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Endothelial Krüppel-Like Factor 4 Mediates the

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Protective Effect of Statins against Ischemic AKI

Tadashi Yoshida,*† Maho Yamashita,* Mieko Iwai,* and Matsuhiko Hayashi*†
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*Apheresis and Dialysis Center, School of Medicine, Keio University, Tokyo, Japan; and †Department of General
Medicine, School of Medicine, Keio University, Tokyo, Japan

ABSTRACT
Endothelial cells participate in the pathophysiology of ischemic AKI by increasing the expression of cell
adhesion molecules and by recruiting inflammatory cells. We previously showed that endothelial Krüppel-
like factor 4 (Klf4) regulates vascular cell adhesion molecule 1 (Vcam1) expression and neointimal formation
after carotid injury. In this study, we determined whether endothelial Klf4 is involved in ischemic AKI using
endothelial Klf4 conditional knockout (Klf4 cKO) mice generated by breeding Tek-Cre mice and Klf4 floxed
mice. Klf4 cKO mice were phenotypically normal before surgery. However, after renal ischemia-reperfusion
injury, Klf4 cKO mice exhibited elevated serum levels of urea nitrogen and creatinine and aggravated renal
histology compared with those of Klf4 floxed controls. Moreover, Klf4 cKO mice exhibited enhanced
accumulation of neutrophils and lymphocytes and elevated expression of cell adhesion molecules, including
Vcam1 and Icam1, in injured kidneys. Notably, statins ameliorated renal ischemia-reperfusion injury in
control mice but not in Klf4 cKO mice. Mechanistic analyses in cultured endothelial cells revealed that
statins increased KLF4 expression and that KLF4 mediated the suppressive effect of statins on TNF-
a–induced VCAM1 expression by reducing NF-kB binding to the VCAM1 promoter. These results provide
evidence that endothelial Klf4 is renoprotective and mediates statin-induced protection against ischemic
AKI by regulating the expression of cell adhesion molecules and concomitant recruitment of inflammatory
cells.

J Am Soc Nephrol 27: 1379–1388, 2016. doi: 10.1681/ASN.2015040460

AKI after ischemia-reperfusion (I/R) is a major
cause of morbidity and mortality in hospitalized
patients.1,2 It frequently occurs in patients in inten-
sive care units suffering from sepsis or after major
surgical interventions. To date, therapeutic ap-
proaches to prevent and/or treat ischemic AKI are
extremely limited. Identification of molecular fac-
tors and mechanisms that help ameliorate ischemic
AKI is, therefore, of considerable interest.
The pathophysiology of ischemic AKI is very
complex and involves multiple cell types, including
tubular epithelial cells, vascular endothelial cells
(ECs), neutrophils, and macrophages.1,2 In partic-
ular, I/R-induced damage in ECs is the key event
leading to increased vascular permeability, en-
hanced endothelium-leukocyte interaction with
concomitant inflammatory cell infiltration, abnor-
to I/R, ECs increase the expression of cell adhesion
molecules, such as vascular cell adhesion molecule
1 (Vcam1) and Icam1, which recruit and activate
circulating inflammatory cells in the kidneys.3–7
The results of previous studies have demonstrated
that administration of blocking antibodies against
Vcam1 or Icam1 ameliorated renal I/R injury in
rodents.3–5 Likewise, antisense oligonucleotides
for Icam1 attenuated ischemic AKI in rats. 6
Received April 29, 2015. Accepted August 2, 2015.
Published online ahead of print. Publication date available at
www.jasn.org.
Correspondence: Dr. Tadashi Yoshida, Apheresis and Dialysis
Center, School of Medicine, Keio University, 35 Shinanomachi,
Shinjuku, Tokyo 160-8582, Japan. Email: tayoshida-npr@umin.
ac.jp

mal coagulation, and vasoconstriction. In response Copyright © 2016 by the American Society of Nephrology

J Am Soc Nephrol 27: 1379–1388, 2016 ISSN : 1046-6673/2705-1379 1379

 BASIC RESEARCH www.jasn.org

increased expression of anti-inflammatory

and antithrombotic factors, including en-
dothelial nitric-oxide synthase and throm-
bomodulin, whereas knockdown of KLF4
led to enhancement of TNF-a–induced ex-
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pression of VCAM1 and tissue factor in

cultured human umbilical vein endothelial
cells (HUVECs). Results from recent stud-
ies have also shown that endothelial Klf4
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was protective against atherothrombosis in

Apoe2/2 -background mice.10 Moreover,
we have previously reported that Tek
promoter–dependent Klf4 deletion in
ECs resulted in enhancement of injury-
induced neointimal formation, in part
through overexpression of cell adhesion mol-
ecules and augmented infiltration of inflam-
Figure 1. Generation of Klf4 cKO mice. (A) Schematic representation of endothelial matory cells into injured carotid arteries.11 As
deletion of the Klf4 gene is shown. The numbers shown represent Klf4 exons. The tri-
such, the results of prior studies provide ev-
angles represent the loxP sites. X represents breeding. (B) Recombination of the Klf4loxP
idence that Klf4 is a critical factor regulating
allele was examined in the kidneys of Klf4 cKO mice and control mice, as determined by
PCR. (C) Klf4 expression was examined by immunohistochemistry in the kidneys of Klf4 the development and progression of multi-
cKO mice and control mice. Klf4 expression was visualized by diaminobenzidine, and ple vascular diseases through its effect on
sections were counterstained with hematoxylin. Representative pictures are shown from endothelial inflammation.
six mice analyzed per each genotype. Magnification 320. Bar: 100 mm. Arrowheads On the basis of the results of the pre-
indicate the glomeruli, and arrows indicate the vessels. Inset: Dotted area is enlarged. viously described studies, it is of significant
interest to determine if endothelial Klf4
contributes to ischemic AKI through mod-
ulation of the expression of cell adhesion
molecules. To address this question, we
derived conditional Klf4-deficient mice in
ECs (Klf4 cKO mice) by breeding Tek-Cre
mice 12 with mice carrying a loxP allele
of Klf4 (Klf4 loxP mice) 13 and analyzed
their phenotype after bilateral renal is-
chemia. In addition, statins, 3-hydroxy-
3-methylglutaryl coenzyme A reductase
Figure 2. Endothelial Klf4 deletion exacerbated renal I/R injury. Klf4 cKO mice and inhibitors, have been shown to improve
14,15 and hu-
control mice were subjected to bilateral renal ischemia for 35 minutes (I/R) or sham ischemic AKI in rodents
16,17
operation, and serum levels of urea nitrogen (A) and creatinine (B) were measured mans. For example, pretreatment with
24 hours after reperfusion. n=5–6 per each group. *P,0.05 compared with sham- cerivastatin for 3 days reduced renal I/R
operated mice. #P,0.05 compared with I/R-injured control mice. injury by preventing inflammatory cell in-
filtration and by inhibiting Icam1 induc-
tion.14 Therefore, we also examined whether
Moreover, Kelly et al. showed that Icam1-deficient mice were endothelial Klf4 mediated the protective effect of statins against
7

protected from I/R injury to the kidneys. These results suggest
that regulation of cell adhesion molecules is critical for con-
trolling ischemic AKI. However, the underlying mechanisms
have not yet been fully elucidated.
Krüppel-like factor 4 (Klf4) is a zinc finger transcription
factor involved in a variety of cellular functions, such as dif-
ferentiation, proliferation, and inflammation, by activating or
repressing the transcriptional activity of multiple genes.8 In
vascular ECs, Klf4 is expressed constitutively and has been
shown to have anti-inflammatory and antithrombotic func-
tions.9 Hamik et al.9 showed that overexpression of KLF4
renal I/R injury.
RESULTS
Endothelial Klf4 Deletion Exacerbated Renal I/R Injury
The results of our previous studies showed that compared with
control mice, Klf4 cKO mice, generated by Tek promoter–
dependent deletion of the Klf4 gene (Figure 1A), exhibited no
differences in multiple parameters, including visible appear-
ance, body weight, systolic and diastolic BP, and heart rate.11

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than control mice. Serum concentrations of

creatinine showed a similar trend (Figure
2B); however, the levels between Klf4 cKO
and control mice after I/R injury did not
reach a significant difference. Proteinaceous
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casts, tubular necrosis, and medullary con-

gestion are established histologic features of
ischemic AKI.1,2,18,19 Histologic analyses re-
vealed that Klf4 deletion significantly exacer-
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bated the formation of proteinaceous casts

and tubular necrosis and the degree of med-
ullary congestion in response to I/R injury
(Figure 3). These results suggest that endo-
thelial Klf4 plays a protective role in ischemic
AKI.

Inflammatory Cell Accumulation and

Expression of Cell Adhesion
Molecules Were Enhanced in Klf4
cKO Mice after I/R Injury
Accumulation of inflammatory cells, in-
cluding neutrophils, lymphocytes, mono-
cytes, and macrophages, was examined by
immunohistochemistry. Results showed
that compared with control mice, I/R injury–
induced accumulation of neutrophils was
significantly increased in the kidneys
of Klf4 cKO mice; however, neutrophils
were not observed in the sham-operated
Figure 3. Endothelial Klf4 deletion exacerbated renal histologic damages after I/R injury. kidneys of both mice (Figure 4, A and B).
Klf4 cKO mice and control mice were subjected to bilateral renal ischemia for 35 minutes I/R injury–induced accumulation of lym-
(I/R) or sham operation, and renal histology was examined 24 hours after reperfusion. phocytes was also enhanced in the kidneys
(A and B) Representative hematoxylin-eosin staining for the outer stripe of outer medulla of Klf4 cKO mice compared with control
(OSOM) and the inner stripe of outer medulla (ISOM) are shown. Bars: 200 mm. (C–E) mice (Figure 4, C and D). By contrast,
Levels of the formation of proteinaceous casts (C), tubular necrosis (D), and medullary monocyte/macrophage accumulation was
congestion (E) were scored semiquantitatively. n=5–6 per each group. Magnification 310. modest and was not different between
*P,0.05 compared with sham-operated mice. #P,0.05 compared with I/R-injured control
Klf4 cKO and control mice after renal I/R
mice.
injury (Supplemental Figure 1).
To determine the mechanisms underly-
ing the enhanced recruitment of neutro-
Partial recombination of the Klf4loxP allele in the whole kidney phils and lymphocytes in Klf4 cKO mice, the expression of
was observed in Klf4 cKO mice but not in control mice, reflect- cell adhesion molecules, such as Vcam1 and Icam1, was ex-
ing the fact that subpopulations of renal cells are ECs (Figure amined in the kidneys. Quantitative analysis using real-time
1B). Klf4 expression was detected in vascular and glomerular RT-PCR showed that Vcam1 expression in injured kidneys
ECs in the kidneys, but it was undetectable in Klf4 cKO mice was much higher in Klf4 cKO mice (2.6-fold increase versus
(Figure 1C). These mice were used to determine if endothelial sham-operated kidneys) than in control mice (1.5-fold in-
Klf4 contributed to renal I/R injury. crease versus sham-operated kidneys) (Figure 5A). Likewise,
Male Klf4 cKO and control mice at 12–14 weeks of age injury-induced Icam1 expression in Klf4 cKO mice (3.9-fold
received bilateral I/R injury for 35 minutes. Twenty-four compared with sham-operated mice) was elevated more than
hours after reperfusion, serum levels of urea nitrogen in- that in control mice (1.7-fold compared with sham-operated
creased in both Klf4 cKO (122639 mg/dl) and control (606 mice) (Figure 5B). Moreover, I/R injury–induced expression
13 mg/dl) mice compared with sham-operated mice (3264 of multiple inflammatory markers, including Cxcl1, Cxcl2,
mg/dl in Klf4 cKO mice and 3364 mg/dl in control mice) Tnf, and Il6, was enhanced in the kidneys of Klf4 cKO mice
(Figure 2A). However, of interest, serum levels of urea nitro- compared with control mice; however, Il10 expression was
gen in Klf4 cKO mice after I/R injury were significantly higher unaltered (Figure 5C). These results suggest that endothelial

J Am Soc Nephrol 27: 1379–1388, 2016 Role of Klf4 in Ischemic AKI 1381
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analyses, and expression of cell adhesion

molecules (Figure 6). However, of impor-
tance, fluvastatin did not attenuate renal I/R
injury in Klf4 cKO mice. Similar results were
obtained from Klf4 cKO mice treated with
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simvastatin (data not shown). These results

suggest that the protective effect of statins
against ischemic AKI is mediated by endothelial
Klf4.
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Results of previous studies suggest that

statin protection against ischemic AKI is medi-
ated in part by heme oxygenase 1 (Hmox1)20
and/or Rho-associated coiled-coil containing
protein kinase 1 (Rock1).21 To evaluate the pos-
sible relationship between Klf4 and these
molecules, we determined the expression of
these proteins in Klf4 cKO and control mice.
Results showed that Hmox1 expression was in-
creased by renal I/R injury and that I/R injury–
induced Hmox1 expression was unaffected
by fluvastatin (Figure 7, A and B). The changes
in Hmox1 expression patterns were similar be-
tween Klf4 cKO and control mice. By contrast,
Rock1 expression was unaltered by I/R injury,
fluvastatin treatment, and/or Klf4 deletion
(Figure 7, A and C). These results do not
eliminate the possible involvement of these
proteins in statin-mediated protection
against ischemic AKI, but they do not
Figure 4. Accumulation of inflammatory cells was enhanced in Klf4 cKO mice after suggest a relationship between Hmox1,
renal I/R injury. Klf4 cKO mice and control mice were subjected to bilateral renal Rock1, and Klf4.
ischemia for 35 minutes (I/R) or sham operation, and the accumulation of neutrophils
(A and B) and lymphocytes (C and D) was examined 24 hours after reperfusion. (A and KLF4 Mediated the Suppressive Effect
C) Representative pictures for immunohistochemical staining for neutrophils (A) and
of Statins on TNF-induced VCAM1
lymphocytes (C) are shown. Neutrophils (A) and lymphocytes (C) were visualized by
Expression by Reducing the
diaminobenzidine, and sections were counterstained with hematoxylin. Bars: 100 mm.
Red arrowheads indicate the positive cells. Insets: Dotted areas are enlarged. (B and Association of NF-kB with the VCAM1
D) The numbers of neutrophils (B) and lymphocytes (D) per five random fields in the Promoter in HUVECs
kidneys were quantified. Magnification 310. n=5–6 per each group. *P,0.05 com- Induction of cell adhesion molecules, such as
pared with sham-operated mice. #P,0.05 compared with I/R-injured control mice. VCAM1 and ICAM1, has been shown to be
regulated by the TNF-NF-kB pathway in
ECs.22 In addition, previous studies have
Klf4 deletion increases injury-induced expression of cell ad- shown that TNF-induced expression of cell adhesion mole-
hesion molecules with concomitant infiltration of inflamma- cules was repressed by statins in cultured ECs.23–25 Moreover,
tory cells, thereby exacerbating renal I/R injury in Klf4 cKO we previously showed that KLF4 inhibited TNF-induced ex-
mice. pression of VCAM1 through blocking the binding of NF-kB to
the VCAM1 promoter in cultured ECs.11 To clarify the mo-
Statins Ameliorated Renal I/R Injury in Control Mice but lecular mechanisms whereby statins attenuated renal I/R in-
Not in Klf4 cKO Mice jury in control mice, but not in Klf4 cKO mice, we examined
We examined if Klf4 mediated the protective effect of statins whether KLF4 contributed to the suppressive effect of statins
against renal I/R injury. Fluvastatin was administered orally to on TNF-induced VCAM1 expression in HUVECs.
Klf4 cKO and control mice for 3 days. On the third day, both HUVECs were transfected with siRNA for KLF4 or a
mice were subjected to bilateral renal I/R injury. Consistent scrambled sequence, and then they were treated with TNF
with the results of previous studies,14,15 treatment with fluvastatin and statins, such as fluvastatin and simvastatin. As shown
ameliorated renal I/R injury in control mice, as assessed in Figure 8A, KLF4 expression was increased by statins in
by serum levels of urea nitrogen and creatinine, histologic HUVECs, whereas it was very low in KLF4 siRNA-transfected

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cell adhesion molecules, such as Vcam1

and Icam1, and enhanced accumulation
of neutrophils and lymphocytes in the in-
jured kidneys. We also showed that the pro-
tective effect of statins against ischemic
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AKI was mediated by endothelial Klf4. In-

deed, pretreatment with statins amelio-
rated renal I/R injury in control mice, but
not in Klf4 cKO mice. Mechanistic studies
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demonstrated that statins suppressed

inflammation-related induction of VCAM1
by inducing KLF4, which inhibited the
binding of NF-kB to the VCAM1 promoter
in ECs. As such, results of this study pro-
v ide ev idence that endothelial Klf4
plays a protective role in ischemic AKI by
regulating the expression of cell adhesion
molecules with concomitant recruitment
of inflammatory cells in the kidneys.
Figure 5. Induction of cell adhesion molecules was augmented in Klf4 cKO mice after
Tek promoter–dependent conditional
renal I/R injury. Klf4 cKO mice and control mice were subjected to bilateral renal Klf4 knockout mice were used in this study.
ischemia for 35 minutes (I/R) or sham operation, and the expression of cell adhesion The Tek promoter is active mainly in ECs,
26
molecules was examined 24 hours after reperfusion. Expression of Vcam1 (A) and but also in some hematopoietic cells.
loxP
Icam1 (B) and Cxcl1, Cxcl2, Tnf, Il6, and Il10 (C) in the kidneys was determined by real- Therefore, the recombination of the Klf4
time RT-PCR. n=5–6 per each group. *P,0.05 compared with sham-operated mice. allele occurred in circulating blood cells
and ECs in Klf4 cKO mice. However, the
#
P,0.05 compared with I/R-injured control mice.
aggravated renal I/R injury in Klf4 cKO
mice was most likely caused by the deletion
HUVECs. Treatment with TNF increased VCAM1 expression of the Klf4 gene in ECs for the following reasons. First, the
by 2.5-fold, whereas fluvastatin and simvastatin, respectively, results of previous studies demonstrated that Klf4 is expressed in
attenuated TNF-induced upregulation of VCAM1 expression monocytes/macrophages, but not in neutrophils and lympho-
in HUVECs (Figure 8B). However, under the condition of cytes.27,28 Second, we previously reported that the number of
suppression of KLF4, TNF-induced VCAM1 expression was neutrophils, lymphocytes, and monocytes in the blood did not
increased 4.0-fold and was not affected by either fluvastatin or differ between the Klf4 cKO and control mice.11 In this study we
simvastatin in HUVECs transfected with KLF4 siRNA. Al- showed that the accumulation of neutrophils and lymphocytes
though VCAM1 expression was regulated by statins and was enhanced in the kidneys at 24 hours after I/R injury in Klf4
KLF4, TNF-induced phosphorylation of p65, a major compo- cKO mice compared with that in control mice. However, these
nent of NF-kB, was unaffected by these factors (Figure 8C). inflammatory cells do not express endogenous Klf4 in either
However, chromatin immunoprecipitation assays revealed Klf4 cKO or control mice. By contrast, although Klf4 is ex-
that TNF-induced increase in p65 binding to the VCAM1 pro- pressed in monocytes/macrophages,27,28 the number of infil-
moter was inhibited by fluvastatin and that the suppressive trated monocytes/macrophages in injured kidneys was very
effect of fluvastatin on TNF-induced p65 binding was abol- low and was not different between Klf4 cKO and control mice
ished by knockdown of KLF4 in HUVECs (Figure 8D). Taken at the early time point examined.19 Accordingly, it is reason-
together, these results suggest that statins suppress inflammation- able to conclude that the phenotype of Klf4 cKO mice after
related induction of VCAM1 by inducing the expression renal I/R injury, including the enhanced accumulation of
of KLF4, which inhibits NF-kB binding to the VCAM1 pro- neutrophils and lymphocytes, is caused by the lack of Klf4
moter in ECs. in ECs.
Results of this study showed that I/R injury–induced accu-
mulation of neutrophils and lymphocytes was enhanced in
DISCUSSION Klf4 cKO mice compared with control mice. The accumula-
tion of inflammatory cells is thought to have an important role
In this study, we showed that deletion of the Klf4 gene in ECs in the pathogenesis of ischemic AKI. In support of this, Kelly
exacerbated renal I/R injury in mice, as assessed by biochem- et al.7 showed the administration of antineutrophil serum
ical parameters and renal histology. Aggravated I/R injury in ameliorated renal I/R injury in mice. However, there is an-
Klf4 cKO mice was accompanied by increased expression of other report showing that the same administration did not

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Figure 6. Fluvastatin ameliorated renal I/R injury in control mice, but not in Klf4 cKO mice. Klf4 cKO mice and control mice were
pretreated with fluvastatin (Fluva) orally for 3 days, and then they were subjected to bilateral renal ischemia for 35 minutes (I/R) or sham
operation. Renal damages were evaluated 24 hours after reperfusion. (A and B) Serum levels of urea nitrogen (A) and creatinine (B) were
measured. (C and D) Levels of the formation of proteinaceous casts (C) and tubular necrosis (D) were scored semiquantitatively. (E and
F) Expression of Vcam1 (E) and Icam1 (F) in the kidneys was determined by real-time RT-PCR. n=5–6 per each group. Data for mice that
were not treated with fluvastatin are reproduced from Figures 2, A and B, 3, C and D, and 5, A and B. *P,0.05 compared with sham-
operated mice. #P,0.05 compared with control mice receiving the same treatment. $P,0.05 compared with I/R-injured mice without
fluvastatin treatment.

alter renal I/R injury in rabbits and rats.29 Because the studies
in rabbits and rats did not examine the local accumulation of
neutrophils in injured kidneys,29 it is uncertain whether renal
I/R injury occurred without neutrophil infiltration in rabbits
and rats. Nevertheless, it is possible that the contribution of
neutrophils to ischemic AKI is different between mice, rabbits,
and rats. Differences between species may explain distinct ex-
perimental results.
Statins are widely used and very potent inhibitors of
cholesterol biosynthesis. In addition to lowering lipids, these
drugs exhibit pleiotropic atheroprotective effects that can modify
inflammatory responses, endothelial function, plaque stability,
and thrombus formation. Regarding the modulation of endo-
thelial function, statins have been shown to induce the expression
of endothelial nitric-oxide synthase and thrombomodulin in
HUVECs.30 They have also been shown to inhibit leukocyte

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Figure 7. Hmox1, but not Rock1, was increased in the kidneys after I/R
injury. Klf4 cKO mice and control mice were pretreated with fluvastatin
(Fluva) orally for 3 days, and then they were subjected to bilateral renal
ischemia for 35 minutes (I/R) or sham operation. Expression of Hmox1,
Rock1, and Gapdh (glyceraldehyde 3-phosphate dehydrogenase) was
determined by Western blotting. (A) Representative pictures are
shown. (B and C) Expression of Hmox1, Rock, and Gapdh was de-
termined by densitometry. n=5–6 per each group. *P,0.05 compared
with sham-operated mice.
adhesion via alteration of expression of cell adhesion mole-
cules24; however, the precise molecular mechanisms are not fully
understood. In this study, we showed that the protective effect of
statins against ischemic AKI was mediated by endothelial Klf4
in mice in vivo. We also showed statins induced KLF4 expression
and siRNA-mediated knockdown of KLF4 abolished the sup-
pressive effect of statins on TNF-induced VCAM1 expression in
HUVECs. These results are consistent with those from previous
J Am Soc Nephrol 27: 1379–1388, 2016
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studies showing simvastatin induced KLF4 expression and a shift
to vasoprotective phenotype via the activation of the MEK5/
Erk5 cascade in human primary ECs.31 Moreover, Maejima
et al.32 also showed that pitavastatin induced KLF4 expression,
accompanied by the chromatin structure changes in the promoter-
enhancer region of the KLF4 gene in HUVECs. Further
studies on the mechanism by which statins induce KLF4 expres-
sion may shed light on novel therapeutic targets for treating and
preventing ischemic AKI and other vascular diseases.
Previous studies have shown that KLF4 can bind to p65
and inhibit the inflammation-associated induction of cell
adhesion molecules in ECs.11 Coimmunoprecipitation as-
says demonstrated that KLF4 and p65 physically interacted
with each other. 11,33 In addition, we showed that TNF-
mediated increases in p65 binding to the VCAM1 promoter
were attenuated in the presence of KLF4, as determined by
chromatin immunoprecipitation assays.11 The inhibitory
action of KLF4 against p65 occurred in the nucleus rather
than in the cytoplasm because phosphorylation and nuclear
translocation of p65 were unaffected by KLF4 overexpres-
sion.11 Overall, results thus far suggest that KLF4 inhibits
NF-kB binding to the promoter region of cell adhesion mol-
ecules through the physical association of KLF4 and p65. In
this study, we showed the suppressive effect of statins on
inflammation-induced VCAM1 expression was mediated
by these KLF4-p65 interactions. These mechanisms are sim-
ply protein-protein interactions and do not involve the tran-
scriptional regulation of KLF4 target genes. However, in most
cases, KLF4 elicits its effects through binding to the consensus
KLF4 binding sites within the promoter-enhancer regions of
its target genes.8 It is of interest to determine whether KLF4-
induced transcriptional alterations also indirectly contribute
to these mechanisms. Moreover, because KLF4 also has an
anti-inflammatory role in macrophages,34 it is of interest
to determine if the mechanisms whereby KLF4 regulates
proinflammatory genes are comparable between ECs and
macrophages.
In summary, we provide evidence that Klf4 in ECs plays a
protective role in ischemic AKI by modulating the expression
of cell adhesion molecules and the accumulation of infiltrating
neutrophils and lymphocytes. Although we found that Klf4
mediated the suppressive effect of statins on the inflammation-
related induction of cell adhesion molecules and subsequent
recruitment of inflammatory cells, further studies are needed
to determine if Klf4 is also a mediator of statins for other
nonlipid-lowering effects.
CONCISE METHODS
Generation of Endothelial Klf4-deficient Mice
Animal protocols were approved by Keio University Animal Care and
Use Committee. Klf4loxP/loxP mice13 were bred with Tek-Cre+/2 mice12
to generate Tek-Cre+/2/Klf4loxP/+ mice. Tek-Cre+/2/Klf4loxP/+ mice
were then bred with Klf4loxP/loxP mice to generate Tek-Cre+/2/Klf4loxP/loxP
Role of Klf4 in Ischemic AKI 1385
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Figure 8. KLF4 mediated the suppressive effect of statins on TNF-induced VCAM1 expression by reducing the association of NF-kB with
the VCAM1 promoter in HUVECs. HUVECs were transfected with siRNAs for KLF4 (siKLF4) or a scrambled sequence (siScramble), and then
they were treated with fluvastatin, simvastatin, or TNF for 24 hours. (A and B) Expression of KLF4 (A) and VCAM1 (B) was determined by real-
time RT-PCR. (C) Expression of VCAM1, phosphorylated p65 (p-p65), p65, and GAPDH (glyceraldehyde 3-phosphate dehydrogenase) was examined
by Western blotting. (D) Association of p65 with the VCAM1 promoter was determined by chromatin immunoprecipitation assays. n=4.
*P,0.05 compared with cells without TNF treatment. #P,0.05 compared with cells transfected with siScramble. $P,0.05 compared with
cells untreated with statins.

(Klf4 cKO) mice and Tek-Cre2/2/Klf4loxP/loxP (control) mice. Both mice Ischemic AKI Model
were on the C57BL/6J background, and littermates were used for Male Klf4 cKO and control mice at 12–14 weeks of age were anesthe-
all comparisons. Genotyping was performed by PCR as described tized with an intraperitoneal injection of pentobarbital sodium. Kid-
previously.35 neys were exposed through flank incisions. Mice were subjected to

1386 Journal of the American Society of Nephrology J Am Soc Nephrol 27: 1379–1388, 2016

35 minutes of bilateral renal ischemia or sham surgery. Ischemia was
induced by clamping both renal pedicles with nontraumatic micro-
vessel clamps. The incisions were temporarily closed during ischemia
or sham surgery. After the clamps were removed, reperfusion of the
kidneys was visually confirmed. Some mice were treated with fluvastatin
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(20 mg/kg per d; Wako Pure Chemical, Osaka, Japan) by gavage
for 3 days before surgery. Twenty-four hours after reperfusion, the
mice were euthanized under pentobarbital anesthesia, and blood
samples and kidneys were harvested.
nYQp/IlQrHD3i3D0OdRyi7TvSFl4Cf3VC1y0abggQZXdgGj2MwlZLeI= on 12/20/2023
Histology and Immunohistochemistry
The kidneys were fixed in 4% paraformaldehyde and embedded into
paraffin. The 5-mm sections were prepared and subjected to hematoxylin-
eosin staining and immunohistochemistry. Histologic analyses were
performed, as previously described.18 Immunohistochemistry was
performed with antibodies for Klf4,36 neutrophil (7/4; Abcam, Inc.,
Cambridge, MA), CD3« (M20; Santa Cruz Biotechnology, Santa Cruz,
CA), Ly6c (ER-MP20; Abcam, Inc.), and F4/80 (CI:A3–1; Abcam,
Inc.). Staining was visualized by diaminobenzidine, and sections
were counterstained by hematoxylin.
Cell Culture
HUVECs (Japanese Collection of Research Bioresources, Osaka,
Japan) were cultured in MCDB107 medium supplemented with EC
growth supplement (Sigma-Aldrich, St Louis, MO) and 10% FBS (Life
Technologies, Carlsbad, CA). One day after plating at 20,000 cells/cm2,
KLF4 siRNA37 or scrambled siRNA were transfected into HUVECs
using Lipofectamine RNAiMAX (Life Technologies). On the next day,
HUVECs were treated with 10 ng/ml human TNF (R&D Systems,
Minneapolis, MN), 1 mmol/L fluvastatin, and/or 1 mmol/L simvastatin
(Sigma) for an additional 24 hours.
Real-Time RT-PCR and Western Blotting
Real-time RT-PCR and Western blotting were performed as described
previously.11,35,38,39
Quantitative Chromatin Immunoprecipitation Assays
Quantitative chromatin immunoprecipitation assays were performed
using anti-p65 antibody as described previously.11
Statistical Analyses
Data are presented as mean6SEM. Statistical analyses were done by
SigmaPlot/SigmaStat9 (Systat Software, San Jose, CA). After confirm-
ing that the data passed the normality test for parametric analyses,
two- or three-way factorial ANOVAs were performed with a post hoc
Fisher protected least significant difference test. The P values ,0.05
were considered significant.
ACKNOWLEDGMENTS
This study was supported by a grant-in-aid for scientific research from
the Ministry of Education, Culture, Sports, Science and Technology of
Japan to T.Y. and M.H. and by Bayer Healthcare Research Fund to T.Y.
DISCLOSURES
None.
J Am Soc Nephrol 27: 1379–1388, 2016
www.jasn.org BASIC RESEARCH
REFERENCES
1. Kinsey GR, Li L, Okusa MD: Inflammation in acute kidney injury.
Nephron, Exp Nephrol 109: e102–e107, 2008
2. Molitoris BA: Therapeutic translation in acute kidney injury: The epithelial/
endothelial axis. J Clin Invest 124: 2355–2363, 2014
3. Kelly KJ, Williams WW Jr, Colvin RB, Bonventre JV: Antibody to in-
tercellular adhesion molecule 1 protects the kidney against ischemic
injury. Proc Natl Acad Sci U S A 91: 812–816, 1994
4. Rabb H, Mendiola CC, Saba SR, Dietz JR, Smith CW, Bonventre JV,
Ramirez G: Antibodies to ICAM-1 protect kidneys in severe ischemic
reperfusion injury. Biochem Biophys Res Commun 211: 67–73, 1995
5. Kapitsinou PP, Sano H, Michael M, Kobayashi H, Davidoff O, Bian A,
Yao B, Zhang MZ, Harris RC, Duffy KJ, Erickson-Miller CL, Sutton TA,
Haase VH: Endothelial HIF-2 mediates protection and recovery from
ischemic kidney injury. J Clin Invest 124: 2396–2409, 2014
6. Haller H, Dragun D, Miethke A, Park JK, Weis A, Lippoldt A, Gross V,
Luft FC: Antisense oligonucleotides for ICAM-1 attenuate reperfusion
injury and renal failure in the rat. Kidney Int 50: 473–480, 1996
7. Kelly KJ, Williams WW Jr, Colvin RB, Meehan SM, Springer TA,
Gutiérrez-Ramos JC, Bonventre JV: Intercellular adhesion molecule-1-
deficient mice are protected against ischemic renal injury. J Clin Invest
97: 1056–1063, 1996
8. Yoshida T, Hayashi M: Role of Krüppel-like factor 4 and its binding
proteins in vascular disease. J Atheroscler Thromb 21: 402–413, 2014
9. Hamik A, Lin Z, Kumar A, Balcells M, Sinha S, Katz J, Feinberg MW,
Gerzsten RE, Edelman ER, Jain MK: Kruppel-like factor 4 regulates
endothelial inflammation. J Biol Chem 282: 13769–13779, 2007
10. Zhou G, Hamik A, Nayak L, Tian H, Shi H, Lu Y, Sharma N, Liao X, Hale A,
Boerboom L, Feaver RE, Gao H, Desai A, Schmaier A, Gerson SL, Wang
Y, Atkins GB, Blackman BR, Simon DI, Jain MK: Endothelial Kruppel-like
factor 4 protects against atherothrombosis in mice. J Clin Invest 122:
4727–4731, 2012
11. Yoshida T, Yamashita M, Horimai C, Hayashi M: Deletion of Krüppel-
like factor 4 in endothelial and hematopoietic cells enhances neo-
intimal formation following vascular injury. J Am Heart Assoc 3:
e000622, 2014
12. Kisanuki YY, Hammer RE, Miyazaki J, Williams SC, Richardson JA,
Yanagisawa M: Tie2-Cre transgenic mice: A new model for endothelial
cell-lineage analysis in vivo. Dev Biol 230: 230–242, 2001
13. Katz JP, Perreault N, Goldstein BG, Lee CS, Labosky PA, Yang VW,
Kaestner KH: The zinc-finger transcription factor Klf4 is required for
terminal differentiation of goblet cells in the colon. Development 129:
2619–2628, 2002
14. Gueler F, Rong S, Park JK, Fiebeler A, Menne J, Elger M, Mueller DN,
Hampich F, Dechend R, Kunter U, Luft FC, Haller H: Postischemic acute
renal failure is reduced by short-term statin treatment in a rat model. J
Am Soc Nephrol 13: 2288–2298, 2002
15. Nesic Z, Todorovic Z, Stojanovic R, Basta-Jovanovic G, Radojevic-Skodric S,
Velickovic R, Chatterjee PK, Thiemermann C, Prostran M: Single-dose in-
travenous simvastatin treatment attenuates renal injury in an experimental
model of ischemia-reperfusion in the rat. J Pharmacol Sci 102: 413–417, 2006
16. Welten GM, Chonchol M, Schouten O, Hoeks S, Bax JJ, van Domburg
RT, van Sambeek M, Poldermans D: Statin use is associated with early
recovery of kidney injury after vascular surgery and improved long-term
outcome. Nephrol Dial Transplant 23: 3867–3873, 2008
17. Molnar AO, Coca SG, Devereaux PJ, Jain AK, Kitchlu A, Luo J, Parikh
CR, Paterson JM, Siddiqui N, Wald R, Walsh M, Garg AX: Statin use
associates with a lower incidence of acute kidney injury after major
elective surgery. J Am Soc Nephrol 22: 939–946, 2011
18. Homma K, Yoshida T, Yamashita M, Hayashida K, Hayashi M, Hori S:
Inhalation of hydrogen gas is beneficial for preventing contrast-induced
acute kidney injury in rats. Nephron, Exp Nephrol 128: 116–122, 2014
19. Kato N, Yuzawa Y, Kosugi T, Hobo A, Sato W, Miwa Y, Sakamoto K,
Matsuo S, Kadomatsu K: The E-selectin ligand basigin/CD147 is
Role of Klf4 in Ischemic AKI 1387
 BASIC RESEARCH www.jasn.org
responsible for neutrophil recruitment in renal ischemia/reperfusion. J
Am Soc Nephrol 20: 1565–1576, 2009
20. Gueler F, Park JK, Rong S, Kirsch T, Lindschau C, Zheng W, Elger M,
Fiebeler A, Fliser D, Luft FC, Haller H: Statins attenuate ischemia-
reperfusion injury by inducing heme oxygenase-1 in infiltrating mac-
rophages. Am J Pathol 170: 1192–1199, 2007
Downloaded from http://journals.lww.com/jasn by BhDMf5ePHKav1zEoum1tQfN4a+kJLhEZgbsIHo4XMi0hCywCX1AW
21. Kentrup D, Reuter S, Schnöckel U, Grabner A, Edemir B, Pavenstädt H,
Schober O, Schäfers M, Schlatter E, Büssemaker E: Hydroxyfasudil-
mediated inhibition of ROCK1 and ROCK2 improves kidney function in
rat renal acute ischemia-reperfusion injury. PLoS One 6: e26419, 2011
22. Collins T, Read MA, Neish AS, Whitley MZ, Thanos D, Maniatis T:
nYQp/IlQrHD3i3D0OdRyi7TvSFl4Cf3VC1y0abggQZXdgGj2MwlZLeI= on 12/20/2023
Transcriptional regulation of endothelial cell adhesion molecules: NF-k
B and cytokine-inducible enhancers. FASEB J 9: 899–909, 1995
23. Kim YS, Ahn Y, Hong MH, Kim KH, Park HW, Hong YJ, Kim JH, Kim W,
Jeong MH, Cho JG, Park JC, Kang JC: Rosuvastatin suppresses the
inflammatory responses through inhibition of c-Jun N-terminal kinase
and Nuclear Factor-kappaB in endothelial cells. J Cardiovasc Phar-
macol 49: 376–383, 2007
24. Eccles KA, Sowden H, Porter KE, Parkin SM, Homer-Vanniasinkam S,
Graham AM: Simvastatin alters human endothelial cell adhesion mol-
ecule expression and inhibits leukocyte adhesion under flow. Athero-
sclerosis 200: 69–79, 2008
25. Wu K, Tian S, Zhou H, Wu Y: Statins protect human endothelial cells
from TNF-induced inflammation via ERK5 activation. Biochem Phar-
macol 85: 1753–1760, 2013
26. Constien R, Forde A, Liliensiek B, Gröne HJ, Nawroth P, Hämmerling G,
Arnold B: Characterization of a novel EGFP reporter mouse to monitor
Cre recombination as demonstrated by a Tie2 Cre mouse line. Genesis
30: 36–44, 2001
27. Feinberg MW, Wara AK, Cao Z, Lebedeva MA, Rosenbauer F, Iwasaki
H, Hirai H, Katz JP, Haspel RL, Gray S, Akashi K, Segre J, Kaestner KH,
Tenen DG, Jain MK: The Kruppel-like factor KLF4 is a critical regulator
of monocyte differentiation. EMBO J 26: 4138–4148, 2007
28. Alder JK, Georgantas RW 3rd, Hildreth RL, Kaplan IM, Morisot S, Yu X,
McDevitt M, Civin CI: Kruppel-like factor 4 is essential for inflammatory
monocyte differentiation in vivo. J Immunol 180: 5645–5652, 2008
29. Thornton MA, Winn R, Alpers CE, Zager RA: An evaluation of the
neutrophil as a mediator of in vivo renal ischemic-reperfusion injury. Am
J Pathol 135: 509–515, 1989
30. Sen-Banerjee S, Mir S, Lin Z, Hamik A, Atkins GB, Das H, Banerjee P,
Kumar A, Jain MK: Kruppel-like factor 2 as a novel mediator of statin
effects in endothelial cells. Circulation 112: 720–726, 2005
31. Ohnesorge N, Viemann D, Schmidt N, Czymai T, Spiering D, Schmolke
M, Ludwig S, Roth J, Goebeler M, Schmidt M: Erk5 activation elicits a
1388 Journal of the American Society of Nephrology
vasoprotective endothelial phenotype via induction of Kruppel-like
factor 4 (KLF4). J Biol Chem 285: 26199–26210, 2010
32. Maejima T, Inoue T, Kanki Y, Kohro T, Li G, Ohta Y, Kimura H, Kobayashi
M, Taguchi A, Tsutsumi S, Iwanari H, Yamamoto S, Aruga H, Dong S,
Stevens JF, Poh HM, Yamamoto K, Kawamura T, Mimura I, Suehiro J,
Sugiyama A, Kaneki K, Shibata H, Yoshinaka Y, Doi T, Asanuma A,
Tanabe S, Tanaka T, Minami T, Hamakubo T, Sakai J, Nozaki N,
Aburatani H, Nangaku M, Ruan X, Tanabe H, Ruan Y, Ihara S, Endo A,
Kodama T, Wada Y: Direct evidence for pitavastatin induced chromatin
structure change in the KLF4 gene in endothelial cells. PLoS One 9:
e96005, 2014
33. Feinberg MW, Cao Z, Wara AK, Lebedeva MA, Senbanerjee S, Jain MK:
Kruppel-like factor 4 is a mediator of proinflammatory signaling in
macrophages. J Biol Chem 280: 38247–38258, 2005
34. Liao X, Sharma N, Kapadia F, Zhou G, Lu Y, Hong H, Paruchuri K,
Mahabeleshwar GH, Dalmas E, Venteclef N, Flask CA, Kim J, Doreian
BW, Lu KQ, Kaestner KH, Hamik A, Clément K, Jain MK: Krüppel-like
factor 4 regulates macrophage polarization. J Clin Invest 121: 2736–
2749, 2011
35. Yoshida T, Kaestner KH, Owens GK: Conditional deletion of Krüppel-
like factor 4 delays downregulation of smooth muscle cell differentia-
tion markers but accelerates neointimal formation following vascular
injury. Circ Res 102: 1548–1557, 2008
36. Yoshida T, Gan Q, Owens GK: Kruppel-like factor 4, Elk-1, and histone
deacetylases cooperatively suppress smooth muscle cell differentia-
tion markers in response to oxidized phospholipids. Am J Physiol Cell
Physiol 295: C1175–C1182, 2008
37. Yoshida T, Yamashita M, Hayashi M: Kruppel-like factor 4 contributes to
high phosphate-induced phenotypic switching of vascular smooth
muscle cells into osteogenic cells. J Biol Chem 287: 25706–25714,
2012
38. Yoshida T, Sinha S, Dandré F, Wamhoff BR, Hoofnagle MH, Kremer BE,
Wang DZ, Olson EN, Owens GK: Myocardin is a key regulator of CArG-
dependent transcription of multiple smooth muscle marker genes. Circ
Res 92: 856–864, 2003
39. Yoshida T, Yamashita M, Horimai C, Hayashi M: Smooth muscle-
selective inhibition of nuclear factor-kB attenuates smooth muscle
phenotypic switching and neointima formation following vascular in-
jury. J Am Heart Assoc 2: e000230, 2013
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org/lookup/suppl/doi:10.1681/ASN.2015040460/-/DCSupplemental.
J Am Soc Nephrol 27: 1379–1388, 2016