Sirt1–hypoxia‐inducible factor‐1α interaction is a key mediator of tubulointerstitial damage in the aged kidney

Abstract Although it is known that the expression and activity of sirtuin 1 (Sirt1) decrease in the aged kidney, the role of interaction between Sirt1 and hypoxia‐inducible factor (HIF)‐1α is largely unknown. In this study, we investigated whether HIF‐1α could be a deacetylation target of Sirt1 and the effect of their interaction on age‐associated renal injury. Five‐week‐old (young) and 24‐month‐old (old) C57Bl/6J mice were assessed for their age‐associated changes. Kidneys from aged mice showed increased infiltration of CD68‐positive macrophages, higher expression of extracellular matrix (ECM) proteins, and more apoptosis than young controls. They also showed decreased Sirt1 expression along with increased acetylated HIF‐1α. The level of Bcl‐2/adenovirus E1B‐interacting protein 3, carbonic anhydrase 9, Snail, and transforming growth factor‐β1, which are regulated by HIF‐1α, was significantly higher in aged mice suggesting that HIF‐1α activity was increased. In HK‐2 cells, Sirt1 inhibitor sirtinol and siRNA‐mediated knockdown of Sirt1 enhanced apoptosis and ECM accumulation. During hypoxia, Sirt1 was down‐regulated, which allowed the acetylation and activation of HIF‐1α. Resveratrol, a Sirt1 activator, effectively prevented hypoxia‐induced production of ECM proteins, mitochondrial damage, reactive oxygen species generation, and apoptosis. The inhibition of HIF‐1α activity by Sirt1‐induced deacetylation of HIF‐1α was confirmed by Sirt1 overexpression under hypoxic conditions and by resveratrol treatment or Sirt1 overexpression in HIF‐1α‐transfected HK‐2 cells. Finally, we confirmed that chronic activation of HIF‐1α promoted apoptosis and fibrosis, using tubular cell‐specific HIF‐1α transgenic mice. Taken together, our data suggest that Sirt1‐induced deacetylation of HIF‐1α may have protective effects against tubulointerstitial damage in aged kidney.

impaired ability to recover from acute kidney injury, contributing to the development of chronic kidney disease (Epstein, 1996). Hypoxia is a well-known inducer of acute kidney injury, and it has also been shown to increase in the aged kidney, especially in the cortex (Tanaka et al., 2006). Since hypoxia-inducible factor-1 α(HIF-1α) is a central regulator of cellular adaptation to hypoxia, it is plausible that chronic activation of HIF-1α can occur in the aged kidney. Although HIF-1α appears to exert a protective effect on renal damage in acute kidney injury (Bernhardt et al., 2006;Ma et al., 2009;Matsumoto et al., 2003;Weidemann et al., 2008), chronic sustained activation of HIF-1α in renal tubular epithelial cells has been found to promote epithelial to mesenchymal transition and kidney fibrosis (Higgins et al., 2007).
Sirtuin 1 (Sirt1), a mammalian homolog of yeast silent information regulator 2 (Sir2), is a survival factor that is involved in lifespan extension (Dali-Youcef et al., 2007;Imai, Armstrong, Kaeberlein, & Guarente, 2000). Previous studies have reported that Sirt1 mediates a wide range of cellular responses through its deacetylation activity targeting numerous transcription factors such as p53, forkhead box O 3 (Foxo3), nuclear factor-κB (NF-κB), and peroxisome proliferatoractivated receptor gamma coactivator-1α (PGC)-1α (Feige & Auwerx, 2008). Recently, it has been reported that Sirt1 binds to HIF-1α and deacetylates its lysine residues (Joo et al., 2015;Laemmle et al., 2012;Lim et al., 2010). However, the effect of Sirt1-HIF-1α interaction on the activity of HIF-1α remains unclear and whether the interplay between Sirt1 and HIF-1α would be protective or harmful in the aged kidney has not been reported. Given that both HIF-1α and Sirt1 play important roles in the cellular response to hypoxic stress in the aged kidney, we propose that the balance between them modulates the acetylation status and activity of HIF-1α by which the latter mediates the progression of age-associated renal damage.

| The aged kidney showed increased extracellular matrix production, macrophage infiltration, and apoptosis
We first examined the functional and structural changes of the aged kidney. As shown in Table 1, aged mice showed higher kidney/body weight ratio and albuminuria as compared with young mice. Glomerular filtration rate (GFR) was significantly lower in the aged mice than in the young control mice. However, blood urea nitrogen (BUN), creatinine, and cystatin C levels were not different between the two groups. Histological analysis exhibited increases in the mesangial matrix area and glomerular size in the aged mice compared to the young mice ( Figure 1a). The expression of type I/IV collagen and fibronectin was significantly higher in the renal cortex of the aged mice compared with the young controls ( Figure 1b). Masson's trichrome staining confirmed a higher deposition of collagen in the aged mice than in the young mice ( Figure 1a). As shown in Figure 1c, the aged mice exhibited a significantly higher number of CD68-positive cells and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-positive apoptotic cells than the young controls.

| The aged kidney showed decreased Sirt1 expression but increased HIF-1α expression and its acetylation
Since both Sirt1 and HIF-1α play critical roles in cellular adaptation to chronic hypoxic stress in the aging process, we examined the expressions of Sirt1 and HIF-1α in the aged kidney. As shown in Figure 1d,e, the aged kidney showed significantly lower expression of Sirt1 but higher expression of HIF-1α, mostly in the acetylated form.

| Sirt1 inhibition increased ECM production and apoptosis in HK2 cells
To investigate whether Sirt1 interacts with HIF-1α in renal tubular epithelial cells, we first examined the effect of Sirt1 expression on extracellular matrix (ECM) production and apoptosis in HK2 cells. As shown in Figure 2a, a Sirt1 inhibitor, sirtinol, significantly induced collagen I and IV production as well as apoptotic markers such as cleaved caspase-3 and cleaved poly (ADP-ribose) polymerase (PARP).
Similar findings were also observed by siRNA-mediated knockdown of Sirt1 (Figure 2b). The role of Sirt1 in these changes was further confirmed by the observation that overexpression of Sirt1 induced T A B L E 1 Characteristics of young (5-week-old) and aged (24month-old) mice

Young Aged
Body weight ( the opposite changes ( Figure 2c). Since we were interested in the interaction of Sirt1 and HIF-1α, we also tested whether modifying Sirt1 could change HIF-1α expression even under normoxia. Interestingly, sirtinol or siRNA-mediated knockdown of Sirt1 significantly enhanced HIF-1α expression in HK2 cells (Figure 2d).

| Hypoxia decreased Sirt1 expression leading to the acetylation and activation of HIF-1α in HK2 cells
We then analyzed the effect of hypoxia on Sirt1 expression and its interaction with HIF-1α. During hypoxia, the expression of Sirt1 was significantly reduced whereas HIF-1α, collagen I/IV, and cleaved PARP were upregulated (Figure 3a,b). The interaction between Sirt1 and HIF-1α was determined by immunoprecipitation. After endogenous HIF-1α was induced by hypoxia, Sirt1-HIF-1α binding was observed (Figure 3c). We next examined whether Sirt1 deacetylates HIF-1α. Lysyl acetylation of HIF-1α was detected by immunoblotting with anti-acetyl-lysine in HIF-1α immunoprecipitates.
As shown in Figure 3d, Sirt1 overexpression significantly decreased HIF-1α acetylation suggesting that Sirt1 regulates lysyl acetylation of HIF-1α. These results suggest that the down-regulation of Sirt1 induced by hypoxia might lead to increased acetylation of HIF-1α.
To test the functional consequences of HIF-1α acetylation, HIF-1α transcriptional activity was measured by luciferase reporter assay and mRNA transcription of HIF-1 target genes such as CA9, Bnip3, Snail, and TGF-β1 as well as ECM-related genes. As shown in Figure 3e, hypoxia-induced lysyl acetylation of HIF-1α was associated with markedly increased transcriptional activity of HIF-1α.

| Sirt1 activation induced deacetylation and inactivation of HIF-1α and subsequently rescued hypoxia-induced tubular changes
To examine whether Sirt1 activation deacetylates HIF-1α and decreases its activity, HK-2 cells were exposed to hypoxia with or without resveratrol treatment. A Sirt1 activator, resveratrol, 0.64 ± 0.08 mg/dl in the older group) was not different between the transgenic and the wild-type mice.

| DISCUSSION
In the current study, we have uncovered that acetylation of HIF-1α increases its activity and exerts proapoptotic and profibrotic effects in the aged kidney model as well as in hypoxia-induced tubular responses in vitro. Furthermore, we demonstrated that chronic activation of HIF-1α specifically in renal tubular epithelial cells promoted apoptosis and fibrosis. Our findings that Sirt1 directly interacts with F I G U R E 3 Hypoxia decreases Sirt1 expression leading to the acetylation and activation of HIF-1α in HK2 cells. Cells were incubated under hypoxia (1% O 2 ) for 6 hr (a) or 24 hr (a-e). Representative western blots show the protein levels of HIF-1α, collagen I and IV, cleaved poly (ADP-ribose) polymerase (PARP), and Sirt1. Equal amounts of protein were subjected to immunoprecipitation with Sirt1 antibody (c) or HIF-1α antibody (d) followed by immunoblotting with antibody against Sirt1, HIF-1α, or acetyl-lysine, n = 6-7. (e) Cells were transfected with luciferase reporter vector for hypoxia-responsive element (HRE), and luciferase activity was measured, n = 3. Real-time RT-PCR was performed to measure the level of mRNA expressions of carbonic anhydrase 9 (CA9), Bcl-2 adenovirus E1B-interacting protein 3 (Bnip3), Snail, TGF-β1, collagen I and IV, and fibronectin. n = 3. *p < 0.001 and † p < 0.01 vs. control HIF-1α, resulting in the deacetylation of HIF-1α establish a critical regulation of HIF-1α by Sirt1. We suggest that Sirt1 represses HIF-1α activity by deacetylation at Lys709. However, under hypoxic stress associated with decreased nicotinamide adenine dinucleotide (NAD + ) level and NAD + /NADH ratio (Braidy et al., 2011;Imai, 2011;Lim et al., 2010), the transcriptional activity of HIF-1α would be further increased with the induction of acetylation by decreased Sirt1 activity (Nakahata et al., 2008). Therefore, the deficiency of Sirt1 expression or its activity with the upregulation of HIF-1α observed in the aged kidney could be a key mediator of kidney injury.
Compelling fore, it appears that the effect of interaction between Sirt1 and HIF-1α on the regulation of HIF-1α activity or its expression is cell typespecific and remains unclear.
HIF-1α is a well-known transcription factor that facilitates adaptive cellular responses to hypoxia. Although HIF-1α is known to play a key role in the protection from acute ischemia (Bernhardt et al., F I G U R E 5 Sirt1 overexpression inhibits HIF-1α activity. (a) Cells were transfected with Sirt1 or empty vector and subjected to 24 hr hypoxia (1% O 2 ), n = 3. (b) Cells were transfected with HIF-1α or empty vector and were pretreated with resveratrol (10 µM) for 30 min or cotransfected with Sirt1, n = 6. (c) WT HIF-1α or K709R HIF-1α mutant was coexpressed with Sirt1, n = 6. Cells were transfected with luciferase reporter vector for hypoxia-responsive element (HRE), and luciferase activity was measured. (a, b) Equal amounts of protein were subjected to immunoprecipitation with HIF-1α antibody followed by immunoblotting with antibody against HIF-1α or acetyl-lysine. (a-c) Realtime RT-PCR was performed to measure the level of mRNA expressions of carbonic anhydrase 9 (CA9), Bcl-2 adenovirus E1B-interacting protein 3 (Bnip3), collagen I and IV, and fibronectin. *p < 0.001, † p < 0.005, ‡ p < 0.01, and § p < 0.05 vs. empty vector, ‖ p < 0.05, ¶ p < 0.001, and # p < 0.005 vs. hypoxia or HIF-1αWT. O/E, overexpression; WT, wild-type 2006; Ma et al., 2009;Matsumoto et al., 2003), recent evidence suggested that prolonged unregulated activation of HIF-1α could enhance maladaptive responses, which lead to glomerulosclerosis and interstitial fibrosis in multiple animal models. Genetic ablation of HIF-1α in the renal tubular epithelium has been shown to inhibit tubulointerstitial fibrosis in UUO kidneys (Higgins et al., 2007). On the other hand, the stable expression of HIF-1α by von Hippel-Lindau deletion promoted interstitial fibrosis (Kimura et al., 2008). In a podocyte ablation model, HIF-1α has been shown to induce glomerulosclerosis through interaction with Smad3 (Baumann, Hayashida, Liang, & Schnaper, 2016). Finally, a recent report has shown that HIF-1α blockade prevented the development of diabetic nephropathy in type 1 diabetic mice (Nayak et al., 2016). These data suggested HIF-1α as a mediator of renal damage. Our observations showing that 8-week activation of HIF-1α, specifically in renal tubular epithelial cells, promoted apoptosis and ECM accumulation strongly support a deleterious effect of HIF-1α in renal tubulointerstitial injury.
Considering that hypoxia is the major mechanism involved in the pathogenesis of renal senescence (Nangaku, Inagi, Miyata, & Fujita, 2008), it is expected that HIF-1α expression or activity would be increased in the aged kidney. Indeed, using pimonidazole F I G U R E 6 Renal tubular cell-specific HIF-1α overexpression (O/E) accelerates renal damage. Six-week-old (c, d) or 12-month-old (e, f) paired box 8-reverse tetracycline-responsive transactivator-based conditional HIF-1α transgenic mice and their littermates were treated with doxycycline for 3 days and sacrificed 8 weeks after HIF-1α induction. (a) Immunofluorescent staining using anti-HA antibody; bars = 50 and 20 µm, respectively. (b, d, f) Bcl-2 adenovirus E1B-interacting protein 3 (Bnip3), collagen I and IV, and fibronectin protein expression levels were analyzed using immunoblot analysis, n = 4-6. (c, e) Representative kidney sections stained with PAS, PicroSirius Red, and TUNEL staining; bars = 100 µm for PAS and PicroSirius Red, 50 µm for TUNEL staining, n = 4-7. *p < 0.005, † p < 0.001, and ‡ p < 0.05 vs. wild-type (WT) immunostaining and hypoxia-responsive reporter of transgenic rats, Tanaka et al. have identified significant hypoxia in all areas of the aged kidney, especially in the cortical area (Tanaka et al., 2006). They showed a significant correlation between the level of hypoxia and the degree of age-related tubulointerstitial injury. In this study, we also found a higher expression of HIF-1α and its downstream targets such as Bnip3, Snail, and TGF-β1 in the aged kidney cortex. Since Bnip3 is a well-known player of hypoxia-induced apoptosis and Snail and TGF-β1 are the key regulators of epithelial to mesenchymal transition and renal fibrosis, the activation of HIF-1α-regulated pathways could mediate renal damage. The significance of our study is highlighted by the findings that acetylated HIF-1α is increased in the aged kidney and that activation of HIF-1α induced by acetylation is associated with the development of tubular apoptosis and interstitial fibrosis. Our observations showing that acetylated HIF-1α was localized in the damaged tubular cells with fragmented nuclei and weak expression of E-cadherin indicate the correlation of Sirt1-HIF-1α interaction to damaged area of the aged kidney.
A limitation of our study is that we did not perform in vivo studies using HIF-1α overexpressed mice to test whether increased Sirt1 activity could rescue the tubulointerstitial damage. Further experiments will need to address this issue.
In conclusion, we have identified a role of Sirt1-HIF-1α interaction as a key mediator of the aged kidney. Our study suggests that deacetylation of HIF-1α induced by Sirt1 activation may have a therapeutic benefit to slow kidney damage in the aging process. Further studies will be needed to determine whether acetylated HIF-1α is also increased in other disease models of kidney injury.

| Animals
Male C57Bl/6J mice were obtained from the Korea Research Institute of Bioscience and Biotechnology (Ochang, Korea). The mice were housed in a pathogen-free facility set on a 12-hr light-dark cycle and given free access to water and regular laboratory chow

| Blood and urine chemistry
Serum and urine creatinine, BUN, and serum cystatin C levels were measured using a colorimetric method with a Cobas 8000 analyzer

| siRNA
An effective predesigned siRNA of Sirt1 (Applied Biosystems, Carlsbad, CA, USA) was selected in a preliminary study. The sequence RYU ET AL.

| Western blot analysis
Tissue and cell lysates were subjected to western blot analysis using

| Statistical analyses
The mean values were compared using ANOVA followed by Fisher's least significant difference method. Unpaired two-tailed Student's t tests were used where appropriate. Data are presented as the mean ± standard error (SE). A p value <0.05 was considered statistically significant.

ACKNOWLEDG MENTS
We

CONF LICT OF I NTEREST
None.

AUTHOR CONTRIBU TI ONS
DRR, MRY, and KHK performed the experimental works. HK, SHK, JSJ, and DCH analyzed the data. HN designed the study, analyzed the data, and wrote the manuscript.