The protein kinase 2 inhibitor tetrabromobenzotriazole protects against renal ischemia reperfusion injury

Protein kinase 2 (CK2) activation was reported to enhance reactive oxygen species production and activate the nuclear factor κB (NF-κB) pathway. Because oxidative stress and inflammation are critical events for tissue destruction during ischemia reperfusion (I/R), we sought to determine whether CK2 was important in the renal response to I/R. Mice underwent 25 min of renal ischemia and were then reperfused. We confirmed an increased expression of CK2α during the reperfusion period, while expression of CK2β remained consistent. We administered tetrabromobenzotriazole (TBBt), a selective CK2α inhibitor before inducing I/R injury. Mice subjected to I/R injury showed typical patterns of acute kidney injury; blood urea nitrogen and serum creatinine levels, tubular necrosis and apoptosis, inflammatory cell infiltration and proinflammatory cytokine production, and oxidative stress were markedly increased when compared to sham mice. However, pretreatment with TBBt abolished these changes and improved renal function and architecture. Similar renoprotective effects of CK2α inhibition were observed for emodin. Renoprotective effects of CK2α inhibition were associated with suppression of NF-κB and mitogen activated protein kinase (MAPK) pathways. Taken together, these results suggest that CK2α mediates proapoptotic and proinflammatory signaling, thus the CK2α inhibitor may be used to prevent renal I/R injuries observed in clinical settings.

Antioxidants and anti-apoptotic therapy have been shown to be protective against I/R-mediated oxidative damage in different experimental models [8][9][10][11] .
Protein kinase 2 (CK2) is a highly conserved and ubiquitously expressed serine/threonine kinase; it is a tetramer composed of two catalytic subunits (α and α ′ ) and two regulatory subunits (β ) in an α 2 β 2 , α α ′ β 2 , or α ′ 2 β 2 configuration 12 . CK2 is upregulated in a variety of human cancers and creates a cellular environment favorable to neoplasia by enhancing cell proliferation and by inhibiting apoptosis 13,14 .
Thus, CK2 has emerged as a promising pharmacological target for anti-cancer therapy 15 . In addition to its apoptotic inhibiting functions, a number of studies have suggested a pro-inflammatory role for CK2. Exogenous expression of CK2α activates inhibitory κ B kinase (IKK)β , which then phosphorylates and degrades inhibitory κ B (Iκ B)α 16 . CK2α is also involved in phosphorylation of p65, which has a synergistic effect on the amplitude of transactivation 17 . Hence, CK2 inhibitors suppress NF-κ B-dependent pro-inflammatory cytokine production and the related inflammatory responses 18-20 . In the last two decades, a number of groups have developed various kinds of CK2 inhibitors. One of the most efficient and selective is 4,5,6,7-tetrabromobenzotriazole (TBBt). The basis for TBBt selectivity is provided by the hydrophobic pocket adjacent to the ATP/GTP binding site which is smaller in CK2, than in the majority of other protein kinases 21 . Treatment of human Jurkat cells with TBBt leads to induction of apoptosis 22 . Another CK2 inhibitor emodin, in comparison with TBBt, is more water soluble and has a limited selectivity for CK2. Besides CK2, emodin inhibits casein kinase 1 23 and receptor tyrosine kinase 24 .
Given that apoptosis and inflammation are critical events for I/R injury, CK2 inhibition may have some role in the pathogenesis of I/R injury. Recently, Kim et al. 25 demonstrated that inactivation of CK2 in the mouse brain enhances production of ROS and neuronal cell death after ischemic injury via increased NADPH oxidase activity. To our knowledge, however, there are no reports about its effects against renal I/R injury. Based on conflicting evidence of apoptotic induction and anti-inflammation of CK2 inhibition, we assessed the effects of TBBt on the intrinsic response to renal I/R injury.

Results
CK2α expression is increased during renal I/R injury. To induce I/R injury, the renal pedicles were bilaterally clamped for 25 min, after which they were reperfused for various time periods (Fig. 1A). This protocol was modified from previously reported methods 4,26,27 . We first determined protein levels of CK2 in reperfused renal tissues (Fig. 1B). The protein levels of CK2α but not of CK2β began to increase  (25 min 1 h after the initiation of reperfusion; they reached their maximum levels at 6 h, remained elevated up to 12 h, and declined thereafter.

CK2α inhibition attenuates renal I/R injury.
To assess the function of CK2α in renal I/R injury, we pretreated mice with TBBt, a CK2α inhibitor, 3 h and 24 h prior to inducing I/R injury, and blood samples were collected 24 h after reperfusion. I/R injury significantly impaired renal function in control mice as BUN and creatinine levels increased from 54.2 ± 6.2 mg/dl and 0.7 ± 0.2 mg/dl, respectively, before I/R to 198.0 ± 25.7 mg/dl and 2.4 ± 0.7 mg/dl, respectively, after reperfusion ( Fig. 2A,B). However, pretreatment with 2 mg/kg TBBt significantly attenuated this impairment; the average BUN and creatinine levels in TBBt-pretreated mice were 135.4 ± 19.4 mg/dl and 1.4 ± 0.4 mg/dl, respectively (p < 0.01).
Macroscopically, kidneys subjected to I/R were clearly enlarged and appeared edematous (data not shown). Upon histological examination, the I/R group showed typical features of severe acute tubular damage, including extensive tubular necrosis, tubular dilatation, and loss of brush border (Fig. 2C). A scoring system was introduced to compare the severity of the histologic signs of I/R, and the tubular injury score of I/R-induced kidneys was markedly increased when compared with that of sham mice (Fig. 2D). Pretreatment with TBBt preserved the normal morphology of the kidney and resulted in slight swelling of the tubular epithelium and a slight loss of brush border. The histopathologic scores supported the histologic findings; TBBt treatment reduced the score from 3.2 ± 0.8 to 1.6 ± 0.8 (p < 0.01). In addition, less severe tubular necrosis was observed in TBBt-pretreated mice (Fig. 2E).

Figure 2. Protection of renal I/R injury by CK2α inhibitor.
Mice were injected with 1 or 2 mg/kg TBBt or 2 or 5 mg/kg emodin via i.p. before I/R injury. After 24-h reperfusion, blood was collected for the measurement of BUN (A) and creatinine (B). (C) Mice were injected with 2 mg/kg TBBt or 5 mg/kg emodin, and kidney tissues were collected 24 h after reperfusion. Tissues were stained with hematoxylin and eosin (H&E, ×200) and periodic acid Schiff (PAS, ×200 or ×400) for light microscopic analysis. Bar = 250 μ m. Histopathologic scoring (D) and quantification of necrotic area (E) were performed in a blind method. Values are mean ± SEM (n = 6 mice per group). ** p < 0.01 vs. sham; ## p < 0.01 vs. VEH. VEH, vehicle; TBBt-1, 1 mg/kg TBBt; TBBt-2, 2 mg/kg TBBt; Emo-2, emodin 2 mg/kg; Emo-5, emodin 5 mg/kg. TBBt decreases apoptosis after I/R injury. Although the major cause of cell death during renal I/R injury is necrosis, apoptotic cell death is also observed during the reperfusion process 7 . The extent of apoptosis was evaluated by TUNEL staining (Fig. 3A). The number of TUNEL-positive apoptotic cells was markedly increased in I/R-injured mice compared to that in sham mice. Increased protein and mRNA levels of proapoptotic caspase-3 and Bax and a decreased protein level of antiapoptotic Bcl-2 were observed in I/R-injured mice (Fig. 3B,C). Pretreatment with TBBt attenuated these changes.

TBBt decreases inflammatory cell infiltration in I/R-injured kidney tissue. The inflammatory
response following I/R injury is related to renal dysfunction 1 . To investigate whether improved renal function in TBBt-pretreated mice was dependent on a suppressed inflammatory reaction, we stained kidney tissues with F4/80 and naphthol AS-D chloroacetate esterase, which are specific for macrophages and neutrophils, respectively. After 24 h of reperfusion, the number of infiltrating macrophages and neutrophils were increased in the I/R-injured kidney tissues, whereas significantly less infiltration of macrophages and neutrophils was observed in TBBt-pretreated mice (Fig. 4A). Real-time RT-PCR analysis confirmed the decreased accumulation of macrophages and suppressed inflammation in TBBt-pretreated mice compared to I/R mice (Fig. 4B). Changes in serum TNF-α and MCP-1 levels were similar to those seen for mRNA expression (Fig. 4C).
TBBt inhibits NF-κB signaling pathway. We next investigated the effects of TBBt on I/R-dependent NF-κ B activation, which is a major signaling pathway of inflammation. In I/R mice, there were increases in nuclear p65 and p50 subunits and cytoplasmic p-Iκ Bα compared to sham mice (Fig. 5A). Accordingly, expression of iNOS, a downstream effector of NF-κ B, was also increased in I/R mice. In contrast, in the presence of TBBt, these changes were almost completely inhibited. Further examination by EMSA clearly showed nuclear translocation of p65 and binding to an NF-κ B consensus sequence in I/R mice but not in TBBt-pretreated mice (Fig. 5B). Our data suggest that TBBt could successfully inhibit NF-κ B activation induced by renal I/R.

TBBt inhibits the MAPK signaling pathway.
To investigate the mechanism by which TBBt suppresses the NF-κ B signaling pathway, we compared mitogen-activated protein kinase (MAPK) signal transduction pathways. I/R injury increased the phosphorylation levels of ERK and p38 MAPK in I/R-injured kidney tissues but not in those of TBBt mice (Fig. 6). Phosphorylation of JNK was not different between groups (data not shown).
TBBt prevents the down-regulation of anti-oxidant potentials. I/R injury is partially mediated by oxidative stress 3 , and CK2 inhibition is known to increase oxidative stress under in vitro cell culture condition 28 . We investigated whether TBBt increases oxidative stress. Surprisingly, TBBt pretreatment caused suppression of ROS production as observed by decrease in 4-hydroxynonenal (4-HNE) positive cells (Fig. 7A). Compared with I/R mice, increases in glutathione levels and superoxide dismutase and catalase enzyme activities were observed in TBBt-pretreated mice (Fig. 7B), suggesting increased     antioxidant potentials. To explain increased anti-oxidant potentials by TBBt, we compared the downstream signaling pathway of NF-E2-related factor-2 (Nrf2), a master regulator antioxidant response, in treated and control mice. Compared to control mice, TBBt pretreatment led to increases in expression levels of NAD(P)H quinone oxidoreductase (NQO1) and hemeoxygenase (HO)-1 (Fig. 7C). These results indicate that TBBt protects kidney I/R injury through preventing the down-regulation of antioxidant genes after I/R injury.

Discussion
In this study, we demonstrated for the first time that pretreatment with a double dose of CK2α inhibitor TBBt significantly protected against renal I/R injury. After bilateral renal I/R injury, TBBt pretreated mice displayed significantly preserved renal function, less histological tubular damage, and reduced inflammatory cell infiltration. The protective effect was associated with ameliorated oxidative stress as manifested by an increase in antioxidant capacity (a higher level of glutathione, higher enzyme activities of SOD and catalase, and lower production of 4-HNE). We further demonstrated that TBBt reduced NF-κ B activation and concomitant inflammatory cytokine production through inhibition of the ERK and p38 MAPK pathways.
I/R-injured kidney tissue is characterized by inflammatory cell infiltration, initially with neutrophils and later with macrophages and lymphocytes 1 . Ischemic injury upregulates the expression of various inflammation-related molecules such as MCP-1 and ICAM-1, which in turn facilitate the recruitment and infiltration of inflammatory cells in the kidney tissue 9,29,30 . Macrophages constitute 40-60% of infiltrating cells during acute allograft rejection. In patients with acute rejection, interstitial macrophage infiltration was significantly higher than in non-rejecting patients 31 . Recently, Kim et al. 18 reported that CK2α is involved in the platelet activating factor-induced NF-κ B activation and that inhibitors for each step block inflammatory cell infiltration into tissues. An increase of CK2α expression and inflammatory cell infiltration in a murine glomerulonephritis model has also been reported to lead to increases in production from genes in the ERK pathway (c-fos, Egr-1, and Elk-1), NF-κ B-regulated inflammatory cytokines (TNF-α and MCP-1), and extracellular matrix proteins (collagen, fibronectin, TGFβ , and PDGF) 32 . Inhibition of CK2α prevented inflammation and decreased expression of the aforementioned genes. These reports point to an important role of CK2α expression for inflammatory cell infiltration during I/R injury. In line with these reports, we observed increased expression of CK2α , MCP-1, and ICAM-1 and activation of the NF-κ B pathway in I/R injured kidney tissues. Because MCP-1 is a potent macrophage chemoattractant, increased levels of MCP-1 with its receptor CCR2 could directly trigger the recruitment of macrophages to kidney tissue. Infiltrated macrophages could, in turn, secrete a variety of cytokines, including TNF-α and IL-6, which would further induce inflammation-related gene expression and promote local inflammatory responses 33 . Expression of the NF-κ B downstream protein iNOS has also been linked to graft rejection in a rat model of allogenic kidney transplantation, where macrophage depletion resulted in a reduction of acute rejection and an upregulation of iNOS expression 34 . In this study, we provided evidence that all of these inflammatory events are associated with CK2α activation, because TBBt effectively suppressed NF-κ B activation, cytokine production, and inflammatory cell infiltration in I/R injured kidney tissues. Ultimately, CK2α suppression seems to contribute to decrease of apoptotic or necrotic cell death.
CK2 is strongly implicated in regulation of cell proliferation and in inhibition of apoptosis 13,14 . However, results vary greatly depending on the cell type and stress model. For example, activation of the proapoptotic pathway by CK2α has been observed in INS-1 cells and isolated islets 35 . Likewise, in our study, CK2α inhibition led to anantiapoptotic effects in renal tubular tissues, which exhibited less tubular injury and enhanced clearance of BUN and creatinine. Since TNF-α has been shown to be an important mediator of tubular epithelial cell apoptosis 36,37 , we suspected that TBBt might have antiapoptotic effects through suppression of the TNF-α -mediated apoptosis pathway. Indeed, we observed significantly suppressed levels of TNF-α mRNA and protein in TBBt-pretreated mice.
Our results are in contrast to those of Kim et al. 25 who demonstrated that CK2α was a negative regulator of NADPH oxidase, an important enzyme for ROS generation in the mouse brain. They further found that the protein levels and enzyme activities of CK2α were significantly reduced after transient focal cerebral I/R injury, and CK2α inhibitor suppressed the death of primary neuronal cells exposed to hypoxia and reoxygenation injury. These opposite conclusions may result from several differences in the two studies. First, each study used a different CK2α inhibitor (TBBt for our study vs. TBCA for theirs). Schneider et al. 38 compared two CK2α inhibitors, 4,5,6,7-tetrabromobenzimidazole (TBBz) and 2-dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole (DMAT) and found that both chemicals inhibited CK2 activity to the same extent, but only DMAT induced ROS generation. Their report suggests that CK2 inhibition is not a prerequisite for ROS generation and that the two closely related CK2α inhibitors may activate different signaling pathways for ROS generation. Second, we used a kidney model with ischemia injury for 25 min using ketamine as an anesthesia, whereas Kim et al. experimented on a brain model with 45 min of ischemia using isoflurane as the anesthesia. Third, even using different strains of mice (C57BL/6 in our study vs. CD1 in theirs) could have contributed to the different outcomes. Fourth, in contrast to the brain I/R model, we did not observe a suppression of CK2α expression after reperfusion; rather, we observed an increase in CK2α (Fig. 1B). Taken together, differences in experimental design could lead to the ultimate beneficial or deleterious outcome for CK2α inhibition in I/R injury. Collectively, our results demonstrate that CK2α inhibition protects against renal I/R injury by the following mechanism: inhibition of p38 and ERK pathways → suppression of NF-κ B pathway → attenuation of inflammation and oxidative stress → prevention of apoptosis and concomitant renal dysfunction. Thus, CK2α may have an aggravating role in the pathogenesis of renal I/R injury.

Materials and Methods
Reagents. TBBt was purchased from Tocris (Bristol, UK) and dissolved at 50 mg/ml in dimethyl sulfoxide. All reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA) unless otherwise noted. Emodin was isolated from R. palmatum as described previously 39 .
Animal experiment. Pathogen-free 7-9-week-old C57BL/6 male mice (Damul Science, Daejeon, Republic of Korea; weight 20-23 g) were maintained on a diet of standard laboratory chow and water ad libitum. Mice were treated with TBBt (1 or 2 mg/kg body weight in 200 μ l of 5% DMSO in saline), emodin (2 or 5 mg/kg body weight 200 μ l of 5% DMSO in saline), or the same volume of vehicle through i.p. injection at 3 h and 24 h before ischemia. Mice were anaesthetized with a ketamine-xylazine mixture through i.m. injection. In the I/R group, both renal pedicles were clamped through midline incisions using a microvascular clip 40 . The kidney was kept moist with gauze soaked in saline, and body temperature was maintained at 37 °C with a warm blanket throughout ischemia. After 25 min of ischemia, the clip was removed to initiate reperfusion. Sham mice underwent the same operation without vascular occlusion. After the desired reperfusion time, mice were killed, and serum and tissue samples were collected and immediately fixed in 10% formalin or stored at − 80 °C until further analysis. Anti-oxidant enzyme activity assay. To analyze the enzymatic activities of superoxide dismutase (SOD), catalase, and glutathione (GSH), kidney tissues were suspended in 10 mM phosphate buffer (pH 7.4), mixed with ice-cold 5% metaphosphoric acid solution and then homogenized. Homogenates were centrifuged at 3000 rpm for 10 min. Enzyme activity in the supernatant was determined using commercial assay kits (Enzo Life Sciences, Plymouth Meeting, PA, USA).
Histology and histopathologic scoring. Kidney tissues were fixed in 10% neutral buffered formalin and then embedded in paraffin. Tissue was sectioned at 4 μ m and then stained with hematoxylin and eosin (H&E) and periodic acid-Schiff reagent (PAS) for light microscopic analyses. Histopathologic damage was defined as tubular epithelial swelling, loss of brush border, vacuolar degeneration, necrotic tubules, cast formation, and desquamation. The degree of tubular injury was estimated at a × 200 magnification using 5 randomly selected fields for each kidney with the following standard; 0, normal; 1, damage involving < 25% of tubules; 2, damage involving 25-50% of tubules; 3, damage involving 50-75% of tubules; and 4, damage involving 75-100% of tubules 40 . Tubular necrosis was quantitated as the percentage of tubules in the outer medulla in which epithelial necrosis or necrotic debris was observed in PAS-stained sections.

Neutrophil infiltration.
A naphthol AS-D chloroacetate esterase kit (Sigma-Aldrich) was used for neutrophil esterase staining of kidney sections. Neutrophils were counted by examining 5-10 viewing fields randomly selected from the outer medulla and corticomedullary junction on each slide at × 400 magnification in a blinded manner. The number of neutrophils were counted by iSolution DT 36 software (Carl Zeiss, Oberkochen, Germany), and results were expressed as percentage of the total cells. Immunohistochemistry. Immunohistochemical staining was performed using the DAKO Envision system (DAKO, Carpinteria, CA, USA), which uses dextran polymers conjugated with horseradish peroxidase to avoid contamination with endogenous biotin. After deparaffinization, tissue sections were treated using a microwave antigen-retrieval procedure in 10 mM sodium citrate buffer. After blocking endogenous peroxidases, sections were incubated with Protein Block Serum-Free (DAKO) to block non-specific staining and were immunostained with antibody against F4/80 or 4-hydroxynonenal (both from Abcam, Cambridge, UK). Stained sections were quantified by iSolution DT 36 software (Carl Zeiss, Oberkochen, Germany), and results were expressed as percentage of the total cells. TUNEL assay. TUNEL  (AI = number of apoptotic bodies/100 cells). Each group was assessed in triplicate, and data were averaged.
Protein extraction. Total protein of kidney tissue was extracted using a tissue protein extraction reagent (Pierce Biotechnology, Rockford, IL, USA). For nuclear protein extraction, kidney tissues were homogenized with a pre-chilled Dounce homogenizer (Wheaton Industries, Millville, NJ, USA) and then extracted with nuclear and cytoplasmic extraction reagents (Pierce Biotechnology). Protein concentration was determined using the Bradford method.
Electrophoretic mobility shift assay (EMSA). Nuclear extracts prepared from the kidney tissues were incubated with proteinase inhibitor cocktail (Calbiochem, Billerica, MA, USA). An oligonucleotide corresponding NF-κ B site (5-CCGGTTAACAGAGGGGGCTTTCCGAG-3) was synthesized and used as a probe for a gel retardation assay. The two complementary strands were annealed and labeled with [α -32 P]dCTP. Labeled probe (10,000 cpm), 10 μ g of nuclear extract, and binding buffer (10 mMTris-HCl, pH 7.6, 500 mM KCl, 10 mM EDTA, 50% glycerol, 100 ng poly(dI·dC), and 1 mM dithiothreitol) were then incubated for 30 min at room temperature in a final volume of 20 μ l. Reaction mixtures were analyzed by electrophoresis on 4% polyacrylamide gels in 0.5× Tris-borate buffer. Gels were dried and examined by autoradiography.

RNA isolation and real-time RT-PCR.
Total RNA was extracted from frozen kidney tissue using the RNA Iso kit (Takara, Shiga, Japan). RNA was precipitated with isopropanol and dissolved in diethylpyrocarbonate-treated distilled water. Total RNA (1 μ g) was treated with RNase-free DNase (Invitrogen), and first-strand cDNA was generated using the random hexamer primer provided in the first-strand cDNA synthesis kit (Invitrogen). Specific primers for each gene were designed using qPrim-erDepot (http://mouseprimerdepot.nci.nih.gov, Supplementary Table 1). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an invariant control. The real-time RT-PCR reaction mixture consisted of 10 ng reverse transcribed RNA, 200 nM forward and reverse primers, and 2× PCR master mixture in a final volume of 10 μ l. The PCR reaction was carried out in 384-well plates using the ABI Prism 7900HT Sequence Detection System (Applied Biosystems, Foster City, CA, USA).

Statistical analysis.
Statistical analyses of the data were conducted using ANOVA and Duncan's tests.
Differences with a p < 0.05 were considered statistically significant.