supplemental figure 1 a · hydroxyproline (ug/10 mg tissue) t etet 0 5 10 15 20 25 supplemental...

Hydroxyproline (ug/10 mg tissue)

PINK1+

/+ Con

trol D

iet

PINK1+

/+ Ade

nine D

iet

PINK1-/

- Con

trol D

iet

PINK1-/

- Ade

nine D

iet

0

5

10

15

20

25

Supplemental Figure 1

Parkin_Parkin_Aden_Diet

Parkin1+

/+Con

trol D

iet

Parkin1+

/+Ade

nine

Diet

0

2

4

6

Park

in/ b

-acti

n

MFN2_Parkin_Aden.Diet

Parkin1+/+Contro

l Diet

Parkin1+/+Aden

ine Diet

Parkin-/-C

ontrol D

iet

Parkin-/-A

denine Diet

0.0

0.5

1.0

1.5

2.0

MFN2

/ b-a

ctin

A

MFN

2/β-

actin

Park

in/β

-act

in

Ctl AD Ctl AD

B

Supplemental Figure 1. PINK1 and Parkin deficiency enhances kidney damage and fibrosis in adenine (AD)model. A) Western blot and densitometry analysis for MFN2, Parkin, and LC3 normalized to β-actin in the kidneyfrom mice fed with control (Ctl, n = 3 per group) or adenine (AD, n = 5 per group) diet for 14 days. Data are mean ±SEM. *P< 0.05, **P<0.01 and ***P<0.001, analyzed by student’s unpaired 2-tailed t-test B) Hydroxyproline contentin the kidney from Pink1+/+ and Pink1-/- mice fed with Ctl (n = 4 per group) or AD (n = 5 per group) diet for 28 days.C) Weight of kidney from Pink1+/+ and Pink1-/- mice (n = 5 per group) fed with Ctl or AD diet for 28 days. D)Circulating chemokine CCL2 levels in the plasma samples obtained from Pink1+/+ and Pink1-/- mice (n = 4 pergroup) fed with Ctl or AD diet for 28 days were determined by ELISA.

Copy of Plasma CCL2 levels (pg/ml)

WT-CONT.D

iet

WT-Ade

n.Diet

PINK1-C

ONT.Diet

PINK1-A

den.D

iet0

500

1000

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C

Plas

ma

CC

L2 (p

g/m

l)

D

Ctl AD Ctl ADPink1+/+ Pink1-/-

Hyd

roxy

prol

ine

(ug/

10 m

g Ki

dney


Copy of Weight of left kidney (mg)

WT-

CONT.DIE

T

WT-

ADEN D

IET

PINK1_

CONT.DIE

T

PINK-K

O ADEN

DIE

T0

50

100

150

200


Wei

ght o

f Kid

ney

(mg)

LC3 II/b-actin Prkn Adenine Sham

Parkin1+/+Contro

l Diet

Parkin1+/+Aden

ine Diet

Parkin-/-C

ontrol D

iet

Parkin-/-A

denine Diet

0.0

0.2

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0.6

0.8

1.0

LC3-

II/β-

actin

Ctl AD

Parkin

MFN2

β-actin

LC3 ILC3 II

AdenineControl

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*

** * ***

******

*

1.5

0.0

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1.0

2.0 6

0

2

4 0.6

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Parkin-ko_Aden-Gal-3/GAPDH

Park

in1+/+

Contro

l Diet

Park

in1+/+

Adenin

e Diet

Park

in-/-C

ontro

l Diet

Park

in-/-A

denin

e Diet

0.0

0.2

0.4

0.6

0.8

1.0

FN/GAPDH

WT-Control

WT-Adenine

Parkin-KO-Control

Parkin-KO-Adenine

0.0

0.5

1.0

1.5

FN

CD206

GAPDH

Col-I

GAPDH

Gal-3

Col-I/GAPDH

WT-Control

WT-Adenine

Parkin-KO-Control

Parkin-KO-Adenine

0.0

0.5

1.0

1.5

E

FN/GAPDH

Col-I/GAPDH

Prkn+/+

Adenine

Prkn+/+ Prkn-/-

Adenine

CD206/GAPDH

WT-Control

WT-Adenine

Parkin-KO-Control

Parkin-KO-Adenine

0.0

0.5

1.0

1.5

CD206I/GAPDH

Gal-3/GAPDH

Prkn-/-

Control Adenine

Control AdenineControl

Control

Ctl AD Ctl ADPrkn+/+ Prkn-/-


Supplemental Figure 1. E) Western blot and densitometry analysis for the expression of fibronectin (FN),Collagen-I (Col-I), CD206, and Galectin-3 (Gal-3) normalized to GAPDH on kidney from Prkn+/+ and Prkn-/- mice fedwith control (Ctl, n = 3 per group) or adenine (AD, n > 5 per group) diet for 14 days. Data are mean ± SEM. *P<0.05, **P<0.01 and ***P<0.001, analyzed by one-way ANOVA (B, C, D, E).

**

******

***

*****

***

**

***

***

Supplemental Figure 1E

1.5

0.0

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1.5

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1.0

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1.0

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Supplemental Figure 1. F and G) PINK1 and Parkin deficiency enhances kidney damage and fibrosis inadenine (AD) model. F) Flow cytometric data showing the numbers galectin-3 (Gal-3)+ F4/80+ cells in the kidneyfrom wild type (n = 6 per group) Pink1-/- (n = 6 per group) and Prkn-/- (n = 3 per group) mice 28 days after control(Ctl) or adenine (AD) diet. The Gal-3+ F4/80+ cells were gated on CD45+ SSC low cells (not shown). G) Flowcytometric data showing the numbers of TGF-β1+ F4/80+ cells in the kidney from wild type (n = 4 per group),Pink1-/- (n = 4 per group) and Prkn-/- (n = 3 per group) mice 28 days after Ctl or AD diet. The TGF-β1+ F4/80+ cellswere gated on CD45+ SSC low cells (not shown). The data are representative of three independent experimentsand are mean ± SEM. *P< 0.05, **P<0.01 and ***P<0.001, analyzed by one-way ANOVA (B-G).

Supplemental Figure 1F and 1G

Gal-3 Counts CD45+F4.80+

WT-CD Counts

WT-AD Counts

Pink1-ko-CD Counts

Pink1-ko-AD Counts

Prkn-ko-CD Counts

Prkn-ko-AD Counts0

500

1000

1500

Gal

-3 o

f F4.

80+

CD

45+

cells

(Cou

nts)

WTCtl AD

*

Pink1-/-

Ctl AD

Prkn-/-

Ctl AD

*****

*

Ctl

AD

Wild Type Pink1-/-

F4/80

Gal

-3F

Prkn-/-C

tlA

D

F4/80

TGF-

β1

G

LAP Counts CD45+ F4.80+

WT-CD Counts

WT-AD Counts

Pink1-ko-CD Counts

Pink1-ko-AD Counts

Prkn-ko-CD Counts

Prkn-ko-AD Counts0

100

200

300

400

500TG

F-β1

of F

4.80

+

CD

45+

cells

(cou

nts)

WTCtl AD

***

Pink1-/-

Ctl AD

Prkn-/-

Ctl AD

***

****

Wild Type Pink1-/- Prkn-/-

23

320 349

22

382

149

70

544223 536

7397

1500

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Ly6c-CD11b+CD45+

PINK1+

/+ Sh

am

PINK1 +

/+ UUO

PINK1 -

/- Sha

m

PINK1 -

/- UUO

01020304050

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Co

un

ts o

f L

y6C

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ells

/50,

000

even

ts

Copy of Ly6c+ CD11b+ CD45+

PINK1

+/+ Sha

m

PINK1

+/+ U

UO

PINK1

-/- Sha

m

PINK1

-/- U

UO

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Copy of CD45+ Flowjo

PINK

1+/+

Sham

PINK

1+/+

UUO

PINK

1-/-

Sham

PINK

1-/- U

UO

0

5000

10000

15000Copy of F4.80+CD45+ new accuri c6

PINK1

+/+ Sha

m

PINK1

+/+ U

UO

PINK1

-/- S

ham

PINK1

-/- U

UO

0

5000

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15000A

D

Kidn

ey C

D45

+ ce

ll co

unts

Kidn

ey F

4/80

+ C

D45

+ co

unts

Bloo

d Ly

6Chi

gh

CD

11b+

cou

nts

Bloo

d Ly

6Clo

w

CD

11b+

cou

nts

Pink1+/+ Pink1-/-Sham UUO Sham UUO


C

B

Supplemental Figure 2. Loss of PINK1 amplifies frequency of circulating Ly6Clow monocytes inexperimental kidney fibrosis. A and B) Quantitative flow cytometric analysis showing the numbers of CD45+mononuclear cells (A) and F4/80+ CD45+ phagocytic population (B) in the kidney from Pink1+/+ and Pink1-/- mice 7days after sham (n = 4 per group) or UUO (n = 3 per group) surgery. C and D) Flow cytometric analysis showingthe numbers of circulating Ly6Chigh CD11b+ (C) and Ly6Clow CD11b+ (D) cells gated from CD45+ cells (not shown)from the peripheral blood from Pink1+/+ and Pink1-/- mice after 7 days of sham (n = 3 per group) or UUO (n = 5 and 3per group) surgery. Data are shown as mean + SEM. ***P<0.001, analyzed by one-way ANOVA.

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Copy of Ly6Clow CD11b+ Counts BLOOD

WT S

ham

WT U

UO

Park

in-KO S

ham

Park

in-UUO

0

50

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Copy of Ly6cHigh CD11b+Counts BLOOD

WT S

ham

WT U

UO

Park

in-KO S

ham

Park

in-UUO

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Copy of F4.80+CD45+ FLOWJO

WT S

ham

WT U

UO

Parkin-

KO Sha

m

Parkin-

UUO

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5000

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Copy of CD45+ FLOWJO

WT S

ham

WT U

UO

Park

in-KO S

ham

Park

in-UUO

0

5000

10000

15000A

Kid

ney

CD

45+

cell

coun

ts

Kid

ney

F4/8

0+

CD

45+

coun

ts

Blo

od L

y6C

high

CD

11b+

cou

nts

Blo

od L

y6C

low

CD

11b+

cou

nts

B

DC

Supplemental Figure 3. Parkin deficiency promotes frequencies of circulating Ly6Clow monocytes inexperimental kidney fibrosis. A and B) Quantitative flow cytometric data showing the numbers of CD45+mononuclear cells (A) and F4/80+ CD45+ phagocytic population (B) in kidney from Prkn+/+ and Prkn-/- mice (n = 4per group) after 7 days of sham or UUO surgery. C and D) Flow cytometric analysis showing the numbers ofcirculating Ly6Chigh CD11b+ (C) and Ly6Clow CD11b+ (D) cells gated from CD45+ cells (not shown) from theperipheral blood from Prkn+/+ and Prkn-/- mice after 7 days of sham (n = 2 per group) or UUO (n = 4 per group)surgery. Data are mean ± SEM. *P< 0.05, and **P<0.01 analyzed by one-way ANOVA.

* **

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*


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Prkn+/+ Prkn-/-Sham UUO Sham UUO




Collagenase Digestion

CD115+ population

Kidney Single Cell Suspension

Renal Mononuclear Cells

Ficoll-Hypaque Density gradient Centrifugation

Mouse Kidney

CD115-Biotin positive selection

Collagenase Digestion

CD68+ population

Kidney Single Cell Suspension

Renal Mononuclear Cells

Ficoll-Hypaque Density gradient Centrifugation

Human Kidney

CD68-PE positive selection

Mouse Renal Macrophage Isolation by MACS® separation

Human Renal Macrophage Isolation by MACS® separation

A B

Anti-CD115-Biotin conjugated antibody

Anti-CD68-PE conjugated antibody

Anti-Biotin MicroBeads Anti-PE MicroBeads

Supplemental Figure 4. Isolation of macrophages from kidney. A) Schema showing the isolation of macrophagesfrom mouse kidney cells using Ficoll-Hypaque density gradient centrifugation followed by CD115 positive selectionthrough magnetic-activated cell sorting (MACS). B) Isolation of macrophages from human kidney using densitygradient centrifugation and subsequently MACS cell separation protocol using CD68 positive selection.


supplemental figure 1 a · hydroxyproline (ug/10 mg tissue) t etet 0 5 10 15 20 25 supplemental...

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