Supplementary Information Titles

Journal: Nature Medicine

Article Title: Podocyte secreted Angiopoietin-like 4 mediates proteinuria in

glucocorticoid sensitive nephrotic syndrome

Corresponding Author: Sumant Singh Chugh

Supplementary Items and Titles:

Supplementary Figure 1: Changes in Angptl4 mRNA expression in experimental

glomerular disease.

Supplementary Figure 2: Experimental studies in Angptl4 -/- mice, Angptl4 transgenic

mice, wild type rats, and Angptl4 transgenic rats.

Supplementary Figure 3: Characterization of Angptl4 transgenic rats.

Supplementary Figure 4: Serum Angptl4 levels in transgenic (TG) rats, and urine
Angptl4 in rats with experimental nephrotic syndrome.

Supplementary Figure 5: Assessment of GBM charge in Angptl4 transgenic (TG) rats

and mice.

Supplementary Figure 6: Assessment of glomerular Angptl4 sialylation.

Supplementary Figure 7: Characterization of Angptl4 secreting HEK293 and mouse

GEC stable cell lines.

Supplementary Figure 8: Albuminuria in ManNAc - treated NPHS2-Angptl4 transgenic

(TG) rats, and oligomerization of recombinant and transgene expressed Angptl4.

Supplementary Figure 9: Confocal assessment of glomerular Angptl4 expression in

human minimal change disease (MCD).

Supplementary Figure 10: Plasma and urine Angptl4 in human primary glomerular
disease.

Supplementary Figure 11: Schematic representation of the role of podocyte secreted

Angptl4 in nephrotic syndrome.

Supplementary Table 1: Brief clinical profile of individuals whose plasma and urine were
assessed for presence and patterns of Angptl4 expression by Western blot.

Supplementary Table 2: List of primers and probes used for Taqman real time PCR.

Supplementary Methods

Supplementary References

Nature Medicine: doi:10.1038/nm.2261

 γ2−NTS
2.5 6
2 5
Fluorescence

Fluorescence
4
1.5
3 Blue Control
1
2 Red NTS
0.5 1
0 0
1 5 9 13 17 21 25 29 1 5 9 13 17 21 25 29 33 37
Cycles Cycles
2.5 7 PAN
6
2

Fluorescence
Blue Control
Fluorescence

5
1.5 4 Red PAN Day 3
3
1 Green PAN Day 6
2
0.5 1 Orange PAN Day 10
0 0
1 4 7 10 13 16 19 22 25 28 1 5 9 13 17 21 25 29 33 37
Cycles Cycles
2 6
5 PHN
Supplementary Figure 1: Changes in Angptl4
Fluorescence
Fluorescence

1.5
4 Blue Control
1 3 Red PHN Day 3 mRNA expression in experimental glomerular
0.5
2 Green PHN Day 6 disease. Real Time PCR tracings for changes in
1 Orange PHN Day 10
0 0 glomerular gene expression shown in Figs. 1b, 1e.
1 5 9 13 17 21 25 29 1 5 9 13 17 21 25 29 33 37
Cycles Cycles
2.5 4
anti-Thy1.1
2
Fluorescence

3
Flourescence

1.5 Blue Control

2
1 Red Thy1.1 Day 1
0.5 1 Green Thy 1.1 Day 3
0 0
1 5 9 13 17 21 25 29 1 4 7 10 13 16 19 22 25 28
Cycles Cycles
1.2 3.5
1 3
CG
Fluorescence

Fluorescence

0.8 2.5
2
Blue Control
0.6 Red CG Day 1
1.5
0.4 1 Green CG Day 3
0.2 0.5 Orange CG Day 10
0 0
1 5 9 13 17 21 25 29 1 5 9 13 17 21 25 29 33 37
Cycles Cycles

18s (control) Angptl4 Nature Medicine: doi:10.1038/nm.2261

 * PAN PAN PAN
a Spot urine albumin / creatinine 60
50
b EFP c
40
30
20
10 FP
0
Angptl4 Angptl4 Angptl4 Angptl4 Antisense Sense Antisense Tubules
EFP
+/+ -/- +/+ -/-
Sheep Sheep NTS NTS Angptl4 +/+ NTS Angptl4 -/- NTS
d Ig Ig
e

Angptl4 mRNA expression

Fold increase in cortical
7 ***
6
5
4
3
Antisense Sense Antisense Tubules
2 Control Control Control
1
Angptl4 CD2AP merged 0 Supplementary Figure 2: Experimental studies in
WT mouse TG mouse
Angptl4 -/- mice, Angptl4 transgenic mice, wild
f 2.5
3
control
7
6 Angptl4 Heterozygous NPHS2- type rats, and Angptl4 transgenic rats. (a)
Angptl4 TG rat Albuminuria in Angptl4 +/+ and Angptl4 -/- mice 48
Fluorescence

Fluorescence

5
2
1.5
4
3
Glomeruli hours after injection of γ2-NTS. (b) Electron
1 2 Red TG rat microscopy of the glomerular capillary loop in NTS
0.5 1 Blue Wild type rat
0 0
injected Angptl4 +/+ and Angptl4 -/- mice. Angptl4
1 5 9 13 17 21 25 29 1 5 9 13 17 21 25 29 33 37 -/- mice had 30.2 + 2.4 % (mean + SE) foot process
Cycles Cycles effacement compared to diffuse effacement in
3 8
2.5 7 Heterozygous aP2- Angptl4 +/+ mice. (c) Digoxigenin labeled in situ
Angptl4 TG rat
Fluorescence

6
hybridization for Angptl4 expression in PAN Day 6
Fluorescence

2 5
White adipose tissue
1.5 4
3
and control rats. Signal for Angptl4 expression (dark
1 Red TG rat
0.5
2
1 Blue Wild type rat
brown / black) in a peripheral distribution is indicated
0 0 by arrows. (d) Confocal imaging of Angptl4 transgenic
1 5 9 13 17 21 25 29 1 5 9 13 17 21 25 29 33 37
mouse glomeruli showing co-localization of Angptl4
Cycles Cycles
3 8 and CD2AP expression. (e) Fold increase in kidney
2.5 7 Heterozygous aP2- cortical mRNA expression in Angptl4 transgenic mice.
Fluorescence
Fluorescence

6
2 Angptl4 TG rat
1.5
5
4 Brown adipose tissue
(f) Real time PCR tracings for studies on glomerular
1 3
Red TG rat
and adipose tissue expression in Angptl4 transgenic
2
0.5 1 Blue Wild type rat rats shown in Fig. 2f. Glomerular control was 18s,
0
1 5 9 13 17 21 25 29
0
1 5 9 13 17 21 25 29 33 37
adipose tissue control was GAPDH. Scale bars (b) 0.5
Cycles Cycles µm (c) 10 µm (d)10 µm.Nature
* P<0.05, ***doi:10.1038/nm.2261
Medicine: P<0.001.
a b NPHS2-Angptl4 TG NPHS2-Angptl4 TG NPHS2-Angptl4 TG

Antisense Antisense Sense

Antisense Antisense Sense

WT WT WT
c d N. S. e

Pulse rate (beats per min) or

(mcg per 18 hours)

NPHS2-Angptl4 TG 1 month * 500

Percentage of intact albumin

250

blood pressure (mm Hg)

100
Albuminuria

200 400
* 80
150 60
(densitometry)

300
100 40
50 20
200 *** *** ***
0 0 100
M

F
M

0
l4

PA e
e
t

t
ra

ra
CD ptl

ps
ps
G
T

pt
G
T

T
W

N
T

ng

la
la

Pulse Systolic Diastolic MAP

W
o.

o.

An

re
re
A
er

om

BP BP
2-

2-

N
et

aP

HS

M
H

WT M Heterozygous TG M
NP

Supplementary Figure 3: Characterization of Angptl4 transgenic rats. (a) Low power electron micrograph of a 5 month old
homozygous NPHS2-Angptl4 transgenic rat. Arrows point towards effaced foot processes. (b) Early exposure in situ hybridization
image for Angptl4 expression in NPHS2-Angptl4 transgenic and wild type rat glomeruli. Arrows point towards a prominent
peripheral pattern in transgenic rats consistent with increased podocyte expression. A faint signal was noted in wild type rats.
Tubular expression was not increased in transgenic rats. (c) Albuminuria in one month old NPHS2-Angptl4 transgenic rats. (d)
Densitometry of the intact albumin 70 kDa band expressed as a percentage of whole lane densitometry in Fig. 3d. Percentage of
intact albumin in urine was significantly higher in NPHS2-Angptl4 transgenic rats compared to all other groups (P < 0.001), except
MCD relapse (N. S. ,not significant). (e) Blood pressure (BP) and pulse rate in 5 month old male wild type and heterozygous NPHS2-
Angptl4 transgenic rats. MAP, mean arterial pressure. Scale bars (a)1 µm (b) 20 µm. * P < 0.05, *** P < Nature
0.001.Medicine: doi:10.1038/nm.2261
 a b 5000
c ***
d

Densitometry units
pI 6.0-6.6 4000
pI 5.4-5.6
3000
2000
pI 8.3-8.5
**
1000
2D gel markers WT rat
2D gel markers 0
WT aP2- NPHS2-
Angptl4 Angptl4
120 120
50 e

Control
Control

NTS
NTS
NTS
50
WT rat aP2-Angptl4 TG rat
WT rat
120 120 120
50
Anti-Angptl4 PI
50
aP2-Angptl4 TG rat NPHS2-Angptl4 TG rat

PAN

PAN
PAN
aP2-Angptl4 TG rat f
120 120
50 120
50 Anti-Angptl4 PI
NPHS2-Angptl4 TG rat
NPHS2-Angptl4 TG rat
Western blot : anti-Angptl4
Supplementary Figure 4: Serum Angptl4 levels in transgenic (TG) rats, and urine Angptl4 in rats with experimental
nephrotic syndrome. (a) 2D gel and Western blot assessment of circulating Angptl4 in serum from wild type and Angptl4
transgenic rats. Area magnified in panel b is demarcated by green rectangles. (b) Magnified images of blots in panel a show
presence of Angptl4 fragments and oligomers in the circulation. (c) Densitometry of circulating Angptl4 seen in panel b in wild
type and Angptl4 transgenic rats. (d) Ponceau red stained nitrocellulose membrane images (equal loading and transfer controls)
taken immediately after transfer of 2D gels shown in panels a and b. (e) Western blot of urine from rats injected with γ2−NTS
showed presence of low order Angptl4 oligomers. (f) Similar oligomers were noted in urine from PAN Day 6 rats. ** P < 0.01,
*** P < 0.001

Nature Medicine: doi:10.1038/nm.2261

a b c

Percentage change in GBM

40
NPHS2-

alcian blue staining

20 TG Angptl4

densitometry
mouse TG rat
0
aP2-
Angptl4
-20
TG rat
-40
*** WT rat NPHS2-Angptl4
WT mouse Angptl4 TG mouse *** TG rat
-60

WT rat NPHS2-Angptl4 TG rat

Supplementary Figure 5: Assessment of GBM charge in Angptl4 transgenic (TG) rats and mice. (a) Alcian blue staining of the
GBM in 3 month old wild type and Angptl4 transgenic mice. (b) Densitometry of glomerular alcian blue staining in 3 month old
Angptl4 trangenic rats and mice, expressed as a percentage difference from their wild type counterparts. (c) Confocal assessment
of GBM heparan sulfate proteoglycan expression in 5 month old wild type and NPHS2-Angptl4 transgenic rats. (d) Assessment of
GBM charge in 5 month old wild type and NPHS2-Angptl4 transgenic rats by the polyethyleneimine method. Arrows in the
electron micrographs point towards the normal GBM charge in the subepithelial and subendothelial regions. Loss of GBM
charge, effacement of foot processes and clustering of cell surface charge were noted in transgenic rat glomeruli. Scale bars (a)
10 µm (c) 10 µm (d) 0.33 µm. *** P<0.001 Nature Medicine: doi:10.1038/nm.2261
 a 4
pI range (non linear)
10
b c d
Neutral pI High pI
220
100

50
40 Control HEK 293 control GEC control
30
Control Anti-Angptl4
220
100

50 PAN HEK 293 ManNAc GEC ManNAc

40 g h
30 3000

Densitometry units
PAN Anti-Angptl4 2500 N. S.
Anti-pThr 2000
1500
220 1000
100 PAN + glucocorticoids 500
0 WT rat
50 WT TG + TG +
control ManNAc
40
30 pI range (non linear)
PAN + glucocort. Anti-Angptl4 f 6 10

e
WT rat NPHS2-Angptl4 TG control fed

Control fed
NPHS2-Angptl4 TG control fed

NPHS2-Angptl4 TG ManNAc fed

NPHS2-Angptl4 TG ManNAc fed
ManNAc fed (antibody blot) ManNAc fed (lectin blot) Supplementary Figure 6 Nature Medicine: doi:10.1038/nm.2261
Supplementary Figure 6: Assessment of glomerular Angptl4 sialylation. (a) 2D gel electrophoresis and Western blot of protein from
perfused glomeruli in control, PAN Day 6, and glucocorticoid treated PAN Day 6 rats revealed presence of small amounts of fragments (red
arrow) and monomers (blue arrow), and larger amounts of low order oligomers (pink, orange arrows) migrating at neutral or high pI.
Magnified images of Angptl4 oligomers enclosed in red boxes are shown in Fig. 4a. Neutral pI Angptl4 fragments, but not oligomers, were
reactive with anti - phosphothreonine antibodies. (b) Ponceau red stained nitrocellulose membrane images (equal loading and transfer
controls) taken immediately after transfer of 2D gels shown in panel a and Fig. 4a. (c) Equal loading and transfer controls for Fig. 4c (as
described for panel b). (d) Equal loading and transfer controls for Fig. 4d (as described for panel b). (e) Equal loading and transfer controls for
Fig. 4e (as described for panel b). (f) 2D gel electrophoresis and Western blots for glomerular podocalyxin expression on Day 12 of experiment
described in Fig. 4e. In addition, podocalyxin expression in wild type rats was also assessed. (g) Densitometry studies from blots in panel f. (h)
Equal loading and transfer controls for panel f (as described for panel b). N. S., not significant, TG, transgenic

Nature Medicine: doi:10.1038/nm.2261

 pI 3 to 11
a b Control HEK293 Control HEK293 c 220 BSA
Conalbumin
120 pI 6 - 6.6
80 pI 5.4 - 5.6
Actin
80000 50 pI 5 - 5.1
GAPDH
Fold increase in Angptl4 mRNA

pI 8.3 - 8.6
*** Carbonic anhydrase
60000
Trypsin inhibitor pI 5.9 - 6.0
expression

20 pI 4.5 Myoglobin
40000 pI 7.0

20000 Angptl4-HEK293 Angptl4-HEK293

0
HEK 293 - HEK 293 - 65-70 kDa
control line Angptl4 line 55 kDa

15 kDa

Western blot Preimmune serum Anti-Angptl4 antibody Western blot & markers superimposed

Clone 17-HD1

Clone 17-HD1

Clone 17-HD1
e

Clone 4-FA3

Clone 4-FA3

Clone 4-FA3
d
Fold increase in Angptl4 mRNA

16
***
14
12
10 220
expression

8 100
80
6 60
4
50
2
0 Anti- Pre- GelCode
Mouse GEC - Mouse GEC - Angptl4 immune blue
antibody serum
control line Angptl4 line
Western blot
Supplementary Figure 7: Characterization of Angptl4 secreting HEK293 and mouse GEC stable cell lines. (a) Analysis of Angptl4 mRNA
upregulation in the Angptl4-HEK293 stable cell line. Expression in the control (empty vector) stable cell line was given an arbitrary value of 1.
(b) 2D gel electrophoresis and Western blot for Angptl4 in supernatant from Angptl4-HEK293 and control-HEK293 stable cell lines collected
under serum free conditions. (c) A molecular weight marker gel (top image) was superimposed with the right lower image from panel b
to generate a composite image (bottom image) using Adobe Photoshop. Most of the intact 70 kDa Angptl4 monomer secreted by the
Angptl4-HEK293 stable cell line migrates between pI 8.0 and 8.5. (d) Analysis of Angptl4 mRNA upregulation in the Angptl4-GEC stable cell
line. (e) Western blot and GelCode blue analysis of recombinant protein secreted by 2 different clones of the Angptl4-GEC stable cell line.
*** P < 0.001 Nature Medicine: doi:10.1038/nm.2261
 a ManNAc dose (mg/ml)
1 2 4 8 b 6000 ManNAc 1 mg/mL 10000 ManNAc 1 mg/mL
140000
6641 6818 6805

(µg per 18 hours)

(µg per 18 hours)

(µg per 18 hours)

Albuminuria
8000
Albuminuria

Albuminuria
120000 4000
6000
100000
4000
2000
80000 2000

60000 0 0

12

12

24
2

3
D 2

D 12
24
D 0
D 3
D 9

D 3
D 2
12

12
0

-1
ay

ay

ay

ay
-1

1
ay

ay

ay

ay

ay

ay
ay
ay

ay

ay

ay

ay
ay
ay

ay

ay

ay
ay

D
ay
D

D
D
D

D
ManNAc dose (mg/ml)
1 2 4 8 ManNAc 1 mg/mL
25000 6576 6000 8000 ManNAc 1 mg/mL
6822 6816

(µg per 18 hours)

(µg per 18 hours)

(µg per 18 hours)

23000

Albuminuria
Albuminuria

6000

Albuminuria
4000
21000
4000
19000 2000
2000
17000
15000 0 0

D 2

D 12
24
D 0
D 3
D 9

D 3
D 2

D 2

D 12
24
D 0
D 3
D 9

D 3
D 2
12

12
0

-1

1
ay
ay
ay

ay

-1

1
ay
ay
ay

ay
ay

ay

ay

ay

ay

ay

ay
ay
ay

ay

ay
ay
ay

ay

ay
D

D
D

D
aP2-
c Angptl4
NPHS2-
Angptl4
aP2-
Angptl4
NPHS2-
Angptl4
Supplementary Figure 8: Albuminuria in ManNAc - treated NPHS2-Angptl4
TG rat TG rat Recomb. TG rat transgenic (TG) rats, and oligomerization of recombinant and transgene
TG rat Recomb.
A2M plasma glom. Angptl4 plasma glom. Angptl4
expressed Angptl4. (a) Representative tracings of albuminuria from pilot studies, in
high order
oligomers
which increasing doses of ManNAc were shown to reversibly reduce albuminuria in
720
NPHS2-Angptl4 transgenic rats with moderate to heavy albuminuria. (b) Individual
tracings of albuminuria from four NPHS2-Angptl4 transgenic rats that competed both
220 middle order the ManNAc and washout phase of the 36 day study. Two urine collections 12 days
oligomers
160 720 apart were conducted prior to the start of the study to ensure that these rats
120 low order developed increasing albuminuria with time. (c) Non-reducing SDS PAGE and
100 oligomers Western blot to assess for oligomerization of circulating and glomerular expressed
80
Angptl4 from transgenic rats, and recombinant Angptl4 from the HEK293 stable cell
Anti-A2M Ab. Anti-Angptl4 Ab. Anti-Angptl4 Ab. line. Gels were run for 1 hour (left) or 2.5 hours (right) to study all oligomeric forms
5% gel, 1 hour 5% gel, 2.5 hours (low, middle and high order oligomers). α2 macroglobulin (A2M) was used as a high
molecular weight market (720 kDa).

Nature Medicine: doi:10.1038/nm.2261

a b
Control Control

Laminin Angptl4 merge High power

MCD 2

Angptl4 Laminin merge

MCD 1

Laminin Angptl4 merge High power

MCD 3

Angptl4 Nephrin merge Laminin Angptl4 merge High power

MCD 1 MCD 4

Nephrin Angptl4 merge High power

MCD 5

Angptl4 Laminin merge

MCD 1
PECAM 1 Angptl4 merge High power
MCD 5 High power

Angptl4 PECAM 1 merge Aquaporin 1 Angptl4 merge Proximal tubule

Supplementary Figure 9: Confocal assessment of glomerular Angptl4 expression in human minimal change disease (MCD). (a) Increased expression
and co-localization of Angptl4 with podocyte (nephrin), GBM (laminin) and endothelial cell (PECAM) markers in the biopsy of an individual with MCD. (b)
Biopsies from 4 additional individuals with MCD were stained for Angptl4 and other glomerular capillary loop markers listed above. Arrows in high power
images point towards overlap of staining patterns. Lowermost panels show presence of Angptl4 in proximal tubules (marked by aquaporin 1), most likely
due to tubular uptake, since upregulation of Angptl4 mRNA expression is not seen in tubules in experimental MCD (puromycin nephrosis) by in situ
hybridization. Scale bars 10 µm. Nature Medicine: doi:10.1038/nm.2261
 a b 220 pI range 5 - 8, non linear pI range 3 - 11, non linear

MCD 7
MCD 6

MCD 8
MCD 6

FSGS 2

FSGS 3
FSGS 1
FSGS 1
Angptl4
oligomer Angptl4
220
220 oligomer

100 Angptl4 100 70

fragments
PI Anti-Angptl4 Western blot PI Anti-Angptl4 50
MCD 9
c pI range (non linear)
6 9
pI range (non linear)
6 9 Albumin (bleed through) Angptl4
MCD 9
160 160 fragments
220
100 100

60 60
50 50
70
40 40

30 50
30
MCD relapse MN FSGS 4
Ig heavy chain Ig heavy chain Angptl4 fragments FSGS 4
160 Western blot with anti-Angptl4
160
100 100 e
60 60
50 50
40 40

30 30
MCD remission FSGS MCD relapse MN
d
Densitometry units

8000 ***
6000
4000
***
2000 ***
0
S
se

on

G
M
ap

si

FS
is
l
re

m
re

MCD remission FSGS

D
C

D
M

C
M

Supplementary Figure 10: Plasma and urine Angptl4 in human primary glomerular disease. Clinical data shown in
Supplementary Table 1. (a) Reducing SDS PAGE and Western blot of urine shows presence of Angptl4 oligomers in individuals with
minimal change disease (MCD), and fragments in focal and segmental glomerulosclerosis (FSGS) patients. (b) 2D gel electrophoresis
and Western blot of urine (left panels) confirms the presence of Angptl4 oligomers and fragments in MCD, and only fragments in
FSGS. Ponceau red stained nitrocellulose membrane images (equal loading and transfer controls) taken immediately after transfer
of 2D gels are also shown (right panels). (c) Representative 2D gel electrophoresis and Western blot of plasma from individuals with
MCD, FSGS and membranous nephropathy (MN). Only individuals with MCD in relapse had elevated circulating levels of 55 - 70 kDa
pI 8 - 8.5 Angptl4 protein (red oval). Increased circulating neutral pI monomers and oligomers were noted in individuals with MCD in
relapse (red arrow), and monomers only in MN (green arrow). (d) Densitometry analysis of circulating Angptl4 represented in panel
c. (e) Equal loading and transfer controls for blots shown in panel c (as described for panel b). *** P < 0.001, comparison with MCD
Nature Medicine: doi:10.1038/nm.2261
remission.
 Foot process GBM Hyposialylated Angptl4

Endo Sialylated Angptl4

Adipose released
Angptl4 oligomers

Angptl4 secretion

Outside - in signal
Podocyte Putative podocyte
receptor
Putative endothelial
receptor

Adipose
tissue

HDL
Effaced
foot process

Supplementary Figure 11: Schematic representation of the role of podocyte secreted Angptl4 in nephrotic
syndrome. Sequence of events arranged from top to bottom. Podocytes secrete neutral and high pI Angptl4 that binds to
the GBM to alter protein - protein interactions, resulting in proteinuria. Over time, Angptl4 reaches up to the endothelial
surface. Progressive accumulation and clustering of Angptl4 in the GBM likely activates signals at the podocyte - GBM
interface, induces foot process effacement and further increase in proteinuria. Circulating Angptl4 secreted from other
organs in disease states e. g. adipose tissue, forms medium and high order oligomers that are bound to HDL particles,
migrate at neutral or low-neutral pI, and do not enter the GBM or cause proteinuria. Nature Medicine: doi:10.1038/nm.2261
 Patient Diagnosis age (years) sex proteinuria primary treatment (initiated or past)
MCD 6 MCD - relapse 14 female nephrotic glucocorticoids
MCD 7 MCD - relapse 14 female nephrotic glucocorticoids + cyclosporin
MCD 8 MCD - relapse 4 male nephrotic glucocorticoids
MCD 9 MCD - relapse 10 male nephrotic glucocorticoids

MCD-R1 MCD remission 12 male < 0.5 gm/24 hours glucocorticoids + cyclophosphamide
MCD-R2 MCD remission 5 male < 0.5 gm/24 hours glucocorticoids + cyclophosphamide
MCD-R3 MCD remission 9 male < 0.5 gm/24 hours glucocorticoids + cyclophosphamide
MCD-R4 MCD remission 3 male < 0.5 gm/24 hours glucocorticoids

FSGS 1 FSGS 49 male sub nephrotic AEC inhibitor

FSGS 2 FSGS 29 female nephrotic ACE inhibitor
FSGS 3 FSGS 9 male sub nephrotic cyclosporin
FSGS 4 FSGS 8 male nephrotic glucocorticoids

MN 1 MN 59 male sub nephrotic ACE inhibitor

MN 2 MN 63 male sub nephrotic ACE inhibitor
MN 3 MN 31 female nephrotic unknown
MN 4 MN 52 male sub nephrotic ACE inhibitor
Supplementary Table 1: Brief clinical profile of individuals whose plasma and urine were
assessed for presence and patterns of Angptl4 expression by Western blot.

Gene / transgene Species Forward primer Reverse primer Taqman probe

Angptl4 rat tctgggatctccaccatttttg tcaccgtccagcctccat caactgtgagatgacttc

Angptl4 rat cgccacccgcttacaca cagaggctggatctggaaaagt tgccaggaactcttt
NPHS2-Angptl4 construct rat tacaggctaccaccctgttgatc aaccgcgggccctctag ccatggaggctacagca
aP2-Angptl4 construct rat tgttgatccagcccatgga agggataggcttaccttcgaatg cagcagcctccc
Prolactin (genomic) rat cttgaagggattgaaaagataattagc ccatgagtcagaaaagcattgaac aggtgagcattttcctg

Supplementary Table 2. List of primers and probes used for Taqman real time PCR.

Supplementary Tables

Nature Medicine: doi:10.1038/nm.2261

 Supplementary Methods
Cloning of full length rat Angptl4, and generation of antibody against full length

recombinant Angptl4: We cloned the full length rat Angptl4 open reading frame from our

previous experiments3, excluding the stop codon, into pcDNA3.1/V5-HisB for eukaryotic

expression, and into pET28a for prokaryotic expression. The E. Coli expressed purified

full length protein was used to generate a polyclonal antibody in rabbits (Proteintech

group) that was tested by ELISA and Western blot. We excised antibody reactive bands

from GelCode blue stained gels and confirmed the presence of Angptl4 peptide

sequences by MALDI-TOF/TOF. Part of the antiserum was affinity purified to the

antigen. All studies described used this antibody. We raised an additional polyclonal

antibody against the N-terminal part of rat Angptl4 (amino acids 7 – 86 excluding signal

peptide) in rabbits, and used this for confirmation studies.

We purchased the following antibodies: goat anti-mouse CD2AP (Santa Cruz

Biotechnology), guinea pig anti-human nephrin (Fitzgerald Industries), mouse anti-rat

β2γ1 laminin (Abcam), mouse anti-rat PECAM-1 (BD Pharmingen), mouse anti-human

aquaporin 1 (Santa Cruz Biotechnology), goat anti-mouse podocalyxin (Alpha

Diagnostic International).

Induction of proteinuria in animal models of human glomerular disease. Induction of

animal models of proteinuria (n = 4 rats/group) in wild type rats are described in

previous publications in parenthesis: Puromycin aminonucleoside nephrosis (PAN)3,

passive Heymann nephritis (PHN)3, PAN with glucocorticoids15, non-HIV collapsing

glomerulopathy14, nephrotoxic serum induced heterologous phase proteinuria3. Anti-

Thy1.1 nephritis was induced by injection of 200 μg of anti-Thy1.1 (Ox-7 hybridoma) or

Nature Medicine: doi:10.1038/nm.2261

control IgG IV into different groups of male Wistar rats (100-125 gm, n = 4 rats/group),

and rats euthanized after 24 and 72 hours.

The following techniques are described in prior publications: Taqman real time PCR24,

confocal imaging3, in situ hybridization25, promoter – reporter studies3, immunogold

electron microscopy using 10nM gold particles24, glomerular extraction and processing

for Western blot24, assessment of charge by polyethyleneimine method26. Real time

PCR studies for screening were performed with a minimum of 3 templates, and if

positive, were confirmed with a minimum of 6 templates. In rat models of proteinuria, 3-

fold change in glomerular gene expression was considered significant3. In the rat model

of non-HIV collapsing glomerulopathy, we assessed glomerular gene expression in

sieved and laser captured glomeruli. Taqman real time PCR primers and probes are

listed in Supplementary Table 2. For in situ hybridization (n = 2 rats / condition), we

generated a digoxigenin labeled probe for rat Angptl4 that included bp 1 to 548 of the

open reading frame. Unless otherwise stated, all 2D gel electrophoresis was performed

using 11 cm Immobiline pH 3–11 strips (GE Healthcare) and Criterion 8–16% Tris HCl

Precast Gels (Bio-Rad Laboratories), using 200 μg protein loaded in the first phase (n =

3 gels / condition). Densitometry of 2D gel Western blots was assessed using Gel-Pro

Analyzer software (Media Cybernetics). For alcian blue staining, the pH of the staining

solution was adjusted to 2.5 using acetic acid, and 0.1% nuclear fast red solution was

used as a counterstain. Densitometry of glomerular basement membrane alcian blue

stain (20 glomeruli / rodent, 3 rodents / group) was assessed using Image-Pro software

(Media Cybernetics).

Nature Medicine: doi:10.1038/nm.2261

Injection of NTS into Angptl4–/– mice: Angptl4–/– mice were provided to Sander Kersten

by Eli Lilly Corporation. The Animal Ethics Committee at Wageningen University

approved the study protocol. We injected 11 week old male Angptl4–/– or Angptl4+/+ mice

(n = 4 mice / group) intravenously with 1.5 mg γ2-NTS or normal sheep serum (Sigma

Aldrich) collected spot urine samples at 48 hours, euthanized the mice at 72 hours,

collected plasma for biochemical measurements, and preserved kidneys for histological

analysis. We assessed urine albumin by ELISA (Bethyl laboratories) and measured

urine creatinine by mass spectrometry. To assess for foot process effacement, the

mean width of foot processes was first measured in electron micrographs of control

treated Angptl4+/+ mice (10 equally spaced readings / loop, 3 loops / glomerulus, 3

glomeruli / kidney, 3 kidneys / group). Effacement was defined as over 2.5 fold increase

in mean width. We assessed the total number of foot processes, and the percentage

effaced foot processes in NTS treated or control treated Angptl4–/– mice.

Injection of lipopolysaccharide (LPS) into Angptl4–/– mice: The study protocol was

approved by the Animal Ethics Committee at Wageningen University. After a baseline

spot urine collection, we injected 12 week old Angptl4–/– and Angptl4+/+ mice (n = 5 mice

/ group) intraperitoneally with 10 μg / gram body weight of ultrapure LPS (Sigma Aldrich,

catalog number L4524) or an equal volume of PBS. Each mouse also received 0.8 ml of

normal saline at 12 and 36 hours after the LPS injection to avoid volume depletion. We

collected urine at the peak of proteinuria 24 hours after LPS injection. Mice were

euthanized 48 hours after LPS injection. We assessed urine albumin and creatinine

concentrations as detailed for the NTS study.

Nature Medicine: doi:10.1038/nm.2261

Measurement of rat blood pressure: Blood pressure and pulse rate were measured in

six 5 month old wild type and proteinuric heterozygous NPHS2-Angptl4 transgenic rats

by the tail cuff method using the SC-1000 apparatus from Hetteras Instruments. A

minimum of 80 reading were analyzed per group.

Studies on oligomerization of Angptl4: Since 2D gel electrophoresis has limitations in

resolving high molecular weight proteins, we performed non-reducing 1D SDS PAGE

and Western blot were performed. We loaded 3 μg of human α2 macroglobulin (A2M,

high molecular weight marker, 720 kDa), 60 μg each of aP2-Angptl4 transgenic rat

plasma and protein from perfused NPHS2-Angptl4 transgenic rat glomeruli, and 35 μg

of concentrated supernatant from HEK293 stable cell line in duplicate into 5% non-

reducing gels, ran the for 1 hour or 2.5 hours, transferred them to PVDF membranes,

and conducted Western blot studies. We also ran standard molecular weight markers in

one lane.

Studies with human samples: We conducted immunostaining and confocal imaging of

human kidney biopsies (n = 5 biopsies per condition) obtained through protocols

approved by the Research Ethics Committee at the Instituto Nacional de Cardiologia,

Mexico City. Control kidney biopsies used for these studies were age, sex and race

matched protocol pre-transplant biopsies. We obtained human sera and urine samples

for 2D gel electrophoresis and Western blot ( n = 4 samples / condition) from a

previously published study27 (select details in Supplementary Table 1).

Nature Medicine: doi:10.1038/nm.2261

 Supplementary References
24. Liu, G. at al. Neph1 and nephrin interaction in the slit diaphragm is an important
determinant of glomerular permeability. J. Clin. Invest. 112, 209-221 (2003).
25. Dijkman, H.B.P.M., Mentzel, S., de Jong, A.S. & Assmann, K.J.M. RNA in situ
hybridization using digoxigenin-labeled cRNA probes. Biochemica 2, 23-27
(1995).
26. Isogai, S., Mogami, K., Shiina, N. & Yoshino, G. Initial ultrastructural changes in
pore size and anionic sites of the glomerular basement membrane in
streptozotocin-induced diabetic rats and their prevention by insulin treatment.
Nephron. 83, 53-58 (1999).
27. Bakker, W.W. et al. Altered activity of plasma hemopexin in patients with minimal
change disease in relapse. Pediatr. Nephrol. 20, 1410-1415 (2005).

Nature Medicine: doi:10.1038/nm.2261