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Figure legends

Figure 1. High-fat diet (HFD)-induced functional and structural glomerular

endothelial cell damage. (A and B) Changes in body weight (A) and fasting blood

glucose levels (B) in mice fed either standard diet (STD) or HFD during the 32-week

experimental period. (C) Urinary nitric oxide (NO) levels in the two groups of mice. (D)

Time-dependent structural changes in glomerular endothelial cells of HFD-fed mice.

Scanning electron microscopy (EM) and immunofluorescence (IF) for isolectin and

Wheat Germ Agglutinin (WGA). Original magnification, ×20,000 for scanning EM,

×1,000 for IF. All results are presented as mean ± SEM. *P < 0.05, **P < 0.01. NS: not

significant.

-/-

Figure 2. Severe podocyte injury in high-fat diet (HFD)-fed iPodo-Atg5 mice. (A)

Representative pictures of immunohistochemistry (IHC) for SQSTM1, a marker of

autophagy insufficiency,

PAS

staining,

scanning

electron microscopy (EM),

immunofluorescence (IF) of podocin and IHC for WT1 in four groups of mice. Original

magnification, ×400 for SQSTM1, PAS staining and WT-1, ×8,000 for scanning EM,

×1,000 for IF of podocin. (B, C) Urinary albumin excretion levels in the indicated mouse

groups at 4 weeks (B) and 8 weeks (C) after the tamoxifen injection. Urinary albumin

excretion levels are expressed as log 10 ratio urinary albumin/creatinine. (D) Quantitation

of WT1-positive podocytes in glomeruli. All results are presented as mean ± SEM. *P <

0.05, **P < 0.01. NS: not significant.

-/-

Figure 3. Severe podocyte injury in iPodo-Atg5 mice with neuraminidase-induced

structural endothelial dysfunction. (A) Scanning electron microscopy (EM) of

f/f

-/-

glomeruli from HFD-fed Atg5 and HFD-fed iPodo-Atg5 mice. Original magnification:

×8,000 and ×20,000 for details. (B) Time-dependent changes in urinary albumin excretion

f/f

level in neuraminidase-injected Atg5

and iPodo-Atg5

-/-

mice. (C) Scanning electron

microscopy (EM) of podocytes. Original magnification: ×8,000. All results are presented

as mean ± SEM. *P < 0.05, **P < 0.01. NS: not significant.

-/-

Figure 4. Enhanced endoplasmic reticulum (ER) stress iPodo-Atg5

mice with

structural endothelial dysfunction. (A) Western blots of Atg7, cleaved caspase 3, and

β-actin in cultured Atg7 and Atg7 podocytes stimulated with/without 5 g/dl bovine

f/f

-/-

serum albumin. (B) Quantitative ratios of cleaved caspase 3 to β-actin (n=4). (C)

Pathological event analysis of data from the proteomic analysis. The details of each

calculated score, (p), (v), and (c), are explained in the Supplemental Method. (D)

Immunohistochemical (IHC) for C/EBP homologous protein (CHOP). Original

magnification: ×400. Semiquantitative measurement of CHOP-positive cells in glomeruli.

(E) Urinary albumin excretion level in vehicle and TUDCA-treated groups. (D) Scanning

electron microscopy (EM), immunofluorescent (IF) for podocin. Original magnification,

×8,000 for scanning EM, and ×1,000 for IF of podocin and. All results are presented as

mean ± SEM. *P < 0.05. NS: not significant.

Figure 1

STD

** ** ** **

**

**

HFD

NS NS

8 weeks

8 12 16 20 24 28 32

STD

8 12 16 20 24 28 32

20

HFD

**

32 weeks

40

STD

STD

Urinary NO3 -/NO2- (mol/day)

60

Scanning EM Isolecctin stain

HFD

250

200

150

100

50

4 weeks

**

Blood glucose (mg/dl)

**

** ** ** ** **

**

HFD

16 weeks

60

50

40

30

20

10

16

32

Weeks after dietary intervention

32 weeks

Body weight (g)

WGA stain

Figure 2

Atg5f/f

IHC : SQSTM1

STD

iPodo-Atg5-/HFD

STD

4 weeks after

TM injection

HFD

Log10 ratio urinary

Albumin / Creatinine a

STD

HFD

Atg5f/f

Log10 ratio urinary

Albumin / Creatinine

WT1-positive cell

number/glomerurus

IHC: WT1

IF: Podocin

Scanning EM

PAS stain

STD

HFD

iPodo-Atg5-/-

8 weeks after

TM injection

STD

HFD

Atg5f/f

STD

HFD

iPodo-Atg5-/-

25

20

15

10

STD

HFD

Atg5f/f

STD

HFD

iPodo-Atg5-/-

Podocyte

damage (-)

Podocyte

damage (-)

Atg5f/f

Log10 ratio urinary

Albumin / Creatinine

3.0

2.5

Neuraminidase

Podocytes

iPodo-Atg5-/-

Endothelial

damage (+)

HFD-fed iPodo-Atg5-/Endothelial

damage (-)

Glomeruli

Scanning EM

Endothelial

damage (+)

Endothelial cells

Podocyte

damage (-)

HFD-fed Atg5f/f

Podocyte

damage (+)

Endothelial

damage (-)

Figure 3

Day 0

**

2.0

Day 1

Atg5f/f

iPodo-Atg5-/-

NS

Days after the injection

NS

1.5

1.0

NS

Days after neuraminidase injection

Day 3

Figure 4

rank

Atg7

Cleaved

caspase 3

β actin

Atg7-/-

1.5

1.0

0.5

Atg5f/f

IHC: CHOP

iPodo-Atg5-/-

6.03E-29

0.133

0.314

Adipogenesis

42.153

2.05E-13

0.084

0.139

Necroptosis

38.921

1.92E-12

0.066

0.193

Excitotoxicity

29.905

9.95E-10

0.054

0.164

Epithelial-Mesenchymal

Transition

28.548

2.55E-09

0.06

0.12

Cell Cycle

25.668

1.88E-08

0.036

0.286

Viral Infection

18.507

2.68E-06

0.042

0.099

Drug Transporter

18.235

3.24E-06

0.042

0.096

17.971

3.89E-06

0.042

0.093

10 Tight Junction

Atg7-/-

Days after

neuraminidase injection

Day 0

93.744

Day 1

20

15

10

Atg5f/f

iPodo

-Atg5-/-

3.0

1.0

0.0

Day 3 after

neuraminidase injection

Scanning EM IF: Podocin

2.0

Vehicle

Endoplasmic Reticulum Stress

iPodo-Atg5-/-

TUDCA

Atg7+/+

TUDCA

0.897

Vehicle

0.157

Log10 ratio urinary

Albumin / Creatinine

0.0

Albumin

score(c)

1.09E-51

Day 1

2.0

CHOP-Positive cell

number In glomeruli

Cleaved caspase3

/β- actin

Atg7+/+

score(v)

169.294

Day 0

score(p)

Apoptosis

Day 1

score

Day 0

Albumin

Pathological event

Protective role of podocyte

dysfunction in diabetes

autophagy

against

glomerular

endothelial

Mamoru Yoshibayashi,1 Shinji Kume,1 Mako Yasuda-Yamahara,1 Kosuke Yamahara,1

Naoko Takeda,1 Norihisa Osawa,1 Masami Chin-Kanasaki,1 Yuki Nakae,2 Hideki Yokoi,3

Masashi Mukoyama,4 Katsuhiko Asanuma,5 Hiroshi Maegawa,1 and Shin-ichi Araki1

Department of Medicine, Shiga University of Medical Science, Otsu, Shiga, Japan

Departments of Stem Cell Biology and Regenerative Medicine, Shiga University of

Medical Science, Otsu, Shiga, Japan

3Department of Nephrology, Graduate School of Medicine, Kyoto University, Kyoto,

Japan

Department of Nephrology, Kumamoto University Graduate School of Medical Sciences,

Kumamoto, Japan

Department of Nephrology, Graduate School of Medicine, Chiba University, Chiba,

Japan.

Supplemental Materials

Supplemental Data. Whole data of the proteomic analysis.

Supplemental Figure 1. Study protocol for high-fat diet (HFD)-induced renal injury

in tamoxifen-inducible podocyte-specific Atg5-deficient mice (iPodo-Atg5-/- mice).

Supplemental Figure 2. Spatial interaction between endothelial dysfunction and

podocyte dysfunction in high-fat diet (HFD)-fed Atg5f/f and HFD-fed tamoxifen

(TM)-inducible podocyte-specific Atg5-deficient mice (iPodo-Atg5-/- mice).

Supplemental Figure 3. No interaction between nitric oxide synthase 3 (NOS3)

dysfunction and autophagy deficiency in albuminuria progression of diabetes.

Supplemental Figure 4. Severe podocyte injury in tamoxifen (TM)-inducible

podocyte-specific Atg5-deficient mice (iPodo-Atg5-/- mice) with neuraminidaseinduced structural endothelial dysfunction.

Supplemental Method.

Supplemental Figure 1

8week-old

STD

HFD

STD

HFD

8 weeks

Tamoxifen

Tamoxifen

Atg5f/f

STD

HFD

iPodo-Atg5-/STD

HFD

Follow up for 8 weeks

Analyze renal phenotype

NS

70

60

50

40

30

20

10

NS

**

**

STD HFD STD HFD

Atg5f/f

iPodo-Atg5-/-

Blood glucose (mg/dl)

Atg5

Podo-CreERT2

-Atg5f/f

f/f

Body weight (g)

NS

250

200

NS

**

**

150

100

50

STD HFD STD HFD

Atg5f/f iPodo-Atg5-/-

Supplemental Figure 1. Study protocol for high-fat diet

(HFD)-induced renal injury in tamoxifen-inducible podocytespecific Atg5-deficient mice (iPodo-Atg5-/- mice). (A) Study

protocol of diet intervention in iPodo-Atg5-/- mice. (B and C)

Comparisons of body weight (B) and fasting blood glucose levels

(C) in the indicated four groups of mice. All results are presented

as mean ± SEM. *P < 0.05, **P < 0.01. NS: not significant.

Supplemental Figure 2

Isolectin stain/IF:Desmin

HFD-fed Atg5f/f

HFD-fed iPodo-Atg5-/-

Supplemental Figure 2. Spatial interaction between endothelial dysfunction and podocyte dysfunction in high-fat diet

(HFD)-fed Atg5f/f and HFD-fed tamoxifen (TM)-inducible podocyte-specific Atg5-deficient mice (iPodo-Atg5-/- mice). Double

immunofluorescent (IF) of desmin and isolectin in glomeruli from the HFD-fed Atg5f/f and HFD-fed iPodo-Atg5-/-. Original

magnification ×1,000. The white boxes indicate the areas for the magnified pictures (A, B, C, D). The green and red color

represents desmin and isolectin, respectively.

Supplemental Figure 3

Podocyte morphology

IF: Podocin

NS

NOS3-/-Atg5f/f

NOS3+/+

-Atg5f/f

Endothelial cell morphology

Scanning EM

Isolectin stain

WGA stain

NOS3-/-iPodo-Atg5-/-

NOS3-/-Atg5f/f

NOS3+/+

-Atg5f/f

Scanning EM

NS

8 weeks

after TM injection

NOS3-/-iPodo-Atg5-/-

Follow up for 8 weeks

Analyze renal phenotype

NOS3-/-iPodo-Atg5-/-

Tamoxifen

NOS3-/-Atg5f/f

NOS3-/NOS3-/-Atg5f/f -iPodo-Atg5-/-

NOS3+/+

-Atg5f/f

NOS3+/+

-Atg5f/f

4 weeks

after TM injection

Log10 ratio urinary

Albumin / Creatinine

12 week-old

Log10 ratio urinary

Albumin / Creatinine

Supplemental Figure 3. No interaction between nitric oxide synthase 3 (NOS3) dysfunction and autophagy deficiency in

albuminuria progression of diabetes. We used NOS3-knockout mice as a model with functional endothelial damage. NOS3deficient mice were purchased from The Jackson Laboratory (Bar Harbor, ME). We crossbred TM-inducible podocyte-specific Atg5deficient mice with NOS3-knockout mice to produce Atg5 and NOS3-double knockout mice (iPodo-Atg5-/--NOS3-/-) (N=5). Agematched Atg5f/f- NOS3-knockout (Atg5f/f-NOS3-/-) mice were used as a simple NOS3-deficient control (N=5), and age-matched

Atg5f/f -NOS3+/+were used as a healthy control (N=5). We intraperitoneally injected TM (75 mg/kg/day for 5 consecutive days) into

these mice at 12 weeks of age. Urinary samples were collected at 4 and 8 weeks after the TM injection. Mice were sacrificed at 8

weeks after the TM injection.

(A) Protocol for generation of podocyte-specific autophagy-deficient mice with systemic NOS3 deficiency (NOS3-/--iPodo-Atg5-/-). (B

and C) Urinary albumin excretion levels in NOS3+/+-Atg5f/f, NOS3-/-- Atg5f/f, and NOS3-/--iPodo-Atg5-/- mice. Urinary albumin

excretion levels are expressed as log10 ratio urinary albumin/ creatinine. (D) Representative images of podocytes and glomerular

endothelial cells. Scanning electron microscopy (EM), and immunofluorescence (IF) of podocin, isolectin, and wheat germ

agglutinin (WGA). Original magnifications, ×8,000 for scanning EM of podocytes, ×20,000 for scanning EM of glomerular

endothelial cells, and ×1,000 for IF of podocin, isolectin, and WGA. The white boxes indicate the areas for the magnified pictures.

All results are presented as mean ± SEM. *P < 0.05. NS: not significant

Supplemental Figure 4

Day 3

Standard diet-fed

iPodo-Atg5-/-

WGA stain

Nephrin

Atg5f/f

Merge

IF: Podocin

Atg5f/f

Neuraminidase

Follow up for 3 days

iPodo-Atg5-/-

Day 0

Day 1

Day 1

Day 0

Days after neuraminidase injection

Days after neuraminidase injection

Day 3

Supplemental Figure 4. Severe podocyte injury in tamoxifen (TM)-inducible podocyte-specific Atg5-deficient mice (iPodoAtg5-/- mice) with neuraminidase-induced structural endothelial dysfunction. (A) Representative pictures of

immunofluorescent (IF) for nephrin and Wheat Germ Agglutinin (WGA). Original magnification: ×1,000. The green and red color

represents nephrin and WGA, respectively. (B) Study protocol of neuraminidase-induced structural endothelial dysfunction on

standard diet-fed Atg5f/f mice and iPodo-Atg5-/- mice. (C) Immunofluorescent (IF) for podocin. Original magnification: ×1,000 for IF

of podocin.

Supplemental Method

HFD-induced diabetic rodent model

Eight-week-old male C57BL/6J mice were obtained from Clea Japan Inc. (Tokyo, Japan). The

mice were fed either a STD (10% of total calories from fat) or a HFD (60% of total calories from

fat). Mice in STD and HFD groups were sacrificed at 4, 8, 16, and 32 weeks, respectively, after

the initiation of dietary intervention.

HFD-induced diabetes in TM-inducible podocyte-specific autophagy-deficient mice

TM-inducible podocyte-specific Atg5-deficient mice (iPodo-Atg5 -/- ) were generated by

crossbreeding Atg5f/f mice with TM-inducible Nphs2-Cre transgenic mice (1). Atg5f/f mice were

used as a control group. Eight-week-old male Atg5f/f mice were fed the STD or HFD, and eightweek-old male iPodo-Atg5-/- mice were fed the STD or HFD. Each group included 6–12 mice. To

induce deletion of the Atg5 gene, we intraperitoneally injected TM (Sigma-Aldrich, St. Louis,

MO) at a dose of 75 mg/kg/day for 5 consecutive days into the mice at 8 weeks after the dietary

intervention (2). Urinary samples were collected at 4 and 8 weeks after the TM injection. Mice

were sacrificed at 8 weeks after the TM injection.

Neuraminidase-induced endothelial damage model

We performed neuraminidase-induced removal of endothelial glycocalyx to establish the

structural endothelial damage model (3, 4). Eight-week-old male Atg5f/f mice and iPodo-Atg5-/mice were injected with neuraminidase via their tail vein at a dose of 0.001 U/gBW

(Neuraminidase from Vibrio cholerae Type III, Sigma-Aldrich). Urinary samples were collected

at the start and following four consecutive days after the neuraminidase injection. Mice were

sacrificed at the start of the study and at 1, 3, and 7 days after the neuraminidase injection. The

numbers of mice were 3–7 at each time point.

TUDCA treatment of neuraminidase-injected iPodo-Atg5-/- mice

iPodo-Atg5-/- mice were allocated into two groups: vehicle administered, as a control, (N=6) and

TUDCA (N=6) groups. TUDCA (500 mg/kg/day, Calbiochem-EMD Millipore, Billerica, MA) was

intraperitoneally administered for 3 consecutive days prior to the neuraminidase injection (5).

Urinary samples were collected at day 1 after the neuraminidase injection.

Bioinformatic analysis of protein expression data

Pathway analysis of the data list from the proteomic analysis was performed using KeyMolnet

(KM Data, Tokyo, Japan) (6). KeyMolnet is a bioinformatics integration platform that enables

analysis of specific pathways based on data collected from recent studies. By importing the list

of Entrez gene IDs, KeyMolnet automatically provides the corresponding molecules in the form

of nodes in a network. Among the various network-searching algorithms, the ‘interaction’ search

identifies molecular networks containing a group of molecules with differential regulation in the

present study. The significance was scored using the following formula in which O = the number

of overlapping molecular relationships between the extracted network and canonical pathway, V

= the number of molecules displayed in the search result, C = the number of molecules

belonging to specific pathways, T = the total number of molecules recorded in KeyMolnet, and X

= the sigma variable that defines incidental agreements.

&'((*,,)

Score(p) = = ∑#$%

𝑓 𝑋

𝑓 𝑋 =CCX • T-CCV-X/TCV,

with Score = −log2 [Score(p)]; Score(v) = O / V; Score(c) = O / C.

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