Endothelial Krüppel-Like Factor 4 Mediates the Protective Effect of Statins against Ischemic AKI

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-α–induced VCAM1 expression by reducing NF-κB 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.

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 intensive care units suffering from sepsis or after major surgical interventions. To date, therapeutic approaches to prevent and/or treat ischemic AKI are extremely limited. Identification of molecular factors 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 particular, I/R-induced damage in ECs is the key event leading to increased vascular permeability, enhanced endothelium-leukocyte interaction with concomitant inflammatory cell infiltration, abnormal coagulation, and vasoconstriction. In response 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 Moreover, Kelly et al.⁷ showed that Icam1-deficient mice were protected from I/R injury to the kidneys. These results suggest that regulation of cell adhesion molecules is critical for controlling 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 differentiation, 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 functions.⁹ Hamik et al.⁹ showed that overexpression of KLF4 increased expression of anti-inflammatory and antithrombotic factors, including endothelial nitric-oxide synthase and thrombomodulin, whereas knockdown of KLF4 led to enhancement of TNF-α–induced expression of VCAM1 and tissue factor in cultured human umbilical vein endothelial cells (HUVECs). Results from recent studies have also shown that endothelial Klf4 was protective against atherothrombosis in Apoe^−/−-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 molecules and augmented infiltration of inflammatory cells into injured carotid arteries.¹¹ As such, the results of prior studies provide evidence that Klf4 is a critical factor regulating the development and progression of multiple vascular diseases through its effect on endothelial inflammation.

On the basis of the results of the previously described studies, it is of significant interest to determine if endothelial Klf4 contributes to ischemic AKI through modulation 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 mice¹² with mice carrying a loxP allele of Klf4 (Klf4^loxP mice)¹³ and analyzed their phenotype after bilateral renal ischemia. In addition, statins, 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors, have been shown to improve ischemic AKI in rodents^14,15 and humans.^16,17 For example, pretreatment with cerivastatin for 3 days reduced renal I/R injury by preventing inflammatory cell infiltration and by inhibiting Icam1 induction.¹⁴ Therefore, we also examined whether endothelial Klf4 mediated the protective effect of statins against 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 appearance, body weight, systolic and diastolic BP, and heart rate.¹¹ Partial recombination of the Klf4^loxP allele in the whole kidney was observed in Klf4 cKO mice but not in control mice, reflecting the fact that subpopulations of renal cells are ECs (Figure 1B). Klf4 expression was detected in vascular and glomerular ECs in the kidneys, but it was undetectable in Klf4 cKO mice (Figure 1C). These mice were used to determine if endothelial Klf4 contributed to renal I/R injury.

Figure 1.

Generation of Klf4 cKO mice. (A) Schematic representation of endothelial deletion of the Klf4 gene is shown. The numbers shown represent Klf4 exons. The triangles represent the loxP sites. X represents breeding. (B) Recombination of the Klf4^loxP 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 cKO mice and control mice. Klf4 expression was visualized by diaminobenzidine, and sections were counterstained with hematoxylin. Representative pictures are shown from six mice analyzed per each genotype. Magnification ×20. Bar: 100 μm. Arrowheads indicate the glomeruli, and arrows indicate the vessels. Inset: Dotted area is enlarged.

Male Klf4 cKO and control mice at 12–14 weeks of age received bilateral I/R injury for 35 minutes. Twenty-four hours after reperfusion, serum levels of urea nitrogen increased in both Klf4 cKO (122±39 mg/dl) and control (60±13 mg/dl) mice compared with sham-operated mice (32±4 mg/dl in Klf4 cKO mice and 33±4 mg/dl in control mice) (Figure 2A). However, of interest, serum levels of urea nitrogen in Klf4 cKO mice after I/R injury were significantly higher 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 casts, tubular necrosis, and medullary congestion are established histologic features of ischemic AKI.^1,2,18,19 Histologic analyses revealed that Klf4 deletion significantly exacerbated the formation of proteinaceous casts and tubular necrosis and the degree of medullary congestion in response to I/R injury (Figure 3). These results suggest that endothelial Klf4 plays a protective role in ischemic AKI.

Figure 2.

Endothelial Klf4 deletion exacerbated renal I/R injury. Klf4 cKO mice and control mice were subjected to bilateral renal ischemia for 35 minutes (I/R) or sham operation, and serum levels of urea nitrogen (A) and creatinine (B) were measured 24 hours after reperfusion. n=5–6 per each group. *P<0.05 compared with sham-operated mice. ^#P<0.05 compared with I/R-injured control mice.

Figure 3.

Endothelial Klf4 deletion exacerbated renal histologic damages after I/R injury. Klf4 cKO mice and control mice were subjected to bilateral renal ischemia for 35 minutes (I/R) or sham operation, and renal histology was examined 24 hours after reperfusion. (A and B) Representative hematoxylin-eosin staining for the outer stripe of outer medulla (OSOM) and the inner stripe of outer medulla (ISOM) are shown. Bars: 200 μm. (C–E) Levels of the formation of proteinaceous casts (C), tubular necrosis (D), and medullary congestion (E) were scored semiquantitatively. n=5–6 per each group. Magnification ×10. *P<0.05 compared with sham-operated mice. ^#P<0.05 compared with I/R-injured control mice.

Inflammatory Cell Accumulation and Expression of Cell Adhesion Molecules Were Enhanced in Klf4 cKO Mice after I/R Injury

Accumulation of inflammatory cells, including neutrophils, lymphocytes, monocytes, 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 kidneys of both mice (Figure 4, A and B). I/R injury–induced accumulation of lymphocytes was also enhanced in the kidneys of Klf4 cKO mice compared with control mice (Figure 4, C and D). By contrast, monocyte/macrophage accumulation was modest and was not different between Klf4 cKO and control mice after renal I/R injury (Supplemental Figure 1).

Figure 4.

Accumulation of inflammatory cells was enhanced in Klf4 cKO mice after renal I/R injury. Klf4 cKO mice and control mice were subjected to bilateral renal 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 C) Representative pictures for immunohistochemical staining for neutrophils (A) and lymphocytes (C) are shown. Neutrophils (A) and lymphocytes (C) were visualized by diaminobenzidine, and sections were counterstained with hematoxylin. Bars: 100 μm. Red arrowheads indicate the positive cells. Insets: Dotted areas are enlarged. (B and D) The numbers of neutrophils (B) and lymphocytes (D) per five random fields in the kidneys were quantified. Magnification ×10. n=5–6 per each group. *P<0.05 compared with sham-operated mice. ^#P<0.05 compared with I/R-injured control mice.

To determine the mechanisms underlying the enhanced recruitment of neutrophils and lymphocytes in Klf4 cKO mice, the expression of cell adhesion molecules, such as Vcam1 and Icam1, was examined in the kidneys. Quantitative analysis using real-time RT-PCR showed that Vcam1 expression in injured kidneys was much higher in Klf4 cKO mice (2.6-fold increase versus sham-operated kidneys) than in control mice (1.5-fold increase versus sham-operated kidneys) (Figure 5A). Likewise, injury-induced Icam1 expression in Klf4 cKO mice (3.9-fold compared with sham-operated mice) was elevated more than that in control mice (1.7-fold compared with sham-operated mice) (Figure 5B). Moreover, I/R injury–induced expression of multiple inflammatory markers, including Cxcl1, Cxcl2, Tnf, and Il6, was enhanced in the kidneys of Klf4 cKO mice compared with control mice; however, Il10 expression was unaltered (Figure 5C). These results suggest that endothelial Klf4 deletion increases injury-induced expression of cell adhesion molecules with concomitant infiltration of inflammatory cells, thereby exacerbating renal I/R injury in Klf4 cKO mice.

Figure 5.

Induction of cell adhesion molecules was augmented in Klf4 cKO mice after renal I/R injury. Klf4 cKO mice and control mice were subjected to bilateral renal ischemia for 35 minutes (I/R) or sham operation, and the expression of cell adhesion molecules was examined 24 hours after reperfusion. Expression of Vcam1 (A) and Icam1 (B) and Cxcl1, Cxcl2, Tnf, Il6, and Il10 (C) in the kidneys was determined by real-time RT-PCR. n=5–6 per each group. *P<0.05 compared with sham-operated mice. ^#P<0.05 compared with I/R-injured control mice.

Statins Ameliorated Renal I/R Injury in Control Mice but Not in Klf4 cKO Mice

We examined if Klf4 mediated the protective effect of statins against renal I/R injury. Fluvastatin was administered orally to Klf4 cKO and control mice for 3 days. On the third day, both mice were subjected to bilateral renal I/R injury. Consistent with the results of previous studies,^14,15 treatment with fluvastatin ameliorated renal I/R injury in control mice, as assessed by serum levels of urea nitrogen and creatinine, histologic analyses, and expression of cell adhesion molecules (Figure 6). However, of importance, fluvastatin did not attenuate renal I/R injury in Klf4 cKO mice. Similar results were obtained from Klf4 cKO mice treated with simvastatin (data not shown). These results suggest that the protective effect of statins against ischemic AKI is mediated by endothelial Klf4.

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.

Results of previous studies suggest that statin protection against ischemic AKI is mediated in part by heme oxygenase 1 (Hmox1)²⁰ and/or Rho-associated coiled-coil containing protein kinase 1 (Rock1).²¹ To evaluate the possible 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 increased 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 between 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 suggest a relationship between Hmox1, Rock1, and Klf4.

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 determined by densitometry. n=5–6 per each group. *P<0.05 compared with sham-operated mice.

KLF4 Mediated the Suppressive Effect of Statins on TNF-induced VCAM1 Expression by Reducing the Association of NF-κB with the VCAM1 Promoter in HUVECs

Induction of cell adhesion molecules, such as VCAM1 and ICAM1, has been shown to be regulated by the TNF-NF-κB pathway in ECs.²² In addition, previous studies have shown that TNF-induced expression of cell adhesion molecules was repressed by statins in cultured ECs.^23–25 Moreover, we previously showed that KLF4 inhibited TNF-induced expression of VCAM1 through blocking the binding of NF-κB to the VCAM1 promoter in cultured ECs.¹¹ To clarify the molecular mechanisms whereby statins attenuated renal I/R injury in control mice, but not in Klf4 cKO mice, we examined whether KLF4 contributed to the suppressive effect of statins on TNF-induced VCAM1 expression in HUVECs.

HUVECs were transfected with siRNA for KLF4 or a scrambled sequence, and then they were treated with TNF and statins, such as fluvastatin and simvastatin. As shown in Figure 8A, KLF4 expression was increased by statins in HUVECs, whereas it was very low in KLF4 siRNA-transfected HUVECs. Treatment with TNF increased VCAM1 expression by 2.5-fold, whereas fluvastatin and simvastatin, respectively, attenuated TNF-induced upregulation of VCAM1 expression in HUVECs (Figure 8B). However, under the condition of suppression of KLF4, TNF-induced VCAM1 expression was increased 4.0-fold and was not affected by either fluvastatin or simvastatin in HUVECs transfected with KLF4 siRNA. Although VCAM1 expression was regulated by statins and KLF4, TNF-induced phosphorylation of p65, a major component of NF-κB, was unaffected by these factors (Figure 8C). However, chromatin immunoprecipitation assays revealed that TNF-induced increase in p65 binding to the VCAM1 promoter was inhibited by fluvastatin and that the suppressive effect of fluvastatin on TNF-induced p65 binding was abolished by knockdown of KLF4 in HUVECs (Figure 8D). Taken together, these results suggest that statins suppress inflammation-related induction of VCAM1 by inducing the expression of KLF4, which inhibits NF-κB binding to the VCAM1 promoter in ECs.

Figure 8.

KLF4 mediated the suppressive effect of statins on TNF-induced VCAM1 expression by reducing the association of NF-κB 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.

Discussion

In this study, we showed that deletion of the Klf4 gene in ECs exacerbated renal I/R injury in mice, as assessed by biochemical parameters and renal histology. Aggravated I/R injury in Klf4 cKO mice was accompanied by increased expression of cell adhesion molecules, such as Vcam1 and Icam1, and enhanced accumulation of neutrophils and lymphocytes in the injured kidneys. We also showed that the protective effect of statins against ischemic AKI was mediated by endothelial Klf4. Indeed, pretreatment with statins ameliorated renal I/R injury in control mice, but not in Klf4 cKO mice. Mechanistic studies demonstrated that statins suppressed inflammation-related induction of VCAM1 by inducing KLF4, which inhibited the binding of NF-κB to the VCAM1 promoter in ECs. As such, results of this study provide evidence 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.

Tek promoter–dependent conditional Klf4 knockout mice were used in this study. The Tek promoter is active mainly in ECs, but also in some hematopoietic cells.²⁶ Therefore, the recombination of the Klf4^loxP allele occurred in circulating blood cells and ECs in Klf4 cKO mice. However, the aggravated renal I/R injury in Klf4 cKO mice was most likely caused by the deletion of the Klf4 gene in ECs for the following reasons. First, the results of previous studies demonstrated that Klf4 is expressed in monocytes/macrophages, but not in neutrophils and lymphocytes.^27,28 Second, we previously reported that the number of neutrophils, lymphocytes, and monocytes in the blood did not differ between the Klf4 cKO and control mice.¹¹ In this study we showed that the accumulation of neutrophils and lymphocytes was enhanced in the kidneys at 24 hours after I/R injury in Klf4 cKO mice compared with that in control mice. However, these inflammatory cells do not express endogenous Klf4 in either Klf4 cKO or control mice. By contrast, although Klf4 is expressed in monocytes/macrophages,^27,28 the number of infiltrated monocytes/macrophages in injured kidneys was very low and was not different between Klf4 cKO and control mice at the early time point examined.¹⁹ Accordingly, it is reasonable to conclude that the phenotype of Klf4 cKO mice after renal I/R injury, including the enhanced accumulation of neutrophils and lymphocytes, is caused by the lack of Klf4 in ECs.

Results of this study showed that I/R injury–induced accumulation of neutrophils and lymphocytes was enhanced in Klf4 cKO mice compared with control mice. The accumulation of inflammatory cells is thought to have an important role in the pathogenesis of ischemic AKI. In support of this, Kelly et al.⁷ showed the administration of antineutrophil serum ameliorated renal I/R injury in mice. However, there is another report showing that the same administration did not 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 experimental 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 endothelial 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 adhesion via alteration of expression of cell adhesion molecules²⁴; 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 suppressive effect of statins on TNF-induced VCAM1 expression in HUVECs. These results are consistent with those from previous 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 expression 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 assays 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 overexpression.¹¹ Overall, results thus far suggest that KLF4 inhibits NF-κB binding to the promoter region of cell adhesion molecules 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 simply protein-protein interactions and do not involve the transcriptional 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. Klf4^loxP/loxP mice¹³ were bred with Tek-Cre^+/− mice¹² to generate Tek-Cre^+/−/Klf4^loxP/+ mice. Tek-Cre^+/−/Klf4^loxP/+ mice were then bred with Klf4^loxP/loxP mice to generate Tek-Cre^+/−/Klf4^loxP/loxP (Klf4 cKO) mice and Tek-Cre^−/−/Klf4^loxP/loxP (control) mice. Both mice were on the C57BL/6J background, and littermates were used for all comparisons. Genotyping was performed by PCR as described previously.³⁵

Ischemic AKI Model

Male Klf4 cKO and control mice at 12–14 weeks of age were anesthetized with an intraperitoneal injection of pentobarbital sodium. Kidneys were exposed through flank incisions. Mice were subjected to 35 minutes of bilateral renal ischemia or sham surgery. Ischemia was induced by clamping both renal pedicles with nontraumatic microvessel 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 (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.

Histology and Immunohistochemistry

The kidneys were fixed in 4% paraformaldehyde and embedded into paraffin. The 5-μm 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), CD3ε (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 μmol/L fluvastatin, and/or 1 μmol/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 mean±SEM. Statistical analyses were done by SigmaPlot/SigmaStat9 (Systat Software, San Jose, CA). After confirming 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.

Disclosures

None.