Smad3 for Renal Fibrosis

Hui Yao Lan, MD, PhD

Department of Medicine & Therapeutics,

and Li Ka Shing Institute of Health Sciences
The Chinese University of Hong Kong
(hylan@cuhk.edu.hk)
TGF-β/Smad signaling is a key pathway leading to CKD

AGEs TGF-β1 IL-1β TNFα

Ang II
TβRII TβRI
AT1/2 RAGE

p
p p p

Smad3

Smad2
p p
p65
p
IκBα

Smad7
Smad4

Sma
d2
Smad4

p
Sma
d3 p p50

Fibrosis Inflammation
The imbalance of Smad signals causes renal inflammation and fibrosis
Relevant Publications
1. Li JH et al: JASN 13:1464-72, 2002
2. Li JH et al: Kidney Int 63:2010-19, 2003
AGEs TGF-β1 IL-1β TNFα
3. Lan HY et al: JASN 14:1535-48, 2003
Ang II 4. Li JH et al: FASEB J 18:176-8,2004
5. Li JH et al: Am J Pathol 164:1389-97, 2004
TβRII TβRI 6. Nakagawa T et al: Kidney Int 66:605, 2004
AT1/2 RAGE 7. Hou CC et al: Am J Pathol 166:761-71, 2005
8. Ng YY et al: Kidney Int 67:s83-91, 2005
p 9. Nakagawa T et al: JASN 16:899-904, 2005
10.Wang W et al: JASN 16:1371-83, 2005
p p p 11.Wang W et al: Cir. Res. 98:1032-9, 2006
__ 12.Wang W et al: Nephrology 10:48-56, 2005
Smad3

Smad3

Smad2
p p 13.Fu P et al: JASN 17:3105-14, 2006
p65 14. Ka SM et al: JASN 18:1777-88, 2007
p 15. Nie J et al: PDI 27:580-8, 2007
IκBα
16. Nie J et al: Kidney Int 72:1336-44, 2007
Smad7 17. Wang W et al: AJP Renal 293:F1123, 2007
+ __ +
18. Guo H et al: JASN 18:2689-703, 2007
Smad4

19: Guo H et al: Gene Ther 14:1712-20, 2007

20. Huang XR et al: JASN 19:233-42, 2008
21. Huang XR et al: AJP-Renal, 295:F118 2008
22. Deng W et al: Cancer Res 68:7200-9, 2008 ,
Sma Smurfs 23. Ng YY et al: Am J Nephrol 29:43-53, 2009
Smad4

d2
p 24. Lan HY: Front Biosci 13:4894-12, 2008
Smurfs 25. Chung ACK et al: NDT 24:1443-54, 2009
Sma
d3
p p50 26. Sun Y et al: Am J Nephrol 30:84-94, 2009
27. Yang F et al: Hypertension 54:877, 2009
28. Chung et al: JASN 21:249-60, 2010
29. Wang X et al: NDT 25:399-12, 2010
30. Zhou L, et al: AJP-Renal 298:1006-17,2010
31. Huang XR, etal:Hypertension55:1165-71, 2010
32. Yang F et al: J Pathol 221:390-401, 2010
33. Chung ACK et al: JASN 21:1317-25, 2010
34. Meng X et al: JASN 21:1477-87, 2010
35. Chen HY et al: Diabetes 60:590-601,2011
36. Lan HY et al: Contrib Nephrol 170:75-82, 2011
Fibrosis Inflammation 37. Qin W et al: JASN 22:1462-74, 2011
38. Zhong X et al: JASN 22:1668-81, 2011
39. Meng X et al: Kidney Int 81:266-79,2012
40. Meng X et al: J Pathol 27:175-88, 2012 …...
Q1: Can restored Smad7 inhibit Smad3-mediated renal fibrosis?
Relevant Publications
1. Li JH et al: JASN 13:1464-72, 2002

AGEs TGF-β1 IL-1β TNFα

2. Li JH et al: Kidney Int 63:2010-19, 2003
3. Lan HY et al: JASN 14:1535-48, 2003
Ang II 4. Li JH et al: FASEB J 18:176-8,2004
5. Li JH et al: Am J Pathol 164:1389-97, 2004
TβRII TβRI 6. Nakagawa T et al: Kidney Int 66:605, 2004
AT1/2 RAGE 7. Hou CC et al: Am J Pathol 166:761-71, 2005
8. Ng YY et al: Kidney Int 67:s83-91, 2005
p 9. Nakagawa T et al: JASN 16:899-904, 2005
10.Wang W et al: JASN 16:1371-83, 2005
p p p 11.Wang W et al: Cir. Res. 98:1032-9, 2006
Smad3 12.Wang W et al: Nephrology 10:48-56, 2005

Smad2
p p 13.Fu P et al: JASN 17:3105-14, 2006
p65 14. Ka SM et al: JASN 18:1777-88, 2007
p 15. Nie J et al: PDI 27:580-8, 2007
IκBα
16. Nie J et al: Kidney Int 72:1336-44, 2007
Smad7 17. Wang W et al: AJP Renal 293:F1123, 2007
18. Guo H et al: JASN 18:2689-703, 2007
Smad4

19: Guo H et al: Gene Ther 14:1712-20, 2007

20. Huang XR et al: JASN 19:233-42, 2008
21. Huang XR et al: AJP-Renal, 295:F118 2008
22. Deng W et al: Cancer Res 68:7200-9, 2008 ,
Sma
d 2 23. Ng YY et al: Am J Nephrol 29:43-53, 2009
Smad4

p 24. Lan HY: Front Biosci 13:4894-12, 2008

Sma 25. Chung ACK et al: NDT 24:1443-54, 2009
d3 p 26. Sun Y et al: Am J Nephrol 30:84-94, 2009
p50
27. Yang F et al: Hypertension 54:877, 2009
28. Chung et al: JASN 21:249-60, 2010
29. Wang X et al: NDT 25:399-12, 2010
30. Zhou L, et al: AJP-Renal 298:1006-17,2010
31. Huang XR, etal:Hypertension55:1165-71, 2010
32. Yang F et al: J Pathol 221:390-401, 2010
33. Chung ACK et al: JASN 21:1317-25, 2010
34. Meng X et al: JASN 21:1477-87, 2010
35. Chen HY et al: Diabetes 60:590-601,2011
Fibrosis Inflammation 36. Lan HY et al: Contrib Nephrol 170:75-82, 2011
37. Qin W et al: JASN 22:1462-74, 2011
38. Zhong X et al: JASN 22:1668-81, 2011
39. Meng X et al: Kidney Int 81:266-79,2012
40. Meng X et al: J Pathol 27:175-88, 2012 ……
 Smad7 Gene transfer Restores Renal Smad7
Expression in the Diabetic Kidney

Renal Smad7 mRNA

0.00014
*
Ratio (Smad7/GAPDH)

0.00012

0.0001

0.00008

0.00006

0.00004

0.00002

Normal DM VC Smad7
Chen HY& Lan HY: Diabetes 60:590-601,2011
 Smad7 Gene Therapy Inhibits
Microalbuminuria in STZ-induced DKD
Urine Albumin
4000 Smad7+DM

VC+DM

DM
3000
ug/24 hrs

*
2000 *
*
**
1000

0
0 w1 w3 w4 w5
Time (Weeks) after disease induction
Chen HY& Lan HY: Diabetes 60:590-601,2011
 Smad 7 Gene Therapy Inhibits Collagen
Accumulation in the Diabetic Kidney
Collagen I mRNA
0.12
Normal DM ***
0.1
***

Relative ratio
0.08
#
0.06

0.04

0.02

0
Normal DM VC Smad7

VC Smad7 Collagen III mRNA

0.00009 * *

Relative ratio
0.00007 #

0.00005

0.00003

0.00001
0

Normal DM VC Smad7
Chen HY& Lan HY: Diabetes 60:590-601,2011
 Smad7 Gene Therapy Inhibits Renal
Inflammation in the Diabetic Rat
10 Macrophages 15 MCP-1 expression
###
cells/mm2

8 ***
10
***

% area
6

4
*** 5 ###
2

0 0

IL-1β mRNA TNFa mRNA

Ratio (mRNA/GAPDH)

Ratio (mRNA/GAPDH)
0.06 0.003 #
#

0.04 0.002

0.02 0.001
* *
0.00 0.000
Normal DM VC Smad7 Normal DM VC Smad7
Chen HY& Lan HY: Diabetes 60:590-601,2011
 Q2: Is Smad3 a therapeutic target for renal fibrosis?

AGEs TGF-β1 IL-1β TNFα

Ang II
TβRII TβRI
AT1/2 RAGE

p
p p
p

Smad2
Smad3
p p
p65
p
IκBα

Smad7
Smad4

Sma
d2
Smad4

p
Sma
d3 p

Smad3 inhibitor (SIS3)

Fibrosis Inflammation
Jinnin M et al. 2006. Mol. Pharmacol. 69, 597
 Smad3 inhibitor (SIS3) inhibits renal fibrosis in the UUO kidney

Day 7 UUO
Normal untreated DMSO SIS3

P-Smad3

Smad3

α-SMA

Col. 1

FN

GAPDH
 Smad3 inhibitor (SIS3) inhibits renal fibrosis in UUO

α-SMA Collagen I
Normal UUO Normal UUO

UUO-DMSO UUO-SIS3 UUO-DMSO UUO(-SIS3

mRNA mRNA
0.05   *** 0.025 ***

 
α-SMA /GAPDH

Col-1/GAPDH

0.04 *** 0.02

0.03   0.015
**

 
##
#
0.02 0.01

0.01   0.005

0.00   0
DMSO
UT DMSO SIS3 Normal UT SIS3
Normal
Day 7 UUO Day 7 UUO
 Smad3 inhibitor halts renal fibrosis in the established UUO kidney

Immunohistochemistry Day 10 UUO (SIS3 treated)

Day 4
Normal UUO UT DMSO SIS3

Col- 1

α-­‐SMA

Real-time PCR Collagen I mRNA α-SMA mRNA

0.00012 0.090
***
α-SMA /GAPDH
*** *** ***
Col-1/GAPDH

***
0.00008 0.060
** #
#
0.00004 0.030

0.00000 0.000
Normal Day4 UUO DMSO SIS3
Normal Day4 UUO DMSO SIS3
UUO
UUO Day 10 UUO
Day 10 UUO
Hypothesis: Smad3 may mediate renal fibrosis via the
Smad3-dependent miRNAs

TGF-β1
AGE
TβRII TβRI
RAGE
Ang II
p p
AT1-R Smad3

HG

p ERK + -
p P38 p
Smad3 p

Fibro-miRNAs

Fibrosis
MicroRNA Profile of Obstructive Nephropathy in Smad3 WT/KO Mice
Fold change P-value
　 WT KO 　
WT KO miR-490 ↓250.00 ↑2.68 *0.004
UUO UUO mir-137 ↓111.11 ↑3.80 0.094 Smad3 WT UUO
vs vs miR-208 ↓111.11 ↓7.04 0.097
Normal Normal
miR-429 ↓3.87 ↑2.18 0.086
miR-200a ↓3.15 ↑3.20 0.082
miR-200b ↓2.32 ↑4.04 0.087
miR-200c ↓1.44 ↑5.01 0.098
miR-15a ↓1.35 ↑3.78 0.885
miR-29a ↓1.73 ↑3.34 *0.039
miR-29b ↓7.29 ↑3.64 *0.030
miR-29c ↓1.96 ↑2.63 *0.019
miR-141 ↓1.97 ↑ 4.81 0.086
miR-192 ↑1.72 ↓2.01 *0.029
miR-216b ↑2.13 ↓4.83 0.162
miR-365 ↑2.37 ↑2.83 0.794
miR-133a ↑3.19 ↑2.86 0.968
miR-190 ↑3.22 ↑2.75 0.999 Smad3 KO UUO
miR-24 ↑3.85 ↑3.33 0.867
miR-10b ↑4.42 ↑3.62 0.930
miR-547 ↑4.91 ↓1.42 *0.023
miR-1 ↑7.46 ↑7.51 1.000
miR-682 ↑11.08 ↓1.19 *0.020
miR-214 ↑17.04 ↑12.37 0.996
miR-133b ↑18.78 ↑46.06 0.659
miR-294 ↑25.95 ↓4.37 *0.029
miR-21 ↑27.77 ↓4.86 *0.030
miR-680 ↑37.13 ↑2.07 0.126
miR-511 ↑40.44 ↑5.21 *0.048
miR-433 ↑44.48 ↓5.99 *0.027
miR-216a ↑55.16 ↑3.16 *0.006
miR-135a ↑64.57 ↑55.38 0.949
miR-669a ↑214.00 ↑17.04 0.986
 Q3: Are Smad3-dependent microRNAs a therapeutic
target for renal fibrosis?
Publications
TGF-β1
1.  Qin W, et al. TGF-β/Smad3 signaling promotes renal
AGE fibrosis by inhibiting miR-29. J Am Soc Nephrol.
TβRII TβRI 2011;22(8):1462-74.
RAGE
Ang II 2.  Chung AC, et al. miR-192 mediates TGF-b/Smad3-
p p driven renal fibrosis. J Am Soc Nephrol.
2010;21:1317-25.
AT1-R Smad3 3.  Zhong X, et al. Smad3-mediated upregulation of
miR-21 promotes renal fibrosis. J Am Soc Nephrol.
HG 2011;22:1668-81.

-­‐  
4.  Zhong X, et al. miR-21 is a key therapeutic target for
p ERK +   renal injury in a mouse model of type 2 diabetes.
Diabetologia. 2013;56(3):663-74 .
p P38 p 5.  Chung AC, et al: Smad7 suppresses renal fibrosis
Smad3 p via altering expression of TGF-β/Smad3-regulated
microRNAs. Mol Ther. 2013 Feb;21(2):388-98
6.  Kantharidis P, et al. Diabetes complications: the
microRNA perspective. Diabetes. 2011;60(7):1832-7
miR-29, 200 miR-21,192,433 7.  Xiao J, et al. miR-29 inhibits bleomycin-induced
pulmonary fibrosis in mice. Mol. Ther 2012,
20:1251-60
8.  Chung AC, et al: MicroRNAs and nephropathy,:
emerging concepts. Int J Nephrol Renovasc Dis.
2013 Sep 25;6:169-179
9.  Li R et al: The microRNA miR-433 promotes renal
fibrosis by amplifying the TGF-β/Smad3-Azin1
pathway. Kidney Int 2013; 84:1129-44.
10.  Chen HY et al: miR-29 inhibits diabetic nephropathy
Fibrosis in db/db mice. Mol Ther 2014 Apr;22(4):842-53
Ultrasound- microbubble -mediated miR-29b
therapy in db/db mice

miR-29b
db/db mice 10 weeks treatment

10-week age Every 3 weeks 20-week age

 Ultrasound-microbubble mediated miR-29b
transfer restores normal levels of renal miR-29b

db/m db/db
miR-­‐29b  expression  

0.015  

miR29/U6 Ct Ratio
0.01  
#  
VC miR-29
0.005   ***   ***  

0  
m   db   m   db   VC   miR-­‐29  

 10  Wks   20  Wks  

miR-29b attenuates microalbuminuria in
db/db mice

Albumin/Creatinine Ratio

1000 db/m
db/db
VC
750
ACR (ug/mg)

miR-29b

500 **

250

0
0 2 4 6 8
Time (weeks) after miR-29 treatment
 miR-29b inhibits collagen I expression in
db/db mice
10  weeks   20  weeks  
m    db   m    db  

Collagen  I  mRNA  
1.2E-­‐04   vc   miR29
*   **  
##  
8.0E-­‐05  
RaKo  

4.0E-­‐05  

0  
m   db   m   db   VC  miR29  
10W   20W  
 miR-29b inhibits collagen IV expression
in db/db mice
10  weeks   20  weeks  
m      db   m   db  

Collagen  IV  mRNA  

vc                  miR29  
4E-­‐04   ***   ***  
3E-­‐04   ##  
RaKo  

2E-­‐04  
1E-­‐04  
0  
m   db   m   db   VC   miR29  
10W   20W  
Q4: Can combined Smad3 inhibitor and Smad7 agonist
produce a better therapeutic effect on renal fibrosis?
AGEs TGF-β1 IL-1β TNFα

Ang II
TβRII TβRI
AT1/2 RAGE

p p p

Smad3

Smad2
p p Asiatic acid
p65
p 积雪草
IκBα

Smad7
Naringenin
Smad4

柚皮素
Sma
d2
Smad4

p
p
Sma p50
d3

Fibrosis Inflammation
Combined Naringenin (Nar, S3i) and Asiatic acid (AA, S7ag) treatment
produces a better inhibitory effect on TG-b-induced Smad3 signaling
by upregulating Smad7 in vitro

DMSO DMSO AA Nar CB

DMSO DMSO AA Nar CB
TGF-β1 - + + + +
TGF-β1 - + + + +
P-Smad3
Smad7
Smad3
GAPDH
GAPDH

P-Smad3 protein Smad7 protein

(Smad7/GAPDH)
1.2
** **
(P-Smad3/GAPDH)

1.2 # # *
©
Ratio
## 0.8
Ratio

0.8

0.4 0.4

0 0
DMSO DMSO AA Nar CB DMSO DMSO AA Nar CB
TGF-β1 - + + + + TGF-β1 - + + + +
Combined Naringenin (Nar, S3i) and Asiatic acid (AA, S7ag) treatment
produces a better inhibitory effect on TGF-β1-induced fibrosis in vitro

Real-time PCR Western blot

Col.I mRNA DMSO DMSO AA Nar CB
Ratio(mRNA/GAPDH)

TGF-β1 - + + + +
0.18 *** # #
©
*** *** ### Col.I
0.12 **
α-SMA
0.06
GAPDH
0 Col.I
1.2

Ratio (Protein/GAPDH)
α-SMA mRNA a-SMA
***
Ratio(mRNA/GAPDH)

# #
*** # ** ** ©
0.006 *** *** ##
0.8
##
*** *
** # #
©
0.003 0.4 ** ** ##

0 0
DMSO DMSO AA Nar CB DMSO DMSO AA Nar CB

TGF-β1 - + + + + TGF-β1 - + + + +
Combined Naringenin (Nar, S3i) and Asiatic acid (AA, S7ag) treatment
rebalances TGF-β/Smad signaling in the UUO kidney

UUO UUO

DMSO DMSO AA Nar CB DMSO DMSO AA Nar CB

P-Smad3 Smad7

Smad3
GAPDH
GAPDH

P-Smad3 protein Smad7 protein

1.5
Ratio (P-Smad3/GAPDH)

** # #

Ratio (Smad7/GAPDH)
© 1.4 ## ##
* ** *** ### ** **
1.0 **
0.7
0.5

0 0
DMSO DMSO AA Nar CB DMSO DMSO AA Nar CB

UUO UUO
Combined AA & Nar treatment produces a better inhibitory effect on
renal fibrosis in mouse model of UUO

Sham UUO UUO+AA UUO+Nar UUO+CB

α-SMA
Collagen I

α-SMA mRNA
Ratio (Col.I/GAPDH)
0.025 Col.I mRNA
Ratio (αSMA /GAPDH)

0.0015
0.020 ***
# ***
*** 0.0010
0.015
*** © # # ©
0.010 ### ###
0.0005
0.005

0 0
Sham DMSO AA Nar CB ShamDMSO AA Nar CB
UUO UUO
Rebalancing SMAD signaling by Smad3 inhibitor and Smad7
agonist may be a new and specific treatment for renal fibrosis

AGEs TGF-β1 IL-1β TNFα

Ang II
TβRII TβRI
AT1/2 RAGE

p p p

Smad3

Smad2
p p Asiatic acid
p65
p 积雪草
IκBα

Smad7
Naringenin
Smad4

柚皮素
Sma
d2
Smad4

p
p
Sma p50
d3

Fibrosis Inflammation
 Supported by
•  973 Program，
• NSFC Major Grant
• RGC CRF Grants
Laboratory of Inflammatory Disease • FIS CUHK