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.
Aged Kidneys Are Susceptible to Cisplatin-Induced AKI
A dose of 20 mg/kg body wt cisplatin successfully induced AKI in 3and 20-month-old 129 mice. Kidney function was evaluated by BUN and serum creatinine (72 hours of cisplatin). BUN and creatinine levels of 20-month-old AKI mice were twice those of 3-month-old mice (Figure 1, A and B). Consistently, the histology study of the kidneys revealed a significantly worse tubular injury in the 20-month-old mice than in the 3-month-old mice, including severe dilation of the proximal tubules, cast formation, and massive detachment and necrosis of the tubular epithelium (Figure 1D). The pathologic analysis assessed by the percentage of damaged tubules at 72 hours of cisplatin in a blind manner further confirmed that aged kidneys are susceptible to toxin insult (Figure 1C). Additionally, electron micrograph showed that cisplatin induced mild swelling of the mitochondria in the proximal tubules in 3-month-old mice, whereas in 20-month-old mice, the same dosage of cisplatin induced mitochondrial fragmentation into spheres and partial ridge breakdown (Figure 1E).
Aging Is Associated with Declined SIRT1 Expression and NAD+ Levels in the Kidney
In the kidney cortex tissue, mRNA and protein expression of SIRT1 and its substrate NAD+ reduced with aging (Figure 2, A–C, Supplemental Figure 1). SIRT1 expression and the NAD+ content in 20-month-old kidneys were approximately one third of those in the 3-month-old kidneys. We then examined the expression of the enzymes of NAD+ biosynthesis: nicotinamide phosphoribosyltransferase (NAMPT), nicotinamide mononucleotide adenylyltransferase1 (NMNAT1), and
NMNAT3.34 Real-time PCR showed that the mRNA levels of all of these enzymes were significantly lower in the 20-monthold kidney cortex compared with the 3-month-old kidney cortex (Figure 2D).
NMN Supplementation Protects the Kidney from Aging-Associated Susceptibility to AKI
NMN is an intermediate product in the NAD+ salvage pathway. Four days of NMN supplementation restored NAD+ levels in both the young and aged kidney cortexes (Figure 3A) and further increased SIRT1 activity as indicated by direct enzymatic activity measurement and reduced acetylation level of Foxo1, a downstream target molecule of SIRT1 (Figure 3, B and C). To explore whether NMN could rescue the mice from aging-associated susceptibility to AKI, we provided both 3and 20-month-old mice with NMN immediately after cisplatin exposure. As shown in Figure 4, the NMN supplement significantly attenuated cisplatin-induced AKI compared with the PBS control. The serum creatinine in 20-month-old mice after cisplatin exposure was reduced by 60% in NMN-treated mice compared with vehicle-treated mice. The histology revealed ameliorated tubular necrosis and cast formation in NMN-treated AKI mice (Supplemental Figure 2). Also, NMN treatment reduced cisplatin-induced apoptosis as assessed by decreased cleaved caspase-3 expression and fewer terminal deoxynucleotidyl transferase–mediated digoxigenindeoxyuridine nick-end labeling (TUNEL)-positive cells.
SIRT1 Deficiency Aggravates Cisplatin-Induced AKI
To determine whether the lower levels of SIRT1 observed in the aged kidney could predispose to worsen kidney injury, studies were performed in SIRT1 heterozygous mice due to embryonic and perinatal lethality of SIRT1 homozygotes.5,35 A loss of one allele of the SIRT1 gene significantly aggravated renal damage compared with in wild-type littermates after cisplatin exposure, resulting in higher BUN and serum creatinine levels, more tubular injury, and more severe mitochondrial fragmentation (Figure 5, A–E). Enhanced tubular cell damage was further confirmed by Western blotting of caspase-3, realtime PCR of apoptosis-associated genes, and TUNEL staining: SIRT1 heterozygotes showed higher cleaved caspase-3 protein levels, BAX, and caspase-3 and -9 mRNA expression as well as many more TUNEL labeling apoptotic cells (Figure 5, F–I).