, Nonclinical Research CenterEﬀects of prednisolone on adriamycin-induced
nephropathy in rats
○ Takumi Yamazawa, Cheng Jun Ma, Kikuyo Nakaoka, Masakazu Imaizumi, Kousuke Morizumi,
Seiichi Katayama, Naoyuki Hironaka, Katsuhide Nishi

Summary in Japanese40

Non-treatment
ADR 3+2 mg/kg
ADR 5 mg/kg
ADR 10 mg/kg

20

0

7

14

21

28

35

3

ADR 3+2 mg/kg
ADR 5 mg/kg&&&&

Day -2

7

14

21

28

35

0

Day 42

800

&&

600

ADR 5 mg/kg

&&

&&

&&

400
&

&&

ADR 3+2 mg/kg

Day -2

7

14

21

28

35

Day 42Day 1-1 W

ADR administration
24hr-urine and blood collection

Administration of 0.5 w/v% CMC-Na or prednisolone(1 or 5 mg/kg), 1 times/day for 35 days

Day 14

Day 21

Day 28

Day 36【Examination items】(1) Urinalysis (Automated analyze method)
Total protein, Albumin (Alb), Creatinine (Cre)(2) Blood chemistry-analysis
Total cholesterol (T-CHO), Creatinine (Cre), Total protein, Albumin (Alb),
Urea nitrogen (UN) (Data of Cre, Total protein and Alb are not shown.)(3) Real-time PCR (RT-PCR)
Collected fresh urine was centrifuged (3,000×g for 15 minutes at r.t.) andsupernatant (cell-free fraction) was transferred to a new tube. Transferred cellfree-fraction was added Urine Conditioning Buﬀer (ZYMO RESEARCH CORP) and wascentrifuged (3,000×g for 15 minutes at r.t.) to form a urine pelleted.
Collected left kidney were embedded with O.C.T compound in a routine method,and stained with toluidine blue. In the toluidine blue microscope slide, glomeruli(15 points per slide) were collected using laser microdissection (LMD，LMD7, Leica
Microsystems GmbH).
Total RNA was extracted from urine pelleted or glomeruli section using themiRNeasy Mini Kit (QIAGEN). Complementary DNA was synthesized using the
TaqMan™ advanced miRNA cDNA synthesis kit (Thermo Fisher scientiﬁc K.K.).
Real-time PCR was carried out with the TaqMan™ advanced miRNA assay (Thermo
Fisher scientiﬁc K.K.) in the LightCycler 480 II instrument (Roche).
Endogenous controls was used miR-361-5p.
The relative expression of target gene (miR-26a) was calculated according to thenext expression.

21

28

35

Day 42

Serum urea nitrogen concentration&&&&

ADR 5 mg/kg&&

ADR 3+2 mg/kg
Non-treatment

Day -2

7

14

21

28

35

Time after administration of adriamycin

Day 420

PDL 5 mg/kg

$$

7

14

21

28

$$

$$

400

Control
PDL 1 mg/kg
PDL 5 mg/kg

$$

300
$$

200
100

Non-treatment
Day -2Urinary albumin excretion500(mg/24hr)(mg/24hr)

$$

$$

$$

PDL 1 mg/kg

200

0

Day 36

Non-treatment
Day -2

7

14

21

28

Day 36Time after administration of adriamycin

Serum total cholesterol concentration

Serum urea nitrogen concentration

30

800
600

PDL 1 mg/kg

500
400

Control
PDL 5 mg/kg

$$

300
200

&

100
0

$$

$$

(mg/dL)

700

7

$$

14

21

28

PDL 1 mg/kg
Control
Non-treatment
PDL 5 mg/kg

20##

Non-treatment
Day -2

25

15

Day 36Time after administration of adriamycin

Day -2

7

14

21

28

Time after administration of adriamycin

Day 36

Kidney weight

2.5

Histopathological analysis of left kidney (H & E staining)

2
$$

1.5
1
0.5

Non-treatment

Control

PDL
1 mg/kg

PDL
5 mg/kg

Each value represents the mean and S.D. of 8 animals.
$$: p<0.01 vs Non-treatment group (Aspin-Welchʼs test)&: p<0.05 vs Control group (Steel test)##: p<0.01 vs Control group (Dunnettʼs test)

Experiment 2 (Real-time PCR)

Relative expression(/Non-treatment group)

Day 7

14

ADR 3+2 mg/kg

&&

600

Control

4000Day -2

&&

&&

7

700

3.0

Urinary miR-26a-5p expression pattern(Real-time PCR)

Relative expression(/Non-treatment group)

Experiment Schedule

Day -2

Non-treatment

20

0Urinary total protein excretion

600(mg/dL)In the day 1, animals were administered ADR intravenously at 5 mg/kg. 24hr-urine andblood were collected once weekly for 6 weeks (Day -2, 7, 14, 21, 28 and 36) for
urinalysis and blood chemistry-analysis.
In the day 35 only, fresh urine was collected for real-time PCR analysis. Prednisolone
was administered orally at 1 or 5 mg/kg once a day for 35 days.
0.5 w/v% carboxymethyl cellulose-Na (0.5 w/v% CMC-Na) was administered orally to
non-treatment and control group.
In the day 36, kidneys were collected and left kidney was used for histopathologicalanalysis (H&E staining) and right kidney was used for real-time PCR analysis.

&&10

Non-treatment

800

Day 42【ADR-induced nephropathy model in experiment 2 and drug administration】&&&&

ADR 5 mg/kg

30

&&

&&

1000

Day 35

Day 42

Experiment 2

ADR administration
24hr-urine and blood collection

Day 2835

Each value represents the mean and S.D. of 7 or 8 animals.
&: p<0.05, &&: p<0.01 vs Non-treatment group (Steel test)(left+right g/100 bW)

Day 21

28

Urinary albumin excretion

40

&&

900

Day 1421

Time after administration of adriamycin

Time after administration of adriamycin

Day 7

14

400

Serum total cholesterol concentration

1000【ADR-induced nephropathy model in Experiment 1】

Day -2

7200

Crlj:WI, ♂, 7w (At the ADR administration)
Experiment 1: N=8×4［Non-treatment, ADR-low dose group (3+2 mg/kg),
ADR-middle dose group (5 mg/kg), ADR-high dose group (10 mg/kg)］
Experiment 2: N=8×4［Non-treatment, Control group, prednisolone-low dosegroup (1 mg/kg), prednisolone-high dose group (5 mg/kg)］

Day 15Day -2

Non-treatment&&

Time after administration of adriamycin

Day 1

&&

600

Time after administration of adriamycin【Animal】(only the 3 mg/kg group)

&&

&&

0

Experiment Schedule&&&&(mg/24hr)&

ADR 3+2 mg/kg

Time after administration of adriamycin

Non-treatment

&

&&

&&

400

800

11

ADR 5 mg/kg

&&

600

42

Urinary creatinine excretion

7

800

0

&&

&&

&&

200

Time after administration of adriamycin

15

Materials and Methods-1 W(mg/24hr)

(%)

60

200

In the day 1, animals were administered ADR intravenously at 3, 5 or 10 mg/kg.
Only the 3 mg/kg group, 2 mg/kg of ADR was additionally administered at day 15.
24 hr-urine and blood were collected once weekly for 6 weeks (Day -2, 7, 14, 21, 28, 35
and 42) for urinalysis and blood chemistry-analysis.

&&

1000

80

0

Urinary total protein excretion

1200(mg/dL)

Chronic kidney disease (CKD) is characterized by progressive and chronicalkidney dysfunction. Although it can be categorized various types withpathogenesis, adriamycin (ADR) induced nephrotic syndrome is considered to bea classic rat model of CKD. By administration of ADR, glomerular ﬁltration barrier
damage and subsequently massive proteinuria are induced. In this study, we
tried to prepare an ADR-induced nephropathy model in rats (Experiment 1) andto validate the usefulness of the model by administration of prednisolone(Experiment 2).

Percent survival

100(mg/24hr)

Objective

Experiment 1(mg/dL)

慢性腎臓病（chronic kidney disease：CKD）は，腎機能が慢性にかつ進行性に悪化する病態の総称である．
抗がん剤の一種であるアドリアマイシン（以下，ADR）を動物に投与することで，大量のタンパク尿を生じる
ネフローゼ症候群を誘導できることが知られている．このような背景から本研究では，ADR 腎症ラットモ
デルについて，モデル確立（実験①）およびプレドニゾロンの薬効薬理試験（実験②）を実施した．
実験①では，Crlj:WI 雄性ラットに ADR を 3 パターンの用量（① 3 mg/kg，② 5 mg/kg，③ 10 mg/kg）で
静 脈 内 投与し，モデルを誘 発した．なお 3 mg/kg 投与 群 のみ，初 回 ADR 投与日の 15 日後に，再 度
2 mg/kg の用量で ADR を投与した．ADR 初回投与日を Day 1として，Day 42 までの間に 1 回／週の頻度で採尿および採血を実施し，生化学的パラメータを測定した．
その結果，5 mg/kg の用量で以降の薬効評価試験を実施することとした．
実験②では，ADR 初回投与日より1 回／日の頻度で 35 日間，プレドニゾロンを1 mg/kg または 5 mg/kg の用量にて経口投与した．その結果，プレドニゾロンを 5 mg/kg の用量で投与した際に，有意ではないものの腎障害の改善が認められた．
また実験②では，Day 35 に採取した新鮮尿，または Day 36 の解剖時に採取した腎臓の薄切切片からRNA を抽出し，糸球体障害マーカーとされるマイクロ RNA（miR-26a）の測定を試みた．なお腎臓の薄切切片からのマイクロ RNA の抽出は，レーザーマイクロダイシェクションを使用して糸球体部分のみを切り出し，
糸球体特異的な RNA を抽出した．RT-PCR 法で正常群，モデル対照群，プレドニゾロン投与群の miR-26aの発現を測定したところ，正常群に対してモデル対照群の miR-26a の発現が有意に低値を示し，（新鮮尿）プレドニゾロン投与群では有意では無いものの，モデル対照群に対して高値を示した（薄切切片）．
本研究の内容は慢性腎臓病に対する薬剤検討を実施するにあたり，有用であると考えられる．

Results

2.5
2.0
1.5
1.0
0.5
0

$
Non-treatment Control

PDL
1 mg/kg

PDL
5 mg/kg

1.6

Glomerular miR-26a-5p expression pattern(Real-time PCR)

1.4

&

1.2
1.0
0.8
0.6
0.4
0.2
0

Non-treatment

Control

PDL
1 mg/kg

PDL
5 mg/kg

Each value represents the mean and S.D. of 8 animals.
$: p<0.05 vs Non-treatment group (Aspin-Welchʼs test)&: p<0.05 vs Control group (Dunnettʼs test)

Laser microdissection (LMD)∆Cp(miR-26a) = Cp(miR-26a) ‒ Cp(miR-361-5p)∆∆Cp(miR-26a) = ∆Cp(miR-26a) ‒ ∆Cp(average of Non-treatment group)
Relative expression to Non-treatment group = 2 ‒ ∆∆Cp(miR-26a)

Conclusion
・ Results of Experiment 1 (the percent of survival, the urinary total protein excretion and the urinary albumin excretion) indicated that ADR at 5 mg/kg was found to besuitable for induction of the nephropathy model rats.
・ By the administration of prednisolone at 1 mg/kg, the urinary total protein excretion, the urinary albumin excretion and serum total cholesterol concentration were not
improved (Experiment 2).
・ The administration of prednisolone at 5 mg/kg was observed to be eﬀective on dysfunction of the kidney by urinalysis and blood chemistry-analysis.
・ As already known, the expression of miR-26a is decreased following nephropathy symptoms progression, and we analyzed the expression miR-26a in urine and theglomerulus in this study.
・ In the ADR administration group (control group), the expression of urinary miR-26a was signiﬁcantly decreased from the non-treatment group.
・ In the PDL 5 mg/kg administration group, the expression of glomerular miR-26a was signiﬁcantly increased from the control group.
・ ADR-induced nephropathy rat model and the measurement of miR-26a are useful in evaluation of developing therapeutic drugs under the present experimental
condition.