Effects of an immunosuppressive agent
on the anti-glomerular basement membrane
nephritis model rat○ Kimihide Okamoto, Shinichi Ozato, Hideaki Kamaga, Kousuke Morizumi,Seiichi Katayama, Naoyuki Hironaka, Katsuhide Nishi

Objective

Results

Anti-glomerular basement membrane (GBM) nephritis is caused by
the production of autoantibody against GBM. The progression of thedisease leads to kidney failure which lowers the QOL of patients
considerably. Therefore, the development of novel therapeuticagents is expected.
The rat model induced by administration of anti-GBM antibody is
considered as one of the experimental models of anti-GBM nephritis.
In the present study, effects of cyclophosphamide (CYP), a typicalimmunosuppressive agent, were evaluated using this model.
The results indicate the animals in Nephritis model group developed
anti-GBM nephritis-like symptoms, and the high dose of CYP
suppressed progression of anti-GBM nephritis-like symptoms by
suppressing renal glomerulus and tubules inflammation.

Photo 1：HE (Glomerulus)

Normal control

CYP 5 mg/kg

Nephritis model

Tuft adhesion/Cellular crescent

Summary in JapanesePhoto 2：HE (Renal tubules)

抗糸球体基底膜腎炎とは，糸球体基底膜（GBM）に対する自己抗体の産生および沈着により誘導され，急速な腎機能の低下が特徴的な疾患である．病態の進行により引き起こされる腎不全は患者のQOLを著しく低下させるため，抗GBM腎炎に対する新規治療薬の開発が待ち望まれている．実験モデルの一つとして抗 GBM 抗体投与による腎炎ラットモデルが知られている．本研 究では，抗 GBM 腎炎モデルを作 製し，免疫 抑 制 剤であるシクロホスファミド（CYP）の効果を検討した．
病態モデル動物は雄性のWKYラットに抗 GBM 抗体を単回静脈内投与することで作製した．
CYPは1日1回，28日間経口投与した．評価は血漿中BUN，体重比腎臓重量，尿中タンパク排泄量，尿潜血スコア，尿中Clusterin，KIM-1，MCP-1濃度および病理組織学的検査にて行った．
病態群では正常群と比較して血漿中BUN，各種尿中パラメーター，尿潜血スコアの増加，体重比腎臓重量の高値および病理組織学的検査では半月体の形成が認められ，病態群における抗 GBM 腎炎様症状が観察された．この病態群の変化に対してCYP 投与群は，血漿中 BUNおよび各種尿中パラメーターの増加を抑制し，体重比腎臓重量で低値を示した．以上より，
CYPは抗GBM腎炎モデル動物の病態進行を抑制することが示唆された．
抗GBM腎炎ラットモデルは新規治療薬の効果を評価するために有用なモデルであり，今後の治療薬開発に貢献しうると考えられる．

Normal control

CYP 5 mg/kg

Nephritis model

Photo 3：IF (Glomerulus)

Normal control

CYP 5 mg/kg

Nephritis model

Materials and Methods● AnimalsMale, WKY rat, 7-week-old (on Day 0)● Induction of anti-GBM nephritis

Endoxan® Oral Powder, Shionogi & Co., Ltd.,administrated orally once a day from Days 0 to 27.

● Sampling and Method

Dissection:● Evaluation

Body weight:
once a week
Plasma blood urea nitrogen (BUN):
7180 Clinical Analyzer, Hitachi High-Tech Corp.
Kidney weight ratio: per 100 g body weight
Urinary protein excretion:
7180 Clinical Analyzer, Hitachi High-Tech Corp.
Score of occult hematuria:
Hemastix®, Siemens Healthcare Diagnostics K.K.
The scoring criteria are shown in the table below.
Urinary clusterin, KIM-1 and MCP-1:
Bio-Plex ProTM RBM Rat Kidney Toxicity Panel 1,
Bio-Rad Laboratories, Inc.
Histopathological analysis:
Hematoxylin-eosin (HE) staining analysis:kidney, formalin-fixed paraffin-embedded block
Immunofluorescent (IF) staining analysis:kidney, frozen block
IgG:
Goat Anti-Rat IgG H&L (Alexa Fluor® 488), Abcam plc.

Normal control
Nephritis model

Distilled water

CYP 2 mg/kg
CYP 5 mg/kg

CYP

Day 0prep.o.
5 mL/kg

N5ScoreCriteria
1
negative
2± (non-hemolysis)
3± (hemomlysis)
41+52+63+

714 1521

CYP administration, p.o., once a day

27 28● Plasma BUN, urinary protein excretion, score of occulthematuria, urinary clusterin, KIM-1 and MCP-1 significantly
increased in the anti-GBM nephritis model animals,
compared with those in the normal control group (Fig. 2 to 8).

● The Nephritis model group showed formation of cellularcrescents (Photo 1), endocapillary hypercellularity (data notshown), hyaline casts and regenerative tubules (Photo 2)
compared with those in the normal control group.

These results indicated glomerular and tubular injury in themodel animals. The tubular injury seen in the Nephritis modelgroup would be mediated by inflammation caused byglomerular injury.

**

##

##

50

500

61320(Day)2628

Fig. 4 Urinary protein excretion(mg/24 hours)

##

7##

##

##

212714(Day)pre15(Day)††0.028

††

††

††

‡‡‡

pre4

714(Day)2127

Fig. 7 KIM-1

1.5

**
**

3**

2600

#400

##

##

200

pre14(Day)Normalcontrol

Nephritis
CYPmodel 2 mg/kg

CYP5 mg/kg

Normal control
Nephritis modelCYP 2 mg/kg
CYP 5 mg/kg

1

**

800

##

0.6

2

Fig. 6 Clusterin

1000

#0.8

3

#1200

0#
#4100

1400

1.00.25****0.46**

200

Fig. 3 Kidney weight ratio

Fig. 5 Score of occult hematuria**pre*

20100**

2510001.2

15300

The score of occult hematuria wasdetermined based on the change
in color of the test paper.
Induction
Plasma collection
Urine collection
Score of occult
hematuria
Dissection###150##**Fig. 2 Plasma BUN

30**

schedule

Dosing solution Administration

200400● Experimental group, scoring criteria of occult hematuria andGroup

250(g)

Plasma collection:

Housed in metabolic cage for 24 hours and urine wascollected.
Blood was collected from subclavian vein using syringewith heparin and centrifugated to obtain plasma.
Measurement of kidney weight was performed.

35300(ng/mL)

Urine collection:

350

Fig. 1 Body weight(g/ 100g body weight)

CYP:

27

● Statistical analysis

1.0

*0.5

10

Fig. 8 MCP-1(ng/mL)● Administration

IgG deposition(mg/dL)

Administration:

Nephritogenic Monoclonal Antibody, Clone b35,
Chondrex, Inc., 100 µg/1.0 mL/bodysingle dose via intravenous injection, Day 0(ng/mL)

Antibody:##pre14(Day)

###

270.0

pre14(Day)

27Fig. 1 to 4, 6 to 8:
Student’s t-test: significantly different from Normal control group (*: P<0.05, **: P<0.01).
Dunnett's multiple comparison test: significantly different from Nephritis model group(#: P<0.05, ##: P<0.01).
Fig. 5:Wilcoxon signed-rank test: significantly different from Normal control group (††: P<0.01).
Steel's multiple comparison test: significantly different from Nephritis model group(‡: P<0.05, ‡‡: P<0.01).
Each symbol represents Mean ± SEM (N=3 to 5)

Conclusion● The high dose of CYP lowered the value ofplasma BUN (Fig. 2) and suppressed cellularcrescent formation (Photo 1) and endocapillary
hypercellularity (data not shown). This result
indicates CYP attenuated glomerular injury via
inflammation suppression.

● The high dose of CYP also lowered the values ofurinary clusterin, KIM-1, MCP-1 and decreased
hyaline casts and regenerative tubules (Fig. 6 to8 and Photo 2). This result indicates CYP
attenuated tubular injury via inflammation
suppression.

● The high dose of CYP decreased urinary protein

excretion from Day 7 (Fig. 4).

These results indicate CYP suppressed progression ofanti-GBM nephritis-like symptoms by suppressing
renal glomerulus and tubules inflammation.
In conclusion, this anti-GBM nephritis model is usefulto evaluate the effects of the novel therapeuticagents.
Glomerulus

Parameter● Plasma BUN● Urinary protein excretion
Histopathological analysis● Cellular crescents● Endocapillary hypercellularity

Renal tubules

Parameter● Clusterin (proximal/distal)● KIM-1 (proximal)● MCP-1 (proximal)
Histopathological analysis● Hyaline casts (proximal/distal)● Regenerative tubules (proximal/distal)