Nicotinamide Mononucleotide, an NAD+ Precursor, Rescues Age-Associated Susceptibility to AKI in a Sirtuin 1–Dependent Manner

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ABSTRACT

The rapid growth of an aging population creates challenges regarding age-related diseases, including AKI, for which both the prevalence and death rate increase with age. The molecular mechanism by which the aged kidney becomes more susceptible to acute injury has not been completely elucidated. In this study, we found that, compared with the kidneys of 3-month-old mice, the kidneys of 20-month-old mice expressed reduced levels of the renal protective molecule sirtuin 1 (SIRT1) and its cofactor NAD+. Supplementation with nicotinamide mononucleotide (NMN), an NAD+ precursor, restored renal SIRT1 activity and NAD+ content in 20-month-old mice and further increased both in 3-month-old mice. Moreover, supplementation with NMN significantly protected mice in both age groups from cisplatin-induced AKI. SIRT1 deficiency blunted the protective effect of NMN, and microarray data revealed that c-Jun N-terminal kinase (JNK) signaling activation associated with renal injury in SIRT1 heterozygotes. In vitro, SIRT1 attenuated the stress response by modulating the JNK signaling pathway, probably via the deacetylation of a JNK phosphatase, DUSP16. Taken together, our findings reveal SIRT1 as a crucial mediator in the renal aging process. Furthermore, manipulation of SIRT1 activity by NMN seems to be a potential pharmaceutical intervention for AKI that could contribute to the precise treatment of aged patients with AKI.

BASIC RESEARCH

It is well documented that the incidence of AKI increases with age.1,2 Multiple studies have shown that aging is an independent risk factor for AKI and its associated mortality and morbidity.1–4 Although aging-associated comorbidities and iatrogenic factors are responsible for these outcomes, age-related cellular deterioration and an impaired response of the renal cells to stress have also been recognized to increase the susceptibility of the kidney to injury.5–8

Recent studies have shown that sirtuin 1 (SIRT1) plays an important role in the cellular response to stress and has been shown to extend the lifespan, at least in lower animals.9–11 SIRT1, an NAD+-dependent deacetylase, is a member of the Sirtuin family, which has seven family members. SIRT1 is primarily localized to the nucleus, where it deacetylates many nuclei proteins and transcription factors. SIRT1 has been shown to underlie calorie restriction–related health benefits, including prolonged lifespan and reduced age-related deterioration.12–14 Consistently, activators of SIRT1, including resveratrol and synthetic SIRT1–activating compounds, have been shown to promote health in experimental animals,15–18 although the results from human studies are somewhat variable, probably because of the complexities of the mechanisms of their action.19–22

NAD+ is known to mediate hydrogen transfer in the intracellular metabolic pathway in mitochondria and the redox reaction. NAD+ has also been found to be an indispensable cofactor for several important enzymatic reactions, including SIRT1, supporting an important role of SIRT1 in modulating the cellular response to metabolism and oxidative stress.23–25 Accumulation studies suggest that cellular NAD+ levels reduce with aging26–28 and are accompanied by reduced SIRT1 activity.5,14 Boosting NAD+ is associated with lifespan extension,26,29 and restoring NAD+ with NAD+ precursors, such as nicotinamide mononucleotide (NMN) and nicotinamide riboside, corrects many metabolic abnormalities.30–33

In this study, we examined the role of NAD+/SIRT1 in the susceptibility to AKI in aged animals. Our study shows that NAD+ and SIRT1 deficits in aged mice kidneys contributed to increased vulnerability to cisplatin-induced AKI; NMN treatment rescued the aged kidneys from cisplatin-induced AKI in an SIRT1-dependent manner. The mechanism by which NAD+/SIRT1 protects the kidney involves epigenetic regulation of the c-Jun N-terminal kinase (JNK) pathway. This study sheds light on the mechanisms underlying age-associated susceptibility to AKI and identifies endogenous NAD+ as a potential therapeutic target for AKI, particularly in the elderly.

RESULTS

 

The Effect of NMN Is Dependent on SIRT1

To determine whether the protective effect of NMN depends on SIRT1, we examined the effect of NMN on SIRT1-deficient mice and also, equivalently damaged wild-type mice treated with higher dose of cisplatin (30 mg/kg body wt) (Supplemental Figure 3). As shown in Figure 6 and Supplemental Figure 4, NMN protected the kidney from severe renal injury in wildtype mice, but this therapeutic effect was substantially attenuated when one allele of the SIRT1 gene was deleted. Consistent in vivo data also supported that NMN rescued cisplatininduced cell death in a SIRT1-dependent manner.

Figure 1. Aging worsens acute kidney injury induced by cisplatin. Renal function evaluated by (A) BUN and (B) serum creatinine in the indicated groups at 72 hours after saline or cisplatin treatment (n=5 for saline-treated groups and n=8 for cisplatin-treated groups). (C) Tissue damage scored by the percentage of damaged tubules in cisplatinor saline-treated 3and 20-month-old mouse kidneys (n=6). (D) Representative hematoxylin and eosin–stained sections at both low and high magnification. (E) EM examination of renal PTCs in the indicated groups is shown and quantified. Data are expressed as mean6SEM and were analyzed using unpaired Student’s t test between two groups and ANOVA between multiple groups followed by post-tests. Scale bars, 2 mm. *P,0.05; **P,0.01.

Figure 2. Decreased SIRT1 expression and NAD+ levels in the aged kidney. (A) Relative mRNA expression of SIRT1 in the kidney cortex of 3and 20-month-old 129 mice (n=6). (B) Representative Western blotting image of SIRT1 protein expression in the kidney cortex. b-Actin was used as the loading control. (C) NAD+ level and (D) the relative mRNA expression of NAMPT, NMNAT1, and NMNAT3 in kidney cortexes of 3and 20-month-old 129 mice (n=6). Data are expressed as mean6SEM and were analyzed by unpaired Student’s t test. *P,0.05; ****P,0.001.

NMN Treatment Benefits Kidney from IschemiaReperfusion Injury

We further verified the therapeutic effect of NMN in an additional AKI model; 22 minutes of bilateral renal ischemia was induced in wild-type mice, and NMN or PBS was administrated before the surgery and 24 hours after reperfusion. Kidney function and histology analysis were performed at 48 hours after reperfusion. As shown in Figure 7, mice treated with NMN had much lower BUN and serum creatinine levels and improved tubular damage compared with mice treated with PBS. Therefore, beneficial effects of NMN on kidney were shown in both cisplatinand ischemia-reperfusion–induced AKI, indicating endogenous NAD+ as a potential target for AKI treatment.

The JNK Signaling Pathway Is Activated in Age-Associated AKI

A microarray study was conducted to explore the molecular mechanism involved in age-associated susceptibility to AKI. Renal cortex samples of SIRT1-intact or -deficient mice subjected to cisplatin or saline as the control were assigned in the mRNA microarray. Each group contained three biologic duplicates. Extensive pathway-network analysis of the array data pointed to MAPK/JNK signaling being responsible for increased acute renal injury in the absence of one allele of

SIRT1 (Supplemental Figure 5). The microarray data were validated by Western blotting of the three main signaling modules in the MAPK pathway. JNK other than p38 or extracellular signal–regulated kinase 1/2 (ERK1/2) showed a robust activation via the enhanced phosphorylation of JNK as well as c-JUN and ATF2 (Figure 8A). In addition, JNK signaling activation was observed in the age-associated injured kidney (Figure 8B).

The JNK Inhibitor Prevents Cisplatin-Induced Apoptosis in Cultured HK-2 Cells

To determine whether JNK activation was associated with SIRT1 deficiency–enhanced cell injury, we examined the effect of JNK inhibitor on cell survival and apoptosis in cells deficient in SIRT1. Consistent with SIRT1 knockout mice, knocking down SIRT1 by siRNA in cultured HK-2 cells sensitized the cells to cisplatin (Figure 9, A and B). Transfection with SIRT1 siRNA or scrambled RNA HK-2 cells showed signs of apoptosis and the phosphorylation of JNK after exposure to cisplatin. Cell viability was decreased and cleaved caspase-3 protein expression was enhanced in cisplatin-treated SIRT1 knockdown cells compared with the control. SP600125, a JNK inhibitor, blocked JNK activation and reduced apoptosis in cisplatin-treated HK-2 cells (Figure 9, A and B). Interestingly, in addition to the protective effect of the JNK inhibitor on HK-2 cells from cisplatin-induced apoptosis, we

Figure 3. NMN supplementation is associated with SIRT1 activation. (A) NAD+ content and (B) the enzymatic activity of SIRT1 in the kidney cortexes of 3and 20-month-old mice given PBS or NMN (n=5–6 for PBS-treated groups and n=3–6 for NMN-treated groups). (C) Western blotting of the acetylation of Foxo1 was performed to assess the enzymatic activity of SIRT1 in the kidney cortex. GAPDH protein expression was used as the loading control. Data are expressed as mean6SEM and were analyzed by ANOVA between multiple groups followed by post-tests. *P,0.05; **P,0.01; ***P,0.001.

 

The relationship between the loss of SIRT1 and the activation of JNK was further examined using two SIRT1 siRNAs targeting different loci of the SIRT1 gene. Transfected with either of these two siRNAs for 48 hours, SIRT1 protein expression was suppressed in HK-2 cells (Figure 9C). JNK was phosphorylated and activated as indicated by Western blotting, which revealed the phosphorylation of ATF2. Because no direct interaction between SIRT1 and JNK was detected in the immunoprecipitation experiment (data not shown), we examined the upstream phosphor-kinase and phosphatase of JNK, because JNK signaling is a phosphorylation-dependent cascade. We investigated MKK7 and MKK4, two major JNK-phosphorkinases, both in the total and phosphorylated form of MKK7 and MKK4 and found no alteration in the deprivation of SIRT1 (Figure 9C). To explore the related phosphatase levels, we constructed a flag-tagged dual-specificity phosphatase16 (DUSP16) plasmid and delivered it into HK-2 cells with SIRT1 siRNA or scrambled RNA, and the phosphorylation and acetylation levels of flag-DUSP16 were determined by immunoprecipitation. When SIRT1 was silenced by siRNA, the phosphorylation of flag-DUSP16 was downregulated, whereas acetylation was upregulated (Figure 9D). Additionally, in HK-2 cells, SIRT1 was coprecipitated with DUSP16 and was not coprecipitated with other JNK phosphatases and phosphor-kinases (Figure 9E). The interaction between SIRT1 and DUSP16 was further confirmed by reverse IP, in which flag-DUSP16 was found to be coprecipitated with SIRT1 (Figure 9F, Supplemental Figure 6A). This finding strongly suggested that, with the lack of SIRT1, DUSP16 would remain acetylated instead of becoming phosphorylated and lose its phosphatase activity, resulting in a constant phosphorylation of JNK. Additional experiment using lysine to arginine mutant DUSP16 plasmids identified lys462 and lys482 as acetylated targets of DUSP16, because SIRT1 deficiency– induced JNK phosphorylation was blunted in HK-2 cells when transfected with lys462 and lys482 mutant DUSP16 plasmid (Supplemental Figure 6B).

 

DISCUSSION

Complex factors contribute to the enhanced susceptibility to AKI with age.3–5,36,37 Comorbidities, such as diabetes, hypertension, hyperlipidemia, vascular diseases, and iatrogenic

factors, such as contrast, medication, and surgical procedures, all may contribute to age-associated susceptibility to AKI. Our previous studies and those of others have shown that the longevity gene SIRT1 plays a critical role in protecting the kidney from injury.5,38–41 This study identified the substrate of SIRT1, NAD+, as an important factor associated with increased susceptibility to AKI among the elderly. We showed that aged kidneys have decayed NAD+ metabolism and reduced SIRT1, and supplementation of NMN, an NAD+ precursor, restored the NAD+ content and SIRT1 activity and rescued cisplatin-induced acute renal damage. SIRT1 deficiency substantially blunted the protective effect of NMN, indicating that the renal protective effect of NMN relied on SIRT1. Thus, increasing endogenous NAD+ levels could become a therapeutic target for AKI, particularly in the elderly.

A reduced level of NAD+ has been detected with aging in worms, skeletal muscles and livers in mice, and skin tissues in human,26–28 although the mechanism remains unclear. The salvage pathway accounts for most of the NAD+ in mammals, which is catalyzed by two rate-limiting enzymes. Briefly, NAD+ is converted from NMN by NMNAT after the conversion of NMN from nicotinamide by NAMPT.34 Thus, a deficit in the salvage pathway could contribute to the insufficient production of NAD+.42 Overconsumption would also cause NAD+ deficiency. PARPs and CD38, two NADases, also use NAD+ as a substrate, thus consuming NAD+. PARP-1 knockout is associated with elevated NAD+ levels.43 During aging, activated PARP-1 due to increased DNA damage has been suggested to contribute to reduced NAD+ levels in aged organs.32,33 A recent study revealed that CD38 level and activity increased in multiple tissues during aging and played an important role in ageassociated NAD+ decline.44 In this study, the message RNA expression levels of both NAMPT and NMNAT were lower in the kidney cortex of 20-month-old mice compared with that in 3-month-old mice, indicating that the decline of the NAD+ expressed in proliferating cells and tissues,45 suggesting that the NMNAT might be decreased as an adaptive response to the reduced metabolism in the aged animals. Notably, two of three isoforms of NMNATs are found in the kidney: NMNAT1, mainly expressed in the nucleus, and NMNAT3, expressed in the mitochondria. Shrinkage of the NAD+ pool in the nucleus and mitochondria during aging was suggested indirectly by decreased NMNAT1 and NMNAT3 expression, but the precise subcellular distribution and alteration of the NAD+ content are not clear. Given that NAD+ is able to travel freely inside the cell, decreased cellular NAD+ levels may result in reduced activity of the enzymes that use NAD+ as a substrate, including SIRT1. This study shows that NMN, an intermediate product of NAD+ biosynthesis in the salvage pathway, replenished NAD+ levels in the aged kidneys and benefited the kidney with cisplatin-induced AKI, suggesting that reduced NAD+ levels are associated with enhanced kidney injury and that NAD+ may be an important intervention target for kidney protection.

NAD+ is required for SIRT1 activity, and SIRT1 has been previously documented as a renal survival factor.38 We examined whether the protective effect of NMN depended on SIRT1. This study shows that the intraperitoneal injection of NMN boosted NAD+ levels in both young and aged kidneys, which was also associated with increased activity of SIRT1, and protected the kidneys from both cisplatinand ischemia-reperfusion–induced AKI, indicating that NMN rescued AKI by activating SIRT1. Importantly, the protective effect of NMN supplementation was substantially blunted in SIRT1-deficient mice, indicating that the protective effect of NMN occurs through SIRT1 activity.

 

The mechanism by which SIRT1 exerts its health benefit has been extensively studied. FOXO, PGC1a, NF-kB, and HIF have been reported to be targets of SIRT1.46 To define the signaling mechanism underlying SIRT1 deficiency–associated AKI, we applied the microarray approach using SIRT1deficient or -intact mice exposed to cisplatin or the vehicle.

 

The array data indicated the activation of JNK signaling. Activation of JNK was then validated through Western blotting. Because JNK phosphorylation is observed in injured tissue, to examine whether the JNK activation is caused by tissue damage, we induced similar kidney injuries among wildtype mice (30 mg/kg cisplatin) and SIRT1-deficient mice (20 mg/kg) and found more intensive JNK phosphorylation in the heterozygotes than in the wild-type mice, indicating that the JNK activation was enhanced by SIRT1 deficiency (Supplemental Figure 3). Additionally, the administration of a JNK inhibitor attenuated cisplatin-induced apoptosis in both SIRT1-intact and -deficient HK-2 cells. In the cell culture study, JNK activation was observed when SIRT1 was knocked down by siRNA in HK-2 cells.

We then examined the mechanism by which SIRT1 influences JNK. Our IP experiment failed to find a direct interaction of SIRT1 and JNK. We then examined the phosphor-kinase and phosphatases of JNK and found that DUSP16 is a potential target of SIRT1. DUSP16, belonging to a family of mitogen-activated protein kinase phosphatases (MKPs), shows a specificity preference for JNK.47 MKPs are inducible and act rapidly as a feedback loop of the MAPK cascade in a cell type–specific manner.48 Furthermore, MKPs can be modified post-transcriptionally. In HK-2 cells transfected with SIRT1 siRNA, we found an enhanced acetylation and reduced phosphorylation of DUSP16. Given that SIRT1 is a deacetylase and targets a variety of proteins in addition to histones, it is plausible that acetylated DUSP16 was present in the absence of SIRT1. More importantly, interaction between SIRT1 and DUSP16 was observed in HK-2 and 293T cells by coimmunoprecipitation. It has been documented that the phosphorylated state of MKPs is associated with their stabilization and prolonged half-life49; therefore, the reduction of phosphorylated DUSP16 in SIRT1 deprivation may reduce their phosphatase activity and lead to the sustained activation of JNK. Two lysines (lys462 and lys482) are closely located near the phosphorylated site, serine446 of the DUSP16 protein, prompting the possibility that acetylation might compete with phosphorylation and influence protein functioning under certain circumstances.

In summary, renal aging is associated with declined NAD+ metabolism and the consequent reduced SIRT1 activity and increased susceptibility to AKI. Supplementation with NMN, an NAD+ precursor, restores NAD+ and SIRT1 levels and protects the kidney from age-related AKI in an SIRT1-dependent manner. This study provides evidence suggesting that approaches to restore endogenous NAD+ levels are a potential therapeutic target for AKI, particularly in the elderly.