Sirt1 Deletion Leads to Enhanced Inflammation and Aggravates Endotoxin-Induced Acute Kidney Injury

Bacterial endotoxin has been known to induce excessive inflammatory responses and acute kidney injury. In the present study, we used a mouse model of endotoxemia to investigate the role of Sirt1 in inflammatory kidney injury. We examined molecular and cellular responses in inducible Sirt1 knockout (Sirt1−/−) mice and wild type littermates (Sirt1+/+) in lipopolysaccharide (LPS)-induced kidney injury. Our studies demonstrated that Sirt1 deletion caused aggravated kidney injury, which was associated with increased inflammatory responses including elevated pro-inflammatory cytokine production, and increased ICAM-1 and VCAM-1 expression. Inflammatory signaling such as STAT3/ERK phosphorylation and NF-κB activation was markedly elevated in kidney tissues of Sirt1 knockout mice after LPS challenge. The results indicate that Sirt1 is protective against LPS-induced acute kidney injury by suppressing kidney inflammation and down-regulating inflammatory signaling.


Introduction
Sepsis arises mostly from bacterial infection which causes multiple organ failure due to excessive systemic inflammation [1,2]. Kidney functions as a natural filter of blood and serves as the first line of defense in our body [3,4]. Unfortunately, it also becomes a direct target of inflammatory injury [5]. Sepsis-induced acute kidney injury (AKI) is very common in the elderly and associated with high mortality [6][7][8]. To date, there has been no effective treatment for this devastating disease [9,10]. Lipopolysaccharide (LPS) challenge is one of the most accepted animal models to explore the underlying mechanisms and potential treatment in sepsis-induced kidney injury [11].
Renal function is significantly compromised during sepsis as indicated by increased blood urea nitrogen (BUN) and urine Kidney injury molecule-1 (KIM-1) levels [12][13][14]. Sepsis also causes renal tubular damage and inflammation as shown by histological analysis [15]. Kidney inflammation is associated with increased production of pro-inflammatory mediators [16], and upregulation of adhesion molecules such as Intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) [17]. Those early inflammatory responses induce leukocyte infiltration during kidney injury [18], which may lead to further damage. Increased cytokine production is a hallmark in many inflammatory diseases including kidney injury [19,20]. Cytokines can induce pro-inflammatory signaling such as activatation of signal transducer and activator of transcription 3 (STAT3) [21], and modulate inflammatory responses through ERK/MAPK cascade [22].
Sirt1, a member of the Sirtuin family [23], is a deacetylase that has been reported to modulate the function of a wide variety of proteins such as NF-kB and p53, through deacetylation of lysine residues [24]. There have been increasing studies suggesting that Sirt1 plays an important role in inflammation, apoptosis, stress resistance, metabolism, differentiation, and aging [8,[25][26][27][28][29][30][31]. In the present study, we investigated the role of Sirt1 in LPS-induced acute kidney injury by inducible deletion of Sirt1 in mice. Our studies demonstrate that Sirt1 knockout mice are highly susceptible to LPS-induced inflammatory kidney injury.

Animal model of acute kidney injury
Mice were housed in cages with free access to food and water in a temperature controlled room with a 12-hour dark/light cycle. All experiments and animal care procedures were approved by the Institutional Animal Care and Use Committee of the University of Texas Health Science Center at Tyler. The generation of inducible Sirt1 knockout mice was described previously [32,33]. Six to seven weeks after the birth, mice were given tamoxifen (100 mg/Kg body weight in corn oil) by intraperitoneal (I.P.) injection daily for 5 days to induce nuclear translocation of Cre recombinase as described previously [33]. Fourteen to fifteen weeks after Sirt1 deletion, age-matched male Sirt1 2/2 and Sirt1 +/ + littermates were used in the studies. Endotoxemia was induced by I.P. injection of 5 mg/kg LPS dissolved in phosphate buffered saline (PBS), control mice were injected with PBS. Experiments were terminated 6 or 24 h after LPS challenge.

Renal function assay and histology analysis
Blood and urine samples were obtained from mice 24 h after LPS challenge, serum BUN and urine KIM-1 levels were examined as markers of renal dysfunction. Paraffin-embedded sections of mouse kidney tissues were stained with hematoxylin and eosin for assessment of renal tubular injury. The histological samples were scored by lab personnel blinded to the samples. The magnitude of tubular injury including tubular dilatation, flattening and vacuolization was scored into five levels (0, none; 1, 0-25%; 2, 25 to 50%; 3, 50 to 75%; and 4, .75%) on the basis of the percentage of affected tubules in a high-power field under light microscope.

ELISA, Immunofluorescence, and Immunoblotting assays
Serum IL-6 and TNF-a levels were determined by ELISA kits (Biolegend). Blood samples were collected 6 and 24 h after LPS challenge. Kidneys samples were obtained 6 h or 24 h after LPS exposure. Immunofluorescence and immunoblotting assays were conducted as described previously [34].

Statistical analysis
Data were analyzed by two-way ANOVA followed by Bonferroni's multiple comparisons tests and expressed as mean 6 SEM. Statistical significance was assigned to P values less than 0.05.

Sirt1 deletion leads to aggravated renal dysfunction after LPS challenge
Serum BUN and urine KIM-1 levels were used as markers of kidney function [14,35]. BUN and KIM-1 levels were increased after LPS challenge, and significantly higher in Sirt1 knockout mice than the wild type littermates (Figure 1 A, B), indicating exacerbated renal dysfunction in Sirt1 knockout mice.
LPS-induced kidney injury was examined in the tubules of the kidney cortex. The mice without LPS challenge showed normal and healthy kidney histology. We detected severe structural damage in the kidneys of Sirt1 2/2 mice (Figure 2). Tubular injury including tubular dilatation, flattening and renal tubular cell vacuolization were markedly increased in Sirt1 2/2 mice when

Sirt1 deletion leads to increased production of proinflammatory cytokines after LPS challenge
The effects of Sirt1 deletion on systemic inflammatory responses were determined by examining serum IL-6 and TNF-a levels. We observed that LPS-induced IL-6 and TNF-a production were significantly increased in Sirt1 2/2 mice when compared with Sirt1 +/+ littermates after LPS exposure (Figure 3), suggesting that Sirt1 modulates systemic production of pro-inflammatory cytokines.

Sirt1 deletion leads to increased neutrophil infiltration in the kidney after LPS challenge
To further assess the effects of Sirt1 deletion on LPS-induced kidney inflammation, neutrophil infiltration into the kidney was examined using neutrophil-specific Gr-1 antibody. No neutrophil infiltration was detected in the control group. LPS challenge led to increased neutrophil infiltration in the kidney, which was much more severe in Sirt1 knockout mice (Figure 4 A, B).

Sirt1 deletion leads to increased ICAM-1/VCAM-1 expression in the kidney after LPS challenge
Adhesion molecules on vascular endothelial cells are major determinants of vascular inflammation [36]. We examined the

Sirt1 deletion leads to enhanced inflammatory signaling in the kidney after LPS challenge
We then conducted experiments to investigate the mechanisms of sirt1 regulation of the kidney inflammation. Inflammatory signaling including Stat3 pathway has been reported to play an important role in the development of kidney injury [37]. Our results indicate that STAT3 phosphorylation was increased in Sirt1 2/2 mice when compared with Sirt1 +/+ littermates ( Figure 6A, C). To confirm Sirt1 regulation of inflammatory signaling, we also examined ERK phosphorylation in the kidney after LPS challenge. The results showed that Sirt1 deletion caused significant increases in ERK1/2 phosphorylation ( Figure 6B, D). Furthermore, Sirt1 deletion led to increased NF-kB activation in kidney tissues after LPS challenge as demonstrated by higher IkBa phosphorylation and degradation (Figure 7), suggesting that Sirt1 down-regulation of inflammatory signaling could be one of the mechanisms contributing to its protection against kidney inflammation.

Discussion
Sepsis remains a devastating disease without cure. While many studies have been done to explore the treatment for the disease [38][39][40][41][42], the mechanisms of septic shock and associated multiorgan failure are yet to be determined [43,44]. Endotoxemia is responsible for sepsis-induced excessive inflammatory injury including acute kidney injury in many clinic settings [45]. The Kidney, as a maintainer of internal environment homeostasis [3,4], is vulnerable to tissue injury caused by toxins and proinflammatory mediators [6], and studies are needed to identify protective mechanisms against sepsis-induced kidney injury.
Altered Sirt1 expression and function have been associated with many pathological changes [46]. We reported previously that Sirt1 plays an important role in regulating lung inflammation and coagulation responses [33]. Interestingly, Sirt1 expression is reduced during aging and in some pre-existing inflammatory diseases such as COPD and alcoholic fatty liver disease [47,48], and the elderly are also highly susceptible to inflammatory disorders including sepsis [23,47,48].
The progression of sepsis is likely associated with two phases. A systemic inflammatory response syndrome (SIRS) phase and a compensatory anti-inflammatory response syndrome (CARS) phase. However, not all patients who suffer from SIRS develop into severe sepsis [49]. Tight control of the balance between the two phases could be an important means of suppressing excessive inflammation [50]. Our studies demonstrate that Sirt1 deletion leads to aggravated inflammatory kidney injury. Given the reduced Sirt1 expression during aging [51], our results may provide an insight on why the elderly are more susceptible to sepsis-associated kidney injury. Therefore, Sirt1 could be considered as a potential target to treat inflammatory kidney injury in the aged population.
Pro-inflammatory cytokines such as TNF-a and IL-6 have been known to play a critical role in sepsis-induced inflammatory injury [19,43]. Our results showed that Sirt1 deletion led to increased LPS-induced IL-6 and TNF-a production, suggesting that Sirt1 acts to suppress inflammatory responses during sepsis. The cytokines is also known to play an important role in kidney injury [37]. Inflammatory signaling such as STAT-3 activation promotes kidney inflammatory responses [52]. In our studies, we showed that Sirt1 deletion led to enhanced pro-inflammatory signaling as demonstrated by increased STAT and ERK phosphorylation. Interestingly, Sirt1 expression in the kidney is higher in young mice and decreased during aging [53]. Even though it will be intriguing to link uncontrolled inflammatory responses in the elderly during sepsis to the reduced Sirt1 expression, more studies are needed to elucidate the role of Sirt1 in aging-related kidney injury.
Excessive neutrophil infiltration often leads to inflammatory tissue injury including acute kidney injury [18]. Leukocyte migration to the injured sites could help to remove dead cells and promote the repair process [54], however, excessive neutrophil accumulation can result in tissue damage [35]. Neutrophil infiltration across the vasculature is a multistep process which requires neutrophil/endothelial interactions through adhesion molecules ICAM-1 and VCAM-1 [36]. Our results demonstrated that Sirt1 deletion led to increased ICAM-1/VCAM-1 expression and neutrophil infiltration in the kidney after LPS challenge, suggesting that Sirt1 modulates some key factors needed for sustained inflammatory responses. Sirt1, a member of Class III Histone Deacetylases [55], modulates transcriptional activities of associated transcriptional factors, cofactors, and histones by controlling their acetylation [56,57]. NF-kB pathway is a central signaling node in inflammatory cytokine production and activation [55]. NF-kB, a master transcription regulator which also controls ICAM-1/VCAM-1 expression [58], is an endogenous substrate of Sirt1 [24]. Deacetylation of NF-kB inhibits its activity and has been linked to the anti-inflammatory function of Sirt1 [59]. Our data indicate that Sirt1 modulates NF-kB pathway in the kidney including IkBa phosphorylation and degradation. In summary, Sirt1 plays a protective role against inflammatory kidney injury in endotoxemia. Sirt1 exerts its function likely through multiple pathways such as suppressing STAT3, ERK1/2, and NF-kB activation. Our studies indicate that Sirt1 is a potential therapeutic target to treat sepsis-induced kidney injury.