Sirtuin 7 Deficiency Ameliorates Cisplatin-induced Acute Kidney Injury Through Regulation of the Inflammatory Response

Cisplatin-induced acute kidney injury (AKI) has been recognized as one of cisplatin’s serious side effects, limiting its use in cancer therapy. Sirtuin 1 (SIRT1) and SIRT3 play protective roles against cisplatin-induced kidney injury. However, the role of SIRT7 in cisplatin-induced kidney injury is not yet known. In this study, we found that Sirt7 knockout (KO) mice were resistant to cisplatin-induced AKI. Furthermore, our studies identified that loss of SIRT7 decreases the expression of tumor necrosis factor-α (TNF-α) by regulating the nuclear expression of the transcription factor nuclear factor kappa B. It has been reported that cisplatin-induced nephrotoxicity is mediated by TNF-α. Our results indicate that SIRT7 plays an important role in cisplatin-induced AKI and suggest the possibility of SIRT7 as a novel therapeutic target for cisplatin-induced nephrotoxicity.

. Expression of SIRT7 in the kidney. (A) SIRT7 expression in mouse kidney, liver, and heart was evaluated. GAPDH and β-actin were used as loading controls. SIRT7 protein expression was deleted in Sirt7 KO mouse kidney. (B) SIRT7 expression in the kidney of Sirt7 FRT/floxed mutant mouse was evaluated by β-galactosidase staining. (C) Representative photomicrographs (×400) of immunohistochemical staining for SIRT7 in kidney sections. Scale bar: 50 µm. (D) Representative photomicrographs (×400) of double immunostaining (lectin and SIRT7). PHA-E was used as a marker of proximal tubules and DBA was used as a marker of collecting tubules. The arrows indicate SIRT7-expressing nuclei. Scale bars: 50 µm.
Scientific RepoRts | (2018) 8:5927 | DOI: 10.1038/s41598-018-24257-7 of the Sirt7 promoter 10 . The inner medullary region and cortical region of the kidney were positive for X-gal staining (Fig. 1B). Parallel immunohistochemical analysis was performed using an anti-SIRT7 antibody. SIRT7 was expressed in the nuclei of renal tubular epithelial cells and glomeruli of the kidney of WT mice, but not in the kidney of Sirt7 KO mice (Fig. 1C). Double immunostaining for SIRT7 and Phaseolus vulgaris erythroagglutinin (PHA-E) lectin (a marker of proximal tubular epithelial cells) revealed that SIRT7 is expressed in proximal tubular cells (Fig. 1D) 16 . Consistent with LacZ staining in the inner medullary region, SIRT7 was also expressed in Dolichos biflorus agglutinin (DBA; a marker of collecting duct cells)-positive collecting duct cells (Fig. 1D) 16 . SIRT7 expression in proximal tubular cells was comparable with that in collecting duct cells ( Supplementary  Fig. S1B,C). No overt histological abnormalities were detected in the kidney of Sirt7 KO mice (Fig. 1C). Moreover, there were no significant differences in body weight, urine volume, and serum components between the 8-weekold WT and Sirt7 KO mice (Supplementary Table 1).

Cisplatin-induced Kidney Injury is Ameliorated in
Sirt7 KO Mice. We investigated the role of SIRT7 in cisplatin-induced AKI. Cisplatin administration led to an increase of Sirt7 mRNA and SIRT7 protein expression in WT mice ( Fig. 2A,B). We next studied the impact of SIRT7 deficiency on cisplatin-induced kidney injury. Interestingly, urinary levels of neutrophil gelatinase-associated lipocalin (NGAL), a biomarker of kidney injury 17 , were significantly lower in Sirt7 KO mice than in WT mice (Fig. 2C). Blood urea nitrogen and serum creatinine levels were significantly lower in Sirt7 KO mice than in WT mice at 72 h after cisplatin injection (Fig. 2D). Furthermore, Sirt7 KO mice showed a significantly higher survival rate than WT mice after cisplatin injection (Fig. 2E). These results indicated that Sirt7 KO mice were resistant to cisplatin-induced AKI. SIRT1 and SIRT3 play protective roles in the development of cisplatin-induced AKI [6][7][8] 6,18,19 was unchanged in the kidney of Sirt7 KO mice after cisplatin administration (Fig. 2F), suggesting that the protective effect of SIRT7 deficiency is independent of these SIRTs.
Cisplatin mainly affects the proximal tubules in the kidney 1 . Accordingly, histological analysis revealed that cisplatin treatment resulted in severe tubular injury reflected by necrosis, cast formation, dilation, and loss of the brush border in the cortical region of WT mice (Fig. 3A). In contrast, the cisplatin-induced damage was significantly ameliorated in the cortical region of Sirt7 KO mice (Fig. 3A). Neither WT nor Sirt7 KO mice showed kidney tissue injury in the inner medullary region by cisplatin ( Supplementary Fig. S2). Cisplatin-induced activated (cleaved) caspase 3 protein expression was detected in the kidney of WT mice, but its expression was lower in Sirt7 KO mice (Fig. 3B). Consistent with this result, the number of TUNEL-positive cells in the injured tubules was significantly decreased in the kidney of Sirt7 KO mice, suggesting that loss of SIRT7 suppresses the apoptosis induced by cisplatin (Fig. 3C).

Inflammation-related Gene Expression and Macrophage Infiltration are Suppressed in the
Kidney of Sirt7 KO Mice. Next, we analyzed the expression of genes involved in cisplatin-induced renal injury. The expression of the gene encoding OCT2 (Slc22a2), which plays a role in cisplatin uptake 20 , and MATE1 (Slc47a1), which regulates the excretion of cisplatin 21 , was unchanged (Fig. 4A). Oxidative stress and inflammation play important roles in the pathophysiology of cisplatin-induced kidney injury; 1-3 therefore, we evaluated oxidative stress and inflammation-related gene expression. The expression of the genes encoding NOX2 (Cybb) and p47phox (Ncf1), which are involved in the production of reactive oxygen species (ROS), was significantly decreased in the cisplatin-treated kidney of Sirt7 KO mice (Fig. 4B). The expression of genes involved in inflammation (Tnfa, Il1b, Il6, Ccl2, and Cxcl2) was also significantly reduced in the cisplatin-treated kidney of Sirt7 KO mice (Fig. 4C), suggesting that SIRT7 plays critical roles in the regulation of oxidative stress and inflammation.
Unilateral ureteral obstruction (UUO) is another experimental model of renal injury 22 . Consistent with the cisplatin model, the expression levels of Tnfa, Il1b, Il6, Ccl2, and Cxcl2 at an acute stage were significantly decreased in the obstructed kidney of Sirt7 KO mice ( Supplementary Fig. S3).
In cisplatin nephropathy, the production of cytokines and chemokines by renal parenchymal cells leads to the recruitment of inflammatory cells 1 . In accordance with the reduced expression of Ccl2 (encoding MCP1) (Fig. 4C), immunostaining for MCP1 showed a decrease of intensity in the cisplatin-treated kidney of Sirt7 KO mice ( Supplementary Fig. S4). The number of F4/80-positive infiltrating macrophages in the interstitium was significantly suppressed in the kidney of Sirt7 KO mice as compared with that in WT mice (Fig. 4D).

SIRT7
Regulates the Expression of TNF-α. We next examined gene expression in control and Sirt7 knockdown (KD) rat renal proximal tubular NRK-52E cells. The introduction of Sirt7 short hairpin RNA (shRNA) markedly reduced the levels of endogenous SIRT7 (Fig. 5A). The expression of Cybb, Ncf1, and Ccl2 mRNA was reduced in the cisplatin-treated kidney of Sirt7 KO mice (Fig. 4B,C), but their expression was not decreased in Sirt7 KD NRK-52E cells after cisplatin exposure (Fig. 5B,C). In contrast, the expression of Tnfa, Il6, and Cxcl2 (encoding MIP-2α) mRNA was significantly decreased in Sirt7 KD NRK-52E cells following cisplatin administration (Fig. 5C). The expression of Il1b mRNA in the cisplatin-treated cells was below the detection limit. These findings suggest that SIRT7 regulates the cisplatin-induced gene expression of Tnfa, Il6, and Cxcl2 via a cell-autonomous mechanism. Cisplatin treatment significantly induced TNF-α protein production in control NRK-52E cells, but resulted in no increase of TNF-α protein production in Sirt7 KD NRK-52E cells ( Supplementary Fig. S5). TNF-α plays a critical role in renal injury and the increase of MIP-2α expression after cisplatin administration 23,24 , whereas it has been reported that IL-6 does not play an injurious role in cisplatin-induced AKI 25 . These results suggest that SIRT7 controls cisplatin-induced nephrotoxicity, at least partly by regulating the expression of TNF-α.
Scientific RepoRts | (2018) 8:5927 | DOI:10.1038/s41598-018-24257-7 SIRT7 Regulates the Inflammatory Response Through Modulating NF-κB p65 Transcription Activity and Nuclear Translocation. The NF-κB transcription factor, composed of a heterodimer of p50 and p65 subunits, is a key mediator of the inflammatory response, including TNF-α, IL-6, and MIP-2α production [26][27][28][29] . In non-stimulated cells, NF-κB largely resides in the cytoplasm where it is bound by its inhibitory IκB family proteins. After stimulation, IκB proteins are phosphorylated by IκB kinase and degraded by the ubiquitin proteasome system 30,31 . Degradation of IκB proteins liberates NF-κB, allowing it to translocate to the nucleus. SIRT1, 2, and 6 regulate the function of NF-κB by interacting with p65 32-34 . Thus, we examined the effect of SIRT7 on NF-κB activity. Control and Sirt7 KD NRK-52E cells were transfected with a p65 expression plasmid together with a pNF-κB luciferase reporter gene. Transfection of p65 activated the reporter gene by 188.9-fold in control NRK-52E cells. However, the transactivation activity of p65 in Sirt7 KD cells was significantly decreased by 41.6% (p < 0.01) (Fig. 6A). Overexpression of SIRT7 in Sirt7 KD NRK-52E cells restored the transactivation of NF-κB activity (Fig. 6B). These results indicate that NF-κB activity is reduced in SIRT7-deficient NRK-52E cells. Previous studies have revealed that FAF1, NME1, SNIP1, ING4, NFκBIL1, and TNFAIP1 regulate the activity of NF-κB in various ways [35][36][37][38][39][40][41] . Since SIRT7 is reported to bind to the proximal promoter regions of these genes 9 , SIRT7 might increase NF-κB activity by affecting their gene expression. However, the expression levels of these genes were similar between control and Sirt7 KD NRK-52E cells ( Supplementary Fig. S6). SIRT1 regulates the activity of NF-κB through the deacetylation of p65. We then examined whether SIRT7 can also deacetylate p65. A co-immunoprecipitation assay revealed that SIRT7 as well as SIRT1 bound to p65 in HEK293T cells (Fig. 6C). Deacetylation of p65 by SIRT1 was detected in these cells (Fig. 6D, lane 3) as described previously 32 , while SIRT7 overexpression did not decrease the acetylation status of p65 (Fig. 6D, lane 2). These results indicate that SIRT7 has no deacetylase activity for p65. Western blot analysis revealed that the expression levels of p65, phospho-p65, and IκBα proteins in whole cell lysates were unchanged between the control and Sirt7 KD NRK-52E cells (Fig. 7A). We next investigated nuclear p65 expression after cisplatin treatment. Exposure of the control NRK-52E cells to cisplatin markedly increased the levels of nuclear p65, whereas this increase was significantly blunted in the Sirt7 KD NRK-52E cells (Fig. 7B). The suppression of increased nuclear p65 expression by cisplatin was also detected in Sirt7 KO mouse embryonic fibroblast cells (Supplementary Fig. S7). We next examined the effect of SIRT7 on the cellular location of p65 by immunofluorescence microscopy. Transiently transfected p65 was mainly cytoplasmic in both untreated control and Sirt7 KD NRK-52E cells (Fig. 7C). After cisplatin stimulation, p65 staining was detected in the nucleus of the control cells, but p65 signals mostly remained cytoplasmic after cisplatin exposure in the Sirt7 KD cells (Fig. 7C). Relative nuclear p65 fluorescence intensity was significantly decreased in Sirt7 KD NRK-52E cells.
Finally, we examined nuclear p65 protein expression in the kidney of Sirt7 KO mice. Western blotting analysis revealed that nuclear p65 protein expression was significantly decreased in the kidney of Sirt7 KO mice compared with that in WT mice after cisplatin administration (Fig. 8A). Immunohistochemical analysis also showed that the number of nuclear p65-positive tubular cells was significantly decreased in the Sirt7 KO mice compared with that in the WT mice (Fig. 8B). These results suggest that the loss of SIRT7 suppresses the nuclear accumulation of p65 after cisplatin exposure both in vitro and in vivo.

Discussion
Inflammation and ROS-generated oxidative stress are involved in cisplatin-induced nephrotoxicity [1][2][3] . Previous studies have indicated that SIRT1 and SIRT3 are protective in cisplatin-induced kidney injury [6][7][8] . Interestingly, SIRT7 apparently plays an opposite role. TNF-α plays a critical role in the pathogenesis of cisplatin-induced AKI 23,24 and stimulates the expression of a number of inflammatory cytokines and chemokines (e.g., MIP-2α). NADPH oxidase is a significant source of ROS. TNF-α also enhances ROS production by increasing the expression of NOX2 and p47phox, which are subunits of the NADPH oxidase complex 42,43 . The expression of Tnfa, Cxcl2 (encoding MIP-2α), Cybb (encoding NOX2), and Ncf1 (encoding p47phox) mRNA was significantly reduced in the cisplatin-treated kidney of Sirt7 KO mice. Thus, SIRT7 deficiency might ameliorate cisplatin-induced renal injury at least in part by reducing the expression of TNF-α. SIRT2 deficiency ameliorates lipopolysaccharide-induced acute tubular injury and suppresses renal failure with decreased renal Cxcl2 mRNA expression 44 . Thus, the reduced expression of Cxcl2 might also be involved in the amelioration of cisplatin-induced kidney injury.
Inflammation is one of the key processes that contributes to renal injury at an acute stage in the UUO model 22 . The expression levels of Tnfa and Cxcl2 were significantly decreased in the obstructed kidney of Sirt7 KO mice. Decreased expression of inflammation-related genes is also detected in the heart of Sirt7 KO mice after myocardial infarction and in the white adipose tissue of high-fat diet-fed Sirt7 KO mice 10,14 . These findings strongly suggest that SIRT7 plays critical roles in the regulation of inflammation. In the present study, we identified that NF-κB Figure 6. Regulation of the transactivation activity of NF-κB by SIRT7. (A,B) The transcriptional activity of NF-κB was examined using the dual luciferase reporter assay. Each Sirt7 KD (shSirt7) and control (shCT) NRK-52E cell was transfected with 80 ng pCI-HA-p65 expression plasmid or pCI-HA-control plasmid as well as 20 ng pNF-κB-Luc plasmid, which contains multiple copies of the NF-κB consensus sequence, and 0.8 ng pRL-TK plasmid (n = 4/group). In addition, Sirt7 KD cells were co-transfected with 100 ng pcDNA3-FLAG-Sirt7 expression plasmid or pcDNA3-FLAG-control plasmid (B) (n = 4/group). (C) HEK293T cells were transfected with 2 μg pcDNA3-HA-Sirt1 expression plasmid or pcDNA3-HA-Sirt7 expression plasmid as well as 2 μg pcDNA3-FLAG-p65 expression plasmid. At 24 h after transfection, an immunoprecipitation assay and western blotting were performed. (D) HEK293T cells were transfected with 1 μg pcDNA3-HA-Sirt1 expression plasmid or pcDNA3-HA-Sirt7 expression plasmid as well as 0.5 μg pcDNA3-FLAG-p65 expression plasmid and 0.5 μg pCMVβ-p300-myc expression plasmid. After 36 h, an immunoprecipitation assay was performed and p65 acetylation was detected by western blotting. *p < 0.05, **p < 0.01. Data are expressed as the mean ± SEM. activity and the expression of TNF-α are decreased in cisplatin-treated Sirt7 KD NRK-52E kidney cells. However, considering the important roles of macrophages in the progression of inflammation and the anti-inflammatory roles of SIRTs in macrophages 45,46 , further investigation is required to clarify the relative contribution of TNF-α production by SIRT7 in infiltrating immune cells using macrophage-specific Sirt7 KO mice.
SIRTs regulate NF-κB function in multiple ways. SIRT6 suppresses NF-κB target gene expression by deacetylating histone H3 lysine 9 and destabilizing the binding of NF-κB to chromatin 34 . Phosphorylation of p65 stimulates acetylation at Lys 310, and this acetylation enhances the transcriptional activity of NF-κB 47 . SIRT1 and SIRT2 inhibit NF-κB activity through deacetylation of p65 at Lys 310 32,33 . We found that total p65 expression levels were unchanged, but nuclear p65 expression after cisplatin exposure was decreased in Sirt7 KD NRK-52E cells. We also found that Sirt7 deficiency in mouse aortic smooth muscle cells led to the suppression of lipopolysaccharide-induced p65 nuclear accumulation (unpublished data). Thus, SIRT7 may regulate the nuclear accumulation of NF-κB p65. SIRT7 is located in the nucleus, while p65 is predominantly present in the cytosol of unstimulated cells. However, there is substantial evidence indicating that NF-κB-IκBα complexes are not static in their localization but are shuttled continuously between the nucleus and cytoplasm [48][49][50] . It may not be unanticipated, therefore, that SIRT7 binds to p65 in the nucleus and regulates this shuttling. Although SIRT7 could not deacetylate p65, recent studies have shown that SIRT7 exhibits desuccinylase or defatty-acylase activity 51,52 . Therefore, SIRT7 might regulate nuclear p65 translocation by regulating non-acetyl-lysine modifications. Lysine acetylation affects protein-protein interactions 53 . Alternatively, SIRT7 might regulate p65 nuclear translocation indirectly by deacetylating p65-interacting proteins. Further studies are necessary to address how SIRT7 regulates p65 translocation to the nucleus.
Several studies have shown that KD of Sirt7 increases apoptosis in vitro 54,55 . We also found that Sirt7 KD promoted apoptosis in NRK-52E cells (data not shown). In contrast, fewer TUNEL-positive cells were noted in the kidney of Sirt7 KO mice compared with WT mice. Cisplatin-induced proinflammatory cytokines increase apoptosis in renal cells 56,57 . Thus, inhibition of the recruitment and accumulation of inflammatory cells by the suppression of cytokines and chemokines in Sirt7 KO mice might be responsible for the overall anti-apoptosis effect in vivo. Further studies are also needed to clarify the relationship between SIRT7 and apoptosis.
In conclusion, these results demonstrated that SIRT7 deficiency ameliorated cisplatin-induced AKI in mice. Our findings suggest that SIRT7 is an important novel therapeutic target for cisplatin-induced AKI.

Methods
Animal Experiments. All experimental procedures were performed in accordance with the guidelines of the Institutional Animal Committee of Kumamoto University and were approved by the Committee on Animal Research at Kumamoto University. Except when indicated, 10-week-old WT and Sirt7 KO mice with a C57/BL6J background were used for this study. The generation of Sirt7 KO mice and Sirt7 FRT/floxed mice was described previously 10,58 . The mice were housed in a temperature-and humidity-controlled environment with a 12:12-h light-dark cycle and were fed a standard normal diet ad libitum with free access to water. Cisplatin (cis-diammineplatinum(II) dichloride; Sigma Aldrich, St. Louis, MO) was dissolved in normal saline at a concentration of 1.0 mg/mL, and was given by intraperitoneal injection of 20 mg/kg body weight as described previously 59 . Metabolic cages were used for 24-h urine collections. Blood and kidneys were harvested at 72 h after cisplatin injection. Serum chemistry analysis was performed by a commercial laboratory (SRL, Tokyo, Japan).
Urinary NGAL concentrations were measured using a mouse NGAL ELISA Kit (BioPorto Diagnostics, Gentofte, Denmark). For creatinine adjustment, urine creatinine levels were measured using a Lab Assay Creatinine Kit (Wako, Osaka, Japan).
Histology and Tubular Injury Score. The kidneys were fixed with 10% formalin neutral buffer solution and embedded in paraffin. Sections (4-μm thick) were stained using Periodic acid-Schiff and examined under a light microscope. The tubular injury score was calculated on a scale of 0-5 on the basis of the percentage of tubules with necrosis, cast formation, dilation, or loss of the brush border: 0, 0%; 1, 1-10%; 2, 11-25%; 3, 26-45%; 4, 46-75%; and 5, 76-100% 60 . A pathologist evaluated 5 randomly selected fields per section of the mouse kidney at a magnification of ×400 in a blind manner. Immunohistochemical Staining. Deparaffinized sections were subjected to microwave pretreatment with a pH 6.0 citrate buffer for p65 immunostaining or pretreatment with proteinase K for F4/80 immunostaining 61 . After the reaction of each primary antibody (anti-p65 antibody, #8242, Cell Signaling Technology, Beverly, MA, and anti-F4/80 antibody clone CI:A3-1, Serotec, Oxford, UK), the samples were incubated with horseradish peroxidase (HRP)-labeled goat anti-rat or goat anti-rabbit antibodies (Nichirei, Tokyo, Japan). The reaction was visualized using the diaminobenzidine system (Nichirei).
Immunofluorescence. Each chamber of a 4-chamber 35-mm glass bottom dish were seeded with 5.0 × 10 3 shRNA-introduced NRK-52E cells and incubated for 24 h. Then, the cells were transfected with 0.5 μg pCI-HA-p65 expression plasmid in each chamber using jetPRIME (Polyplus-transfection, Strasbourg, France). At 24 h after transfection, the cells were treated with or without 30 μM cisplatin for 1 h. Then, the cells were washed with PBS and fixed in a 10% formalin neutral buffer solution for 15 min. The fixed cells were incubated with 0.1% Triton X-100 in PBS for 10 min to permeabilize the cells. In the blocking step, the cells were covered with Blocking One (Nacalai Tesque) for 20 min. The cells were incubated at 4 °C overnight with primary antibodies against the hemagglutinin (HA) tag (1:500, Roche Diagnostics). To detect the primary antibody, the sections were incubated with anti-rat Alexa Fluor 555-conjugated antibodies (1:1000, Life Technologies). Hoechst 33342 (Dojindo, Kumamoto, Japan) was used for nuclear staining. The fluorescence intensity in the regions of interests (ROI) in the nucleus and cytoplasm was measured using ImageJ software, as described previously 62 . Relative fluorescence intensity is represented by the ratio of nuclear fluorescence intensity to cytoplasmic fluorescence intensity.
Luciferase Assay. The NF-κB luciferase reporter plasmid (pNF-κB-Luc, Takara) was used for measuring NF-κB activity, and the pRL-TK plasmid was used as an internal control. The pCI-HA-p65 expression plasmid was used for examining the expression of p65, and the pCI-HA-control plasmid was used as a control. The pCI-HA-p65 expression plasmid was constructed as follows: the HA tag was inserted into the pCI-p65 expression plasmid (gifted from Dr. Takashi Minami) by PCR using HA-containing primers. The pcDNA3-FLAG-Sirt7 expression plasmid was used for the expression of Sirt7, and the pcDNA3-FLAG-control plasmid was used as a control.
The shRNA-introduced NRK-52E cells were seeded in 12-well plates at 7.5 × 10 4 cells/well and maintained for 24 h. Subsequently, the pNF-κB-Luc plasmid and pRL-TK plasmid were co-transfected into the cells with expression plasmids using jetPRIME (Polyplus-transfection). The luciferase assay was performed 24 h later with the Dual-Luciferase reporter assay system (Promega, Madison, WI).