Salutary Effect of Kallistatin in Salt-Induced Renal Injury,
Inflammation, and Fibrosis via Antioxidative Stress
Bo Shen, Makoto Hagiwara, Yu-Yu Yao, Lee Chao, Julie Chao
Downloaded from http://hyper.ahajournals.org/ by guest on June 18, 2017
Abstract—An inverse relationship exists between kallistatin levels and salt-induced oxidative stress in Dahl-salt sensitive
rats. We further investigated the role of kallistatin in inhibiting inflammation and fibrosis through antioxidative stress
in Dahl-salt sensitive rats and cultured renal cells. High-salt intake in Dahl-salt sensitive rats induced elevation of
thiobarbituric acid reactive substances (an indicator of lipid peroxidation), malondialdehyde levels, reduced
nicotinamide-adenine dinucleotide phosphate oxidase activity, and superoxide formation, whereas kallistatin gene
delivery significantly reduced these oxidative stress parameters. Kallistatin treatment improved renal function and
reduced kidney damage as evidenced by diminished proteinuria and serum urea nitrogen levels, glomerular sclerosis,
tubular damage, and protein cast formation. Kallistatin significantly decreased interstitial monocyte-macrophage
infiltration and the expression of tumor necrosis factor-␣, intercellular adhesion molecule-1, and vascular cell adhesion
molecule-1. Kallistain also reduced collagen fraction volume and the deposition and expression of collagen types I and
III. Renal protection by kallistatin was associated with increased NO levels and endothelial NO synthase expression and
decreased p38 mitogen-activated protein kinase, extracellular signal-regulated kinase phosphorylation, and transforming
growth factor-␤1 expression. Moreover, kallistatin attenuated tumor necrosis factor-␣–induced intercellular adhesion
molecule-1 and vascular cell adhesion molecule-1 expression via inhibition of reactive oxygen species formation and
p38 mitogen-activated protein kinase and nuclear factor-␬B activation in cultured proximal tubular cells. Kallistatin
inhibited fibronectin and collagen expression by suppressing angiotensin II–induced reactive oxygen species generation
and transforming growth factor-␤1 expression in cultured mesangial cells. These combined findings reveal that
kallistatin is a novel antioxidant, which prevents salt-induced kidney injury, inflammation, and fibrosis by
inhibiting reactive oxygen species–induced proinflammatory cytokine and transforming growth factor-␤1
expression. (Hypertension. 2008;51:1358-1365.)
Key Words: Dahl salt-sensitive rat 䡲 kallistatin 䡲 reactive oxygen species 䡲 inflammation 䡲 fibrosis
K
allistatin is a plasma protein that belongs to the serine
protease inhibitor family.1,2 Although most serine protease inhibitors are synthesized primarily in the liver, kallistatin is widely expressed in organs such as the kidney, heart,
and blood vessel.3– 6 Kallistatin is a negative acute-phase
protein, because its expression in the liver is rapidly reduced
after lipopolysaccharide-induced inflammation.7 Conversely,
transgenic mice overexpressing human kallistatin are resistant to lipopolysaccharide-induced mortality.8 Local delivery
of the kallistatin gene inhibited inflammatory responses and
reduced joint swelling in a rat model of arthritis.9 Furthermore, our recent study showed that kallistatin gene transfer
protected against acute myocardial ischemia-reperfusion injury by inhibition of cardiomyocyte apoptosis and inflammatory cell recruitment.10 Whether kallistatin plays a protective
role against renal injury has not been investigated.
A strong correlation has been observed between oxidative
stress and immune cell infiltration in salt-sensitive hyperten-
sion. In fact, oxidative stress is considered to be a major
contributing factor in the development of renal injury, because it can stimulate the expression of proinflammatory and
profibrotic molecules.11 Inflammation is crucial to the subsequent development of fibrosis, the final contributing factor to
kidney failure. Dahl salt-sensitive (DSS) rats develop progressive and sclerotic renal lesions after salt loading, making
them a popular model of human salt-sensitive hypertension.12,13 High-salt loading in DSS rats increases inflammatory cell infiltration, glomerular enlargement, and extracellular matrix protein accumulation in association with increased
oxidative stress in the kidney.14 In this study, we investigated
the mechanisms of kallistatin in inflammatory cell accumulation and renal fibrosis during the progression of renal injury
in DSS rats after salt loading, as well as in cultured renal
proximal tubular and mesangial cells. Our data demonstrate
that kallistatin has a novel role as an antioxidant in the
protection against renal injury by inhibiting salt-induced
Received December 7, 2007; first decision December 27, 2007; revision accepted February 26, 2008.
From the Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston.
Correspondence to Julie Chao, Department of Biochemistry and Molecular Biology, Medical University of South Carolina, 173 Ashley Ave,
Charleston, SC 29425. E-mail [email protected]
© 2008 American Heart Association, Inc.
Hypertension is available at http://hypertension.ahajournals.org
DOI: 10.1161/HYPERTENSIONAHA.107.108514
1358
Shen et al
Reduction of Oxidative Stress by Kallistatin
1359
Table 1. Effect of Kallistatin Gene Delivery on Renal Damage, Blood Pressure, and TBARS Levels in DSS
Rats After Salt Loading
Parameters
NS, Mean⫾SEM
KW/BW, g/100 g of BW
BP, mm Hg
0.59⫾0.02*
138.7⫾3.5*
HS⫹Ad.Null, Mean⫾SEM
HS⫹Ad.HKS, Mean⫾SEM
0.93⫾0.04
0.80⫾0.02*†
217.8⫾1.9
198.2⫾2.6*†
TBARS, ␮mol/L
1.77⫾0.01*
Urinary protein, mg/d/100 g of BW
66.3⫾4.4*
Serum urea nitrogen, mg/mL
0.55⫾0.03*
0.80⫾0.10
Creatinine clearance, mL/min
1.15⫾0.04*
0.51⫾0.09
1.07⫾0.09*†
0*
2.84⫾0.44
1.49⫾0.15*†
0.33⫾0.10*
2.42⫾0.28
1.8⫾0.12*†
0*
3.29⫾0.18
2.33⫾0.21*†
Glomerular sclerotic score
Arterio-arteriolar sclerotic score
Tubulointerstitial injury score
2.70⫾0.21
2.03⫾0.11*
155.8⫾8.2
125.3⫾7.8*†
0.57⫾0.06*
NS indicates normal salt; HS, high salt; BW, body weight; KW, kidney weight; BP, blood pressure. n⫽7.
*P⬍0.05 vs HS⫹Ad.Null; †P⬍0.05 vs NS.
Downloaded from http://hyper.ahajournals.org/ by guest on June 18, 2017
inflammatory cell recruitment and renal fibrosis in vivo and
in cultured renal cells.
parameters were considered significantly different at a value of
P⬍0.05.
Methods
Results
For an expanded Methods section, please see http://hyper.ahajournals.org.
Preparation of Replication-Deficient Adenoviral
Vectors Containing Human Kallistatin and
Purification of Recombinant Kallistatin
Adenoviral vectors carrying the human kallistatin cDNA under the
control of the cytomegalovirus enhancer-promoter (Ad.HKS) or the
adenoviral vector alone (Ad.Null) were constructed and prepared as
described previously.10 Expression and purification of recombinant
human kallistatin were performed as described previously.15
Animal Treatments
All of the procedures complied with the standards for care and use of
animal subjects as stated in the Guide for the Care and Use of
Laboratory Animals (Institute of Laboratory Resources, National
Academy of Sciences). Four-week– old, male DSS rats (SpragueDawley Harlan) were used. Rats were fed either a normal-salt (0.4%
NaCl) or high-salt (4% NaCl) diet for 2 weeks, and those on the
high-salt diet were injected IV via the tail vein with either Ad.Null
or Ad.HKS, both at a dose of 5⫻109 plaque forming units per rat.
Four weeks after gene delivery or 6 weeks after high-salt diet,
animals were euthanized, and kidneys were examined for histological and biochemical analyses.
Cell Culture and Detection of Reactive Oxygen
Species Formation
Immortalized rat proximal tubular cells16 were incubated with tumor
necrosis factor (TNF; 10 ng/mL) in the absence or presence of
kallistatin (0.2 to 0.4 ␮mol/L) or SB202190 (a p38 mitogen-activated protein kinase [p38MAPK] inhibitor; 5 ␮mol/L) for 15
minutes for Western blot analysis or 24 hours for quantitative
RT-PCR. Rat glomerular mesangial cells were incubated with
angiotensin II (50 nM) or transforming growth factor (TGF-␤1; 5
ng/mL) in the absence or presence of kallistatin (0.2 and 0.4 ␮mol/L)
or apocynin (a reduced nicotinamide-adenine dinucleotide phosphate
[NADPH] oxidase inhibitor; 500 ␮mol/L) for 24 hours. The medium
was collected for TGF-␤1 ELISA. Intracellular production of reactive oxygen species (ROS) was measured by using 2⬘,7⬘dichlorofluorescein diacetate (Molecular Probes).17
Statistical Analysis
Data were analyzed using standard statistical methods, ANOVA, and
unpaired Student t test, followed by the Bonferroni posthoc test.
Group data were expressed as means⫾SEMs. Values of all of the
Inverse Relationship Between Kallistatin Levels
and Oxidative Stress
Serum concentrations of thiobarbituric acid reactive substances (TBARS) are an index of lipid peroxidation, which is
caused by oxygen-free radical interaction with the polyunsaturated fatty acids of cell membranes. Significant elevation of
circulating TBARS levels was observed in DSS rats fed a high-salt
diet for 6 weeks (2.70⫾0.21 versus 1.77⫾0.01 ␮mol/L; n⫽6 to 8;
P⬍0.01) with a concomitant decrease in serum kallistatin
levels (103.6⫾6.0 versus 167.5⫾4.2 ␮g/mL; n⫽6 to 8;
P⬍0.01). Kallistatin levels in the kidney were also reduced in
DSS rats after a high-salt diet compared with a normal diet
(149.3⫾8.2 versus 272.4⫾5.3 ng/mg of protein; n⫽6 to 8;
P⬍0.01). These results indicate that oxidative stress is
inversely correlated with kallistatin levels under pathological
conditions.
Renal Kallistatin Expression After Gene Delivery
Improves Renal Function
Immunoreactive human kallistatin was expressed in glomerular and tubular cells after kallistatin gene delivery. No
specific staining was found in the kidney with control
adenovirus injection (data not shown). Human kallistatin
levels in renal extracts after gene delivery were 82.9⫾18.1
ng/mg of protein (n⫽3) but were not detectable in the rats
receiving Ad.Null. Kallistatin gene delivery significantly
reduced circulating TBARS levels and improved renal function (Table 1). High-salt loading for 6 weeks resulted in a
dramatic increase in urinary protein excretion, and kallistatin
administration significantly decreased urinary protein levels.
Moreover, kallistatin treatment completely reversed saltinduced elevation of serum urea nitrogen levels and creatinine clearance. Blood pressure markedly increased after salt
loading, and kallistatin administration resulted in a slight but
significant reduction of blood pressure (Table 1). However,
blood pressure was still significantly higher in the kallistatin
group compared with DSS rats on a normal-salt diet. DSS rats
Hypertension
May 2008
A
B
ED-1
Monocytes/Macrophages (/mm 2)
1360
HS+Ad.Null
NS
HS+Ad.HKS
350
*P<0.05 vs. HS+Ad.Null; # P<0.05 vs. NS.
300
*#
250
200
150
*
100
50
0
NS
NS
HS+Ad.Null HS+Ad.HKS
NS
D
HS+Ad.Null HS+Ad.HKS
E
NS
HS+Ad.Null HS+Ad.HKS
GAPDH
3.0
* P<0.05 vs. HS+Ad.Null; #P<0.05 vs. NS.
GAPDH
4
* P<0.05 vs. HS+Ad.Null
GAPDH
5
* P<0.01 vs. HS+Ad.Null
1.5
*#
*
1.0
0.5
0
NS
HS+Ad.Null
HS+Ad.HKS
*
2
*
1
0
NS
HS+Ad.Null
HS+Ad.HKS
4
3
2
*
2.0
3
Relative VCAM-1 mRNA Levels
VCAM-1
Relative ICAM-1 mRNA Levels
TNF-α
ICAM-1
2.5
HS+Ad.HKS
*
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Relative TNF-α mRNA Levels
C
HS+Ad.Null
1
0
NS
HS+Ad.Null HS+Ad.HKS
Figure 1. Kallistatin prevents salt-induced inflammatory cell infiltration. A, Representative images of immunohistochemical staining for
ED-1, a specific marker for monocytes-macrophages, in the cortex. Original magnification is ⫻200. B, Quantification of monocytesmacrophages in the renal cortex. Representative Western blot and real-time PCR analysis of the proinflammatory mediators (C) TNF-␣,
(D) ICAM-1, and (E) VCAM-1. Values are expressed as means⫾SEMs; n⫽7.
on a normal-salt diet were normotensive throughout the
experimental period.
Kallistatin Reduces Salt-Induced Renal Injury and
Inflammatory Response
The morphology of renal injury induced by high-salt intake
was evaluated by Periodic acid-Schiff staining. Kidneys of
DSS rats on a normal-salt diet had normal structure. However, DSS rats on a high-salt diet for 6 weeks exhibited
tubular dilatation, glomerular sclerosis, and extensive protein
cast formation. Kallistatin gene transfer in DSS rats attenuated tubular damage and also resulted in fewer protein casts
and sclerotic glomeruli compared with rats in the Ad.Null
group. Quantitative analysis indicated that kallistatin significantly reduced glomerular sclerotic, arterio-arteriolar sclerotic, and tubulointerstitial injury scores (Table 1). Accumulation of monocytes/macrophages was determined by
immunohistochemistry staining against ED-1. DSS rats fed a
normal-salt diet had a small number of ED-1–positive cells.
In contrast, significant accumulation of monocytes/macrophages was observed in DSS rats a high-salt diet for 6 weeks
(Figure 1A). Kallistatin gene delivery significantly inhibited
salt-induced monocytes/macrophages accumulation in the
kidney (Figure 1A and 1B). Kallistatin significantly reduced
salt-induced protein and gene expression of TNF-␣, intercellular adhesion molecule (ICAM-1), and vascular cell adhesion molecule (VCAM-1; Figure 1C through 1E).
Kallistatin Attenuates Salt-Induced Renal Fibrosis
and Collagen Expression
Kidney sections were stained with Sirius red for the determination of total collagen in DSS rats (Figure 2A). Rats fed a
normal-salt diet exhibited a small amount of collagen in the
interstitial space and glomeruli. Although high-salt loading
increased the accumulation of collagen in the interstitium and in
glomeruli, kallistatin gene transfer attenuated collagen deposition (Figure 2B). Immunohistochemical staining of collagen
types I and III indicated that kallistatin gene transfer reduced
salt-induced collagen expression in the interstitial space and
periglomeruli (Figure 2A). Furthermore, kallistatin significantly
inhibited collagen types I and III mRNA levels in the kidney
compared with the high-salt group (Figure 2C and 2D).
Kallistatin Restores Renal Endothelial NO
Synthase Expression and Nitrogen Oxide Levels
and Reduces Oxidative Stress
Kallistatin gene transfer significantly increased urinary nitrogen oxide levels and reduced oxidative stress compared with
DSS rats on a high-salt diet (Table 2). Kallistatin administration also significantly restored endothelial NO synthase
expression in high-salt–loaded DSS rats (see the online data
supplement). Renal MDA levels were increased after the
high-salt diet compared with the normal diet but were
lowered by kallistatin gene delivery. Moreover, a high-salt
diet induced a significant upregulation of the expression of
NADPH oxidase subunit-p47phox expression (see the online
Shen et al
NS
A
Reduction of Oxidative Stress by Kallistatin
HS+Ad.Nul l
1361
HS+Ad.HK S
Sirius Red
Collagen I
Collagen III
10
0
NS
HS+Ad.Null HS+Ad.HKS
D
5
* P<0.05 vs. HS+Ad.null
4
3
*
2
1
*
0
NS
HS+Ad.Null HS+Ad.HKS
Relative Collagen III mRNA Levels
20
Relative Collagen I mRNA Levels
* P<0.01 vs. HS+Ad.Null
*
Downloaded from http://hyper.ahajournals.org/ by guest on June 18, 2017
Collagen Fraction Volume (% )
C
30
*
B
5
* P<0.05 vs. HS+Ad.Null
4
3
*
2
1
*
0
NS
HS+Ad.Null
HS+Ad.HKS
Figure 2. Kallistatin prevents salt-induced renal fibrosis. A, Representative images of histological staining with Sirius red and immunohistochemical staining for collagen types I and III. Original magnification is ⫻200. B, Quantification of collagen fraction volume. Sirius
red stains collagen fibers red and cytoplasm gray. Real-time PCR analysis of mRNA levels for collagen (C) type I and (D) type III. Values
are expressed as means⫾SEMs; n⫽7.
data supplement) and elevated NADPH oxidase activity
compared with DSS rats on a normal diet. Kallistatin gene
delivery significantly reversed these increases. Superoxide
formation paralleled NADPH oxidase activity. Superoxide
levels were elevated in the Ad.Null group above those in the
normal-salt group. Kallistatin gene transfer significantly lowered salt-induced superoxide formation. These results indicate that kallistatin increases NO production and suppresses
oxidative stress in salt-loaded DSS rats.
ically elevated in the high-salt group compared with the
normal-salt group and were diminished in DSS rats injected
with Ad.HKS (Figure 3B). Similarly, real-time PCR showed
that kallistatin reduced salt-induced TGF-␤1 mRNA levels
(Figure 3C).
Kallistatin Inhibits p38MAPK and Extracellular
Signal-Regulated Kinase Activation and
TGF-␤1 Expression
Kallistatin significantly inhibited TNF-␣–induced ROS formation in cultured proximal tubular cells in situ as detected by
elevated intensity of 2⬘,7⬘-dichlorofluorescein diacetate fluorescence (Figure 4A and 4B). Moreover, kallistatin reduced
p38MAPK and I␬B␣ phosphorylation in a dose-dependent
manner (Figure 4C). Inhibition of p38MAPK activation by
kallistatin led to inhibition of TNF-␣–induced ICAM-1 and
Western blot analysis showed that kallistatin gene delivery
markedly reduced salt-induced phosphorylation of p38MAPK
and extracellular signal-regulated kinase (Figure 3A). Renal
TGF-␤1 protein levels, determined by ELISA, were dramatTable 2.
Kallistatin Inhibits TNF-␣–Induced ROS
Formation, p38MAPK, and I␬B␣ Phosphorylation
and ICAM-1 and VCAM-1 Expression in Proximal
Tubular Cells
Effect of Kallistatin Gene Delivery on Urinary NO Content and Oxidative Stress
Parameters
NS, Mean⫾SEM
HS⫹Ad.Null, Mean⫾SEM
HS⫹Ad.HKS, Mean⫾SEM
Urinary nitrogen oxide levels, ␮mol/d/100 g of BW
1.65⫾0.03*
0.86⫾0.13
1.38⫾0.03*
Renal MDA levels, ␮mol/mg of protein
0.35⫾0.04*
0.69⫾0.03
0.55⫾0.04*†
NADPH oxidase activity, rlu/min/mg of protein
0.27⫾0.02*
0.37⫾0.03
0.30⫾0.01*
Superoxide formation, nmol/min/mg of protein
0.30⫾0.07*
0.99⫾0.13
0.40⫾0.07*
NS indicates normal salt; HS, high salt; MDA, malondialdehyde; rlu, relative light units. n⫽7.
*P⬍0.05 vs HS⫹Ad.Null; †P⬍0.05 vs NS.
1362
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A
May 2008
NS
HS+Ad.Null
HS+Ad.HKS
P-p38MAPK
p38MAPK
P-ERK
Figure 3. Effect of kallistatin gene delivery on p38MAPK and extracellular
signal-regulated kinase (ERK) activation
and TGF-␤1 expression. A, Representative Western blots of phosphorylated
and total p38MAPK and ERK. B, ELISA
for renal TGF-␤1 levels. C, Real-time
PCR analysis of mRNA levels for renal
TGF-␤1. Values are expressed as
means⫾SEMs; n⫽7.
ERK
C
700
* P<0.05 vs. HS+Ad.Null; #P<0.05 vs. NS.
600
#
500
*#
400
300
200
100
0
Relative TGF-β mRNA Levels
HS+Ad.Null HS+Ad.HKS
NS
2.5
* P <0.05 vs. HS+Ad.Null
#P <0.07 vs. HS+Ad.Null
2.0
#
1.5
*
1.0
0.5
0
NS
HS+Ad.Null HS+Ad.HKS
VCAM-1 expression. TNF-␣–induced proinflammatory mediator expression was mediated by p38MAPK activation, as the
effect of TNF-␣ was blocked by SB202190, a p38MAPK
inhibitor (see the online data supplement). Taken together,
kallistatin, through inhibition of ROS formation, attenuated
TNF-␣–induced inflammatory cytokine expression by suppressing p38MAPK and nuclear factor ␬B (NF-␬B) activation.
ment with kallistatin or apocynin, an NADPH oxidase inhibitor, significantly blocked Ang II–induced ROS production
(Figure 5A and 5B). Kallistatin and apocynin also abrogated
Ang II–stimulated TGF-␤1 protein and mRNA levels (Figure
5C and 5D). Furthermore, kallistatin abolished TGF-␤1–
induced fibronectin and collagen I expression in mesangial
cells (see the online data supplement). These results indicate
that kallistatin inhibited TGF-␤1 expression through suppression of NADPH oxidase activity and ROS formation.
Kallistatin Inhibits Angiotensin II–Induced ROS
Formation, TGF-␤1, Fibronectin, and Collagen
Expression in Mesangial Cells
Discussion
Angiotensin (Ang) II treatment significantly increased ROS
formation in cultured mesangial cells (Figure 5A). PretreatA
This is the first study to demonstrate that kallistatin levels are
inversely correlated with oxidative stress and that kallistatin
B
TNF-α
Control
DCF Fluorescence (fold of Control)
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TGF-β
β Levels (pg/mg protein)
B
15
* P<0.05 vs. TNF- α group
12
TNF-α +HKS(0.2 µM)
TNF-α +HKS(0.4 µM)
9
6
*
3
*
0
Control
*
TNF- α
HKS
(0.2µM)
HKS
(0.4µM)
TNF-α
C
Control
P-p38 MAPK
p38 MAPK
P-I κ B α
I κ Bα
TNF-α
TNF-α+
TNF-α+
HKS (0.2µM) HKS (0.4µM)
Figure 4. Effect of kallistatin on TNF-␣–
induced ROS formation and ICAM-1 and
VCAM-1 expression in cultured proximal
tubular cells. A, Representative images
of ROS-induced 2⬘,7⬘-dichlorofluorescein
fluorescence in proximal tubular cells. B,
Quantification of ROS production. Values
are expressed as means⫾SEMs; n⫽8.
C, Representative Western blots of phosphorylated and total p38MAPK and
I␬B␣.
Shen et al
DCF Fluorescence (fold of Control)
Ang II +Apocynin
3.0
* P<0.05 vs. Ang II group
2.5
2.0
*
Ang II +HKS
1.5
*
1.0
0.5
0
Control Ang II
HKS
Apocynin
Ang II
β1 Levels in Culture Medium (pg/ml)
TGF-β
800
*
*
400
0
Control
3
2
1
0
Ang II
HKS Apocynin
Ang II
*P<0.05 vs. Ang II group
*
*
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1200
4
Figure 5. Effect of kallistatin on ROS
generation and TGF-␤1 expression in
cultured rat mesangial cells. A, Representative images of ROS-induced 2⬘,7⬘dichlorofluorescein fluorescence in
cultured rat mesangial cells. B, Quantification of ROS production. Values as
expressed as means⫾SEMs; n⫽8. C,
TGF-␤1 levels in the culture medium
were measured by ELISA. The results
were normalized by total protein concentration in the medium. D, TGF-␤1 levels
were measured by real-time PCR and
normalized with GAPDH. Values are
expressed as means⫾SEMs; n⫽3.
*
*P<0.01 vs. Ang II group
Relative TGF- β1 mRNA Levels
D
C
1600
1363
B
Ang II
Control
*
A
Reduction of Oxidative Stress by Kallistatin
*
Control
Ang II
supplementation by gene delivery reduced oxidative stress in
the serum and kidney. The antioxidative activity of kallistatin
inhibited inflammation and fibrosis in DSS rats and on
cultured proximal tubular and mesangial cells. The antiinflammatory effect of kallistatin observed in this study is
consistent with our previous reports that kallistatin gene
transfer reduces inflammatory cell recruitment and TNF-␣
levels in animal models of rat arthritis and myocardial
ischemia-reperfusion.9,10 Our results showed that kallistatin
inhibited proinflammatory cytokine and TGF-␤1 expression
through suppression of NADPH oxidase activity, ROS formation, and, thus, p38MAPK and NF-␬B activation in
cultured renal cells. The results obtained from the in vivo and
cell culture studies provide strong evidence that kallistain, as
a potent antioxidant, protects against salt-induced renal
injury.
High-salt loading in DSS rats induced renal injury and
blood pressure elevation in association with increased circulating ROS levels and reduced NO bioavailability. Kallistatin
treatment protected against salt-induced renal dysfunction, as
evidenced by reduced serum urea nitrogen and urinary
protein levels. Although kallistatin administration partially
lowered salt-induced blood pressure rise, it almost completely
prevented renal injury. Moreover, kallistatin treatment restored endothelial NO synthase-nitrogen oxide levels and
reduced salt-induced superoxide formation. Furthermore, previous studies showed that hypotensive treatment with amlodipine and hydralazine did not prevent glomerulosclerosis
and proteinuria in DSS rats despite the reversal of systemic
high blood pressure to the normal level.18,19 In addition,
treatments with antioxidant or antiinflammatory agents are
HKS
Apocynin
Ang II
capable of reducing arterial pressure, as well as improving
renal function in salt-sensitive hypertension.20,21 Therefore,
we can conclude that the renoprotective effects of kallistatin
are not primarily attributed to its blood pressure–lowering
ability. A modest reduction in blood pressure after kallistatin
treatment may be related to a combination of NO-mediated
vasodilation and amelioration of ROS generation.
Oxidative stress is a major contributing factor in inducing
renal injury, because it can stimulate the expression of
proinflammatory and profibrotic molecules.12 High-salt intake has been shown to increase renal NADPH activity,
urinary H2O2, 8-isoprostane, and thromboxane B2 excretion.22 NO, a potent antioxidant, is capable of inhibiting
neutrophil superoxide anion production via a direct action on
the membrane components of NADPH oxidase and the
assembly of reduced nicotinamide-adenine dinucleotide/
NADPH oxidase subunits.23 In DSS rats fed a high-salt diet,
NO production is impaired because of a significant reduction
in NO synthase activity.24 Consistent with previous findings,
significantly increased serum TBARS levels and renal ROS
formation, as well as decreased endothelial NO synthase
expression, were observed in DSS rats after high-salt loading.
Kallistain administration increased nitrogen oxide levels
partly by restoring endothelial NO synthase expression and
also effectively decreased NADPH oxidase activity and
superoxide production in the kidney. Increased NO formation
and lowered oxidative stress after kallistatin treatment are
crucial in renal protection for suppressing inflammation and
fibrosis.
It is well known that inflammation and oxidative stress are
intricately interrelated. In fact, ROS can trigger an inflamma-
1364
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tory response through activation of the TNF-␣ pathway.25
ROS activates p38MAPK and the transcription factor NF-␬B,
which leads to proinflammatory cytokine release and inflammatory cell accumulation in the kidney.26,27 Sustained inflammatory responses may contribute to the progressive renal
injury.28 Moreover, inflammation of renal cells in culture is
associated with increased oxidative stress.29 We observed that
kallistatin gene delivery effectively blocked high-salt–induced inflammatory cell infiltration into the renal interstitium. The inhibitory effect of kallistatin may be, in part,
because of the reduced expression of the proinflammatory
mediators TNF-␣, ICAM-1, and VCAM-1. Inhibition of
NF-␬B, an upstream signaling molecule of ICAM-1 and
VCAM-1, led to strongly reduced immune cell infiltration in
the interstitial tissues and ameliorated hypertensive-induced
renal damage.30 In agreement with the in vivo study, we
found that recombinant kallistatin inhibited TNF-␣–induced
ROS formation, I␬B␣ degradation (an inhibitor of NF-␬B
activation), and inflammatory cytokine expression in cultured
proximal tubular cells. These results indicate that kallistatin
exerts antiinflammatory effects via suppression of oxidative
stress and NF-␬B activation.
The development of fibrosis, the final contributing factor to
kidney failure, is preceded by oxidative stress and inflammation. Oxidative stress has been shown to promote inflammation and to increase the release of active TGF-␤1 via
activation of mitogen-activated protein kinase pathways.31,32
Activated TGF-␤1 participates in the development of renal
failure in controlling extracellular matrix deposition and
remodeling by stimulating collagen and fibronectin synthesis.33,34 Despite the positive-feedback loop between TGF-␤1
and NO under physiological conditions, the inhibitory effect
of NO on TGF-␤1 production was found to be reduced after
salt intake in DSS rats.35,36 In this study, we showed that
kallistatin administration restored NO levels and significantly
prevented salt-induced TGF-␤1 expression. Recombinant
kallistatin also reduced Ang II–induced ROS generation and
TGF-␤1 secretion and expression in cultured mesangial cells.
Ang II–induced effects were abrogated by apocynin, an
inhibitor of NADPH oxidase. Moreover, kallistatin inhibited
TGF-␤1–induced collagen and fibronectin gene expression in
mesangial cells. These findings indicated that the antifibrotic
effect of kallistatin is mediated by inhibition of oxidative
stress and TGF-␤ expression.
Perspectives
Oxidative stress is a major contributing factor in the development of renal injury by the stimulation of proinflammatory
and profibrotic molecule expression. This study demonstrates
an inverse relationship of kallistatin with oxidative stress
parameters and identifies kallistatin as a novel antioxidant in
preventing salt-induced renal injury, inflammation, and fibrosis in DSS rats, as well as in cultured proximal tubular and
mesangial cells. Inhibition of ROS formation by kallistatin
leads to lower proinflammatory cytokine and profibrotic
mediator expression and, thus, protection against oxidative
kidney damage. Our study reveals that kallistatin, as a potent
antioxidant, may have therapeutic potential for the treatment
of oxidative organ failure.
Sources of Funding
This work was supported by National Institutes of Health grants
HL-44083 and C06 RR015455 from the National Center for Research Resources Extramural Research Facilities Program.
Disclosures
None.
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Salutary Effect of Kallistatin in Salt-Induced Renal Injury, Inflammation, and Fibrosis via
Antioxidative Stress
Bo Shen, Makoto Hagiwara, Yu-Yu Yao, Lee Chao and Julie Chao
Downloaded from http://hyper.ahajournals.org/ by guest on June 18, 2017
Hypertension. 2008;51:1358-1365; originally published online April 7, 2008;
doi: 10.1161/HYPERTENSIONAHA.107.108514
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Salutary Effect of Kallistatin in Salt-Induced Renal Injury, Inflammation
and Fibrosis via Anti-Oxidative Stress
Bo Shen, Makoto Hagiwara, Yu-Yu Yao, Lee Chao, and Julie Chao
Department of Biochemistry and Molecular Biology, Medical University of South Carolina
Running title: Reduction of oxidative stress by kallistatin
Text words: 6250
Abstract words: 248
Figure numbers: 7
Correspondence to: Julie Chao, Ph.D.
Department of Biochemistry and Molecular Biology
Medical University of South Carolina
173 Ashley Avenue, Charleston, SC 29425
Phone: (843) 792-9927
FAX: (843) 792-4850
E-mail: [email protected]
Methods
Blood Pressure Measurement
Systolic blood pressure was measured with DASYlab 5.5 software (Kent Scientific
Corporation) by the tail-cuff method.1 Unanesthetized rats were placed in a plastic
holder resting on a warm pad maintained at 37oC during the measurements. Average
readings were taken for each animal after the animals had become acclimated to the
environment.
Assays for TBARS, Malondialdehyde (MDA), Blood Urea Nitrogen (BUN) and
Urinary Protein Levels
Serum lipid peroxidation, an indicator of oxidative stress, was determined by measuring
TBARS levels at 535 nm using MDA standards (0 to 3 µM) (Sigma).2 MDA levels in
renal extracts were measured in a similar fashion with cytosolic protein from renal
extracts. BUN levels were determined using a modified urease-indophenol method.3
Protein levels in urine were measured by Bio-Rad DC Protein Assay (Bio-Rad
Laboratories).
Morphological and Histological Investigations
Four-μm-thick sections were obtained from each sample. Sections were stained with
periodic acid-Schiff (PAS) for morphometric analysis and with Sirius red to determine
the extent of fibrosis.4,5 Renal glomerulosclerosis, arterio-arteriolar sclerosis, and
tubulointerstitial damage were scored as described previously.6,7 Immunohistochemistry
1 was performed using the Vectastain Universal Elite ABC Kit (Vector Laboratories),
following the supplied instructions. Kidney sections were incubated at 4oC overnight with
antibodies against the monocyte/macrophage marker ED-1 (1:100; Chemicon Inc.),
collagen type I (1:100; Sigma) and collagen type III (1:400; Sigma). Quantification of
monocytes/macrophages was determined by counting ED-1 positive cells in 10 fields at
400x magnification.
Enzyme-Linked Immunosorbent Assay (ELISA)
Human kallistatin levels in the serum were determined using an ELISA specific for
human kallistatin as described previously.8 Renal TGF-β1 levels were determined using
ELISA kit (R & D Systems) and following the supplied instructions.
Western Blot Analysis
Renal tissues were homogenized in lysis buffer containing 1:100 protease inhibitor
cocktail (Sigma) and centrifuged at 15,000 x g at 4oC for 30 min. After centrifugation, the
cytosolic fraction protein concentration was measured Bio-Rad DC Protein Assay kit
(Bio-Rad Laboratories). Western blot analysis of tissue extracts was performed for
p38MAPK, phospho-p38MAPK, ERK and phospho-ERK (Cell Signaling Technology
Inc). Cell culture lysates also underwent western blot for p38MAPK, phosphop38MAPK, IκBα and phospho-IκBα (Cell Signaling Technology Inc).
Measurement of NOx Levels, NADPH Oxidase Activity and Superoxide Formation
2 Nitric oxide levels in urine samples collected at 3 weeks after gene delivery were
measured by a fluorometric assay for nitrite/nitrate.9 NADPH oxidase activity in the
renal extracts was measured in the presence of NADPH substrate (100 μM) and
lucigenin (75 μM) by chemiluminescence assay.10 Light emission levels were expressed
as relative light units (rlu) per min per mg of protein. Superoxide production was
measured using a ferricytochrome c reduction assay according to a modified previous
protocol.11
Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR)
Total RNA was extracted from kidney and cultured cells using Trizol reagent
(Invitrogen). cDNA was transcribed from 2 μg of RNA using a high cDNA archive kit
(Applied Biosystems) following the manufacturer’s instructions. The qRT-PCR reaction
was carried out using the Gene Expression Assay and running on 7300 real-time PCR
system (Applied Biosystems, Foster, CA). Quantification was determined by Relative
Quantification Software (Applied Biosystems).
Proximal Tubular Cell Culture
Immortalized rat proximal tubular cells, a generous gift from Dr. Julie Ingelfinger from
Harvard Medical School, were cultured as previously described.12 Cells were cultured in
Dulbecco’s Modified Eagle’s Medium (DMEM) (Invitrogen) at 34°C in a humidified
5% CO2 atmosphere. Cells were cultured in 12-well plates until they achieved 80%
confluence, then the culture medium was replaced with serum-free DMEM for 16 hour.
Cells were then incubated with TNF-α (10 ng/ml) in the absence or presence of
3 kallistatin (0.2-0.4 µM) or SB202190 (a p38MAPK inhibitor, 5 μM) for 15 min for
western blot analysis or 24 hour for qRT-PCR.
Glomerular Mesangial Cell Culture
Rat glomerular mesangial cells were purchased from American Type Culture Collection
(ATCC). The cells were cultured in DMEM with 0.4 mg/ml G418 at 37°C in a
humidified 5% CO2 atmosphere. Cells were cultured in 12-well plates until they
achieved 80% confluence, after which the culture medium was replaced with serum-free
DMEM for 16 hour. Cells were then incubated with angiotensin (Ang) II (50 nM) or
TGF-β1 (5 ng/ml) in the absence or presence of kallistatin (0.2 and 0.4 µM) or apocynin
(a NAPDH oxidase inhibitor, 500 μM) for 24 hour. The medium was then collected and
centrifuged, and the supernatant was frozen at -80°C for subsequent assay of TGF-β1.
qRT-PCR was performed to determine TGF-β1, fibronectin and collagen expression.
Detection of Reactive Oxygen Species (ROS) Formation
Intracellular production of ROS was measured by using 2',7'-dichlorofluorescein
diacetate (DCF-DA; Molecular Probes).13 Rat renal cells were grown in 6 well plates.
When the cells reached 80% confluence, they were incubated for 30 min with 50 pM
DCF-DA diluted in DMEM medium. The cells were then pretreated with human
kallistatin or apocynin (500 µM) for 30 min before addition of Ang II for 1 hour or
TNF-α for 15 min. After incubation the cells were washed twice with PBS and imaged
using fluorescence microscope. To measure ROS levels, the cells were seeded to a 96well plate, and were treated as described above. Relative fluorescence was measured
4 using a fluorescence plate reader Victor3TM (Perkin Elmer Life Science) at excitation
and emission wavelengths of 485 and 528 nm, respectively.
Reference:
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293:F1889-F1897.
6 Figures:
Figure S1: Effect of kallistatin gene delivery on renal eNOS and NADPH subunit
p47phox expression. (A) Real-time PCR analysis of mRNA levels for renal eNOS. (B)
Real-time PCR analysis of mRNA levels for renal p47phox. Values are expressed as
mean ± SEM, n=7.
7 Figure S2: Effect of kallistatin on renal cells. Kallistatin treatment inhibited TNF-αinduced ICAM-1 and VCAM-1 expression in cultured proximal tubular cells.
(A)
ICAM-1 and (B) VCAM-1 levels were measured by real-time PCR and normalized with
GAPDH. SB: SB 202190, a p38MAPK inhibitor. Kallistatin treatment inhibited TGF-β1
induced fibronectin and collage I expression in cultured rat mesangial cells. (C)
fibronectin and (D) collagen I levels were measured by real-time PCR and normalized
with GAPDH. Values are expressed as means ± SEM, n=3.
8