Epigenetics and CKD

Masaomi NangakuDivision of Nephrology and Endocrinology

the University of Tokyo Graduate School of Medicine

Nephrology and Transplantation Update Course

I have the following relationships to disclose.Potential Financial Conflicts of Interest

(1)Employment: No

(2)Stock ownership or options: No

(3)Patent royalties/licensing fees: No

(4)Honoraria and advisory fees: Kyowa-Hakko-Kirin, Astellas, Chugai,

GSK, JT, Tanabe-Mitsubishi

(5)Research funding: Kyowa-Hakko-Kirin, Daiichi-Sankyo, Alexion,

Astellas, Takeda, JT

ＣＯＩ disclosure

presenter： Masaomi Nangaku

Mimura, Tanaka, Nangaku. CEPP 2016

Epigenetics: the study of persistent

changes in gene expression that does not

involve mutations of the underlying DNA

Metabolic memory

Epigenetic changes

Long-term benefits of intensive glucose control for

preventing ESKD: ADVANCE-ON

Muh Geot Wong et al. Dia Care 2016

5th September

Seminar 2: Basic Science; Diabetes

Chairs: Josephine Forbes & Melinda Coughlan

Karin Jandeleit-Dahm

Masaomi Nangaku

Carol Pollock

Hypoxic memory

Epigenetic changes

GF

R

AKI: No CKD Progression

AKI: CKD Progression

AKI on CKD:

CKD Progression

VAP-1 in pericytes enhances neutrophil infiltration

into the IR-injured kidney by generating H2O2

Tanaka, Nangaku et al. Kidney Int 2017

Basile, Nangaku et al for the ADQI XIII Work Group. JASN 2016

Basile, Nangaku et al for the ADQI XIII Work Group. JASN 2016

Renal hypoxia in the pathophysiology of

the AKI-to-CKD transition

Nangaku et al. Nephron 2017

Hypoxia

Uremia

Modified from Mimura, Tanaka, & Nangaku. Semin Nephrol 2013

Hypoxia as the final common pathway to ESKD

Nangaku. J Am Soc Nephrol 2006

Peritubular ischemia contributes more to tubular damage than

proteinuria in immune-mediated glomerulonephritis

Muh Geot Wong et al. JASN 2008

Double immunostaining showed

pimonidazole-positive tubules

(brown) located close to Kim-1–

positive tubules (pink).

pimonidazole

Hirakawa, Tanaka, Nangaku. J Diabetes Investig 2017

HIF

Nrf2

Defense against hypoxia

Defense against

oxidative stress

Oxygen sensing –

an essential process for

survival

2016 Albert Lasker Basic

Medical Research Award

Gregg L. SemenzaWilliam G. Kaelin, Jr. Peter J. Ratcliffe

Hypoxia

Uremia

Modified from Mimura, Tanaka, & Nangaku. Semin Nephrol 2013

D-serine accelerates tubular senescence

SA-βG staining

D-serineControl

γ-H2AX staining

Okada, Nangaku et al.

Sci Rep in press

Chirality

Okada, Nangaku et al.

Sci Rep in press

Indoxyl sulfate suppresses HIF activity

Tanaka, Nangaku et al.

FASEB J 2013

reporters

HIF targets

IS: indoxyl sulfate

Suppression of HIF and its targets by indoxyl sulfate

Huang et al. Kidney Int 2016

IL-10

VEGF

Indoxyl sulfate inhibits binding of HIF to its co-factor,

p300

Tanaka, Nangaku et al.

FASEB J 2013

Indoxyl sulfate induces HIF-inhibiting CITED2 protein

Tanaka, Nangaku et al. FASEB J 2013

Increased CITED2 mRNA

stability by IS

CITED2 mRNA

CITED2 protein

The efficient displacement of HIF-1α from its complex with

CBP/p300 by CITED2 is kinetically driven and proceeds

through a transient ternary intermediate

Berlow et al. Nature 2017

Hypoxia

Uremia

Modified from Mimura, Tanaka, & Nangaku. Semin Nephrol 2013

Hirakawa, Nangaku et al. Sci Rep 2015

Measurement of oxygen tension in the kidney

utilizing a novel phosphorescence probe

Hirakawa, Nangaku et al. Sci Rep 2015

Ph

os

ph

oflu

ore

sc

en

ce

life tim

e (µ

s)

Phosphofluorescent probe to detect hypoxia

S1 segment has higher oxygen tension compared

with S2 segment

Hirakawa & Nangaku.

manuscript in submission

U: upstream tubules

D: downstream tubles

Epigenetics

DNA methylation

miRNA and lncRNA

Histone modification

Chromosomal conformational change

Maternal protein restriction diet induced less weaning weight

and hypermethylation of ribosomal DNA in offspring

Holland et al. Science 2016

Ischemia-reperfusion injury decreased the genome-

wide methylation level and the CpG methylation level

Zhao et al. Gene 2017

Aberrant Rasal1 promoter methylation contributes to

sustained fibroblast activation and AKI-to-CKD progression

Tampe et al. Kidney Int 2017

Rasal1: Ras-Gap–like protein-1

Epigenetics

DNA methylation

miRNA and lncRNA

Histone modification

Chromosomal conformational change

A clear advantage of targeting lncRNA rather than epigenetic-

related enzymes or other non-coding RNA such as miRNA is the

direct sequence specificity of the target site.

RNA-seq revealed DARS-AS1 as a hypoxia-induced lncRNA

Mimura, Nangaku et al.

Physiol Rep 2017

DARS-AS1 is induced by HIF-1

Mimura, Nangaku et al.

Physiol Rep 2017

Knockdown of DARS-AS1 aggravates apoptotic cell death

Mimura, Nangaku et al.

Physiol Rep 2017

Epigenetics

DNA methylation

miRNA and lncRNA

Histone modification

Chromosomal conformational change

Maternal H3K27me3 controls DNA methylation-independent

imprinting

Inoue et al. Nature 2017

H3K4me1 normoxia

H3K4me1 hypoxia

H3K27ac normoxia

H3K27ac hypoxia

H3K4me3 normoxia

H3K4me3 hypoxia

HIF1α normoxia

HIF1α hypoxia

-35kb-24kbGLUT3

Epigenetic modification of expression of

glucose transporter 3 (GLUT3) by hypoxia

enhancer mark

active mark

Mimura, Nangaku et al. Mol Cell Biol 2012

0

200

400

600

800

1000

1200

1400

0 h 1 hr 2 hrs 4 hrs 8 hrs 12hrs 24hrs

Histone demethylation by hypoxia

Upregulation of KDM3A

ON

OFF supprssive

H3K9me2

0 1 2 4 8 12 24 hr

1400

1200

1000

800

600

400

200

0

KDM3A = Jmjd1a

demethylase of

H3K9me2

Mimura, Nangaku et al. Mol Cell Biol 2012

Regulation of GLUT3 expression by hypoxia

normoxia hypoxia

GLUT3 GLUT3

Up-regulation

Mimura, Nangaku et al. Mol Cell Biol 2012

Cross-enhancement of ANGPTL4 transcription

by HIF1 and PPAR β/δ

Inoue, Nangaku et al. Genome Biol 2014

HIF1α

PPARβ/δ

Inoue, Nangaku et al. Genome Biol 2014

OMX super-resolution microscopy showed changes of the

distribution of H3K9Me3 marks in NRK-52e cells by TGF-β1

H3K9Me3: repressive histone mark

NUP62: nuclear envelope protein

nucleoporin 62

Hewitson et al.

Front Pharmacol 2017

curcumin improves nephrosclerosis via suppression of

histone acetylation

Normal

saltHigh

salt

High salt +

curcuminacetylated H3K9

Serum creatinine

Muta et al. NDT 2016

ChIP using anti-acetyl-H3K9

increased EZH2 in the human fibrotic kidney

Zhou et al. JASN 2016

EZH2 (Enhancer of zeste

homolog 2):

histone methyltransferase of

H3K27me

EZH2 mediates kidney

fibrosis by downregulating

expression of Smad7 and

PTEN

Zhou et al. JASN 2016

Dznep: inhibitor of EZH2

Dznep attenuates renal fibrosis in obstructed

kidneys

αSMADznep

I/R

0

10

20

Vehicle群 阻害薬投与群

Control I/RArea(%)

* p<0.001

**

*

0

0.5

1

Vehicle群 阻害薬投与群

*

WB

αSMA Ab

Β-Actin Ab

Vehicle Dznep

Amelioration of AKI-to-CKD transition by Dznep

DznepVehicle

DznepVehicle

Mimura, Hirakawa, Nangaku. manuscript in submission

Vehicle

Vehicle DznepH

3K

27

me

3 （

DA

B)

Nuclear staining of tubular

cells showed decreased

staining of H3K27me3.

Suppression of H3K27me3 by Dznep

Mimura, Hirakawa, Nangaku. manuscript in submission

DZnep 2 control 1 control 2 hypoxia 1 hypoxia 2 DZnep 1 DZnep 20.000.02

2.901.19

3.88

0.000.00

4.2715.59

3.97

47.99

0.040.00

2.80

9.630.040.55

0.00

4.250.96

11.765.80

8.56

3.523.237.89

0.02

0.000.760.00

0.00

0.0013.64

13.716.74

5.52218.61

9.249.84

35.99

46.9910.37

6.17

24.23

88.3937.73

17.98

109.343.977.17

6.84

116.5149.21

7.7017.29

91.66

51.6158.1121.35

11.53

13.385.78

197.57

60.10

12.3528.63

12.4126.92

8.57

8.167.08

13.68

13.22

21.8112.47

16.40

7.99

57.777.09

10.77

4.0918.8014.19

6.68

23.2878.79

20.635.79

4.27

28.2118.047.63

339.67

40.745.267.37

15.75

5.7917.21

55.7115.43

7.06

2.907.795.01

8.86

288.829.85

3.81

7.61

9.2525.67

11.181.28

24.20

501.8812.58

13.420.00

102.5113.56

113.84

84.2429.9419.94

16.34

7.955.80

28.08

14.88

3.066.52

6.924.62

9.62

0.0160.3211.09

25.15

19.516.37

16.45

19.97

5.216.86

34.148.54

4.40

4.1414.68

11.9521.28

11.4516.52

10.97

25.8947.2727.59

14.13

12.84103.2645.78

23.80

14.538.93

90.3422.30

11.36

26.499.90

22.56

37.31

10.1713.61

8.83

19.81

11.7611.54

16.99

32.5810.41

34.2523.25

44.7414.55

16.0515.71

10.84

39.9320.5914.90

25.00

20.029.48

17.40108.37

20.3211.26

11.2811.77

28.32

40.4216.8431.89

14.44

15.9210.83

14.13

27.55

30.6413.63

27.3715.51

23.89

19.6816.34

13.04

12.31

22.1414.20

40.84

22.4119.5411.54

20.84

26.6515.82

21.4823.22

11.809.05

11.7617.69

13.68

11.7113.6731.30

41.88

12.1427.25

13.0514.86

58.76119.59

14.7316.84

14.09

12.4962.31

11.67

18.28

78.039.22

6.80

19.95

5.388.14

26.23

109.7752.51

8.8336.62

9.79

17.204.00

15.41

13.86

9.025.29

18.61

8.56

5.0010.64

3.7756.94

9.269.57

8.168.48

9.59

3.7317.27

13.50

15.81

26.4010.63

8.834.69

7.139.10

11.29

4.75429.31

11.6513.44

21.87

34.6314.6312.47

15.80

9.5812.8924.34

45.73

17.819.11

4.188.06

6.1310.05

5.895.87

7.35

11.273.81

211.48

190.60

1.5211.53

8.460.00

10.078.95

0.02

0.0051.63

9.047.28

8.60

243.61

13.6410.60

3.41

9.4518.385.56

11.24

7.0227.39

186.796.22

9.98

11.6023.306.56

16.43

7.545.38

169.20

6.54

26.8825.34

4.6310.15

4.4623.19

17.89

2.9813.23

10.65284.93

10.86

3.52

15.605.10

77.47

5.0514.123.29

10.98

14.6477.52

5.699.67

136.40

773.160.01

10.02

12.31

4.4193.867.77

9.91

4.9014.36

0.0517.91

4.88

4.7925.31

0.000.43

18.540.34

11.41

4.74

0.006.59

6.70

23.937.342.93

5.40

2.1811.51

9.3010.11

8.19

5.120.006.67

13.16

18.775.21

16.57

4.14

27.6524.95

9.1412.84

7.32

5.317.17

18.7944.52

13.289.98

8.06

0.003.68

17.74

40.05

0.005.07

12.971.70

37.979.89

2.499.58

13.62

5.394.636.72

7.12

10.471.005.09

44.13

8.1013.20

3.1171.86

5.99

1.236.238.11

6.50

5.8522.43

8.69

15.71

0.0014.46

0.00

9.1834.85

7.179.49

5.787.20

10.54

2.94

23.51

6.91

0.00

2.92

16.21

478 g

en

es

VehicleControl

VehicleI/R injury

DznepI/R injury

Group1

Group2

Group3

149 g

en

es

103 g

en

es

226 g

en

es

Low

High

RNA-seq of tubules isolated by laser capture

microdissection

Dznep suppressed TIMP2 expression in the kidney

RNA-seq of the kidney

0

5

10

15

20

Vehicle Ctl Vehicle IR Dznep IR

Timp2

RN

A-s

eq

(rp

km

)

Mimura, Hirakawa, Nangaku. manuscript in submission

0

100

200

300

400

Normoxia

Dznep(-)

Hypoxia

Dznep(-)

Hypoxia

Dznep(+)

TIMP2R

NA

-seq

(rp

km

)

HK2

0

20

40

60

80

Normoxia

Dznep(-)

Hypoxia

Dznep(-)

Hypoxia

Dznep(+)

TIMP2

RPTEC

HK2: human kidney-2 RPTEC: renal proxymal tubular epithelial cell(human primary culture cells)

RNA-seq: in vitro

Mimura, Hirakawa, Nangaku. manuscript in submission

Ischemia/reperfusion Fibrosis↑

Dznep Novel microRNA targets

TIMP2↓MMP↑

TIMP2↑Collagen↑

Fibrosis↓

MMP↓

Collagen↓

Dznep suppresses expression of TIMP2 via microRNA

Identified by small RNA-seq

Ischemia/reperfusion

Mimura, Hirakawa, Nangaku. manuscript in submission

Mimura, Tanaka, & Nangaku. Semin Nephrol 2013

ISN President

LOC chair

Meetings Committee chair

William

Couser

Richard

JohnsonStuart

ShanklandMark

Okusa

David

HarrisKai-Uwe

Eckardt

Juergen

Floege

Motoko

Yanagita

Tetsuhiro

Tanaka(Nephrology &

Endocrinology)

Reiko

Inagi(CKD Pathophysiology)