P . .1.. Hill, e.M .. Hof'linan. E.O .. Hunsicker, L.G. ,

Lindblad, AS, Marcussen, N., Mihatseh, M .. I .. Nadasdy, T ..

Nickerson, P .. Olscn. T.S .. Padadimitriou, .I.e., Randhall'a, P.S ..

Rayner. D.e., Roberts, I. , Rose , S., Rush, D., Salinas-Madrigal. L ..

Sa lol11 on, D.K., Sund, S. , Taskincn, E. , Trpkov, K. and Yamagu­

chi. Y. (1999): The Banff 97 work ing class ilica tion of renal

allograft pathology. Kidney. Int. 55: 7 13- 723.

13. Rice. J.e.. Pcng. T .. Kuo, Y.F. , Pendyala, S. , Simmons, L ..

Boughton . .I .. Ishihara, K .. Nowicki , S. and Nowicki. 13 . .1. (2006):

Renal allograft injury is associated with urinary tract infection

caused by Escherichia coli bearing adherence factors. Am . ./.

Transplant. 6: 2375- 2383.

14. Rinkardt. N.E. and Kruth, S.A. (1996): Azathioprine-induced bone

marrow toxicity in four dogs. The Canadian vetcrinary journal 37:

612- 613.

15. Solez, K. , Colv in , R.B., Racusen, L.C .. Haas. M .. Sis, B .. Mcngel.

M. , Iialloran. P.F. , Baldwin, W., l3anfi , G. , Collins, A.B .. Cosio.

F. , David, D.S., Drachenberg, e. , Einecke. G., Fogo. A.B .. Gib­

son, I.W .. Glot?, D. , Iskandar. S.S. , Kraus. E .. Lerut, E .. Mannon.

R.B., Mihatsch, M., Nanki ell , 13..1., Nickeleit. V., Papadimitriou .

.I.e., Randha wa. P .. Regclc, H., Renaudin. K., Roberts , I .. Seron.

D., Smith. R.N. and Valcnte, M. (2008): l3anlT 07 classification of

renal allograft pathol ogy: updates and future directions. Am . .I.

Transplant. 8: 753- 760.