Organ protection by caloric restriction depends on activation of the de novo NAD+ synthesis pathway

Therapeutic strategies to treat acute kidney injury (AKI) are lacking in clinical practice. Interestingly, preconditioning by hypoxia (HP) and caloric restriction (CR) is highly protective in rodent AKI models. However, the underlying molecular mechanisms of this process are unknown. A comparative transcriptome analysis of murine kidneys after HP and CR identified Kynureninase (KYNU) as a common downstream target. Using a newly generated KYNU-deficient mouse line, we show that KYNU strongly contributes to the protective effect of preconditioning. Metabolome, transcriptome and proteome analyses reveal the KYNU-dependent de novo nicotinamide adenine dinucleotide (NAD+) biosynthesis pathway as necessary for CR-associated maintenance of NAD+ levels. Importantly, the impact of CR on the de novo NAD+ biosynthesis pathway can be recapitulated in humans. These findings provide a valuable insight into the molecular mechanisms mediating protection upon preconditioning and point towards the de novo branch of NAD+ biosynthesis as a conserved target in nephroprotection.

Knockdown of Quinolinate Phosphoribosyltransferase Results in Decreased Salicylic Acid-Mediated Pathogen Resistance in Arabidopsis thaliana

Nicotinamide adenine dinucleotide (NAD) is a pivotal coenzyme that has emerged as a central hub linking redox equilibrium and signal transduction in living cells. The homeostasis of NAD is required for plant growth, development, and adaption to environmental stresses. Quinolinate phosphoribosyltransferase (QPRT) is a key enzyme in NAD de novo synthesis pathway. T-DNA-based disruption of QPRT gene is embryo lethal in Arabidopsis thaliana. Therefore, to investigate the function of QPRT in Arabidopsis, we generated transgenic plants with decreased QPRT using the RNA interference approach. While interference of QPRT gene led to an impairment of NAD biosynthesis, the QPRT RNAi plants did not display distinguishable phenotypes under the optimal condition in comparison with wild-type plants. Intriguingly, they exhibited enhanced sensitivity to an avirulent strain of Pseudomonas syringae pv. tomato (Pst-avrRpt2), which was accompanied by a reduction in salicylic acid (SA) accumulation and down-regulation of pathogenesis-related genes expression as compared with the wild type. Moreover, oxidative stress marker genes including GSTU24, OXI1, AOX1 and FER1 were markedly repressed in the QPRT RNAi plants. Taken together, these data emphasized the importance of QPRT in NAD biosynthesis and immunity defense, suggesting that decreased antibacterial immunity through the alteration of NAD status could be attributed to SA- and reactive oxygen species-dependent pathways.

P–175 Possible role of NAD+ metabolism in oocyte aging: insights from gene expression profiles in reproductive aged oocytes

Study question Does reproductive aging alter the expression of genes involved in NAD+ metabolism in the mammalian oocyte? Summary answer We found that aging alters the expression of thirty genes encoding for NAD+-producing and NAD+-consuming enzymes pathway in mouse MII oocytes. What is known already NAD, a multifunctional metabolite in living cells, is known to convert between its oxidized NAD+ and reduced NADH forms during nutrients breakdown; the intracellular NAD+/NADH redox state reflects cell ability in generating ATP energy. NAD+ is utilized by proteins that control gene expression, DNA repair, apoptosis, mitochondrial biogenesis (i.e. sirtuins). Raising NAD+ by inducing its biosynthesis leads to sirtuins activation, so directly linking the cellular redox state with signalling events. The NAD+ pool is set by a critical balance between NAD+ biosynthetic and NAD+ consuming pathways. NAD(P)H levels declined in aged oocytes and NAD+ precursors seem to counteract ovarian aging. Study design, size, duration Pools of 25 oocytes at MII stage were obtained from young (4–8 weeks) and reproductively aged (48–52 weeks) CD–1 mice and processed for the analysis of 41 genes participating in NAD+ biosynthetic and NAD+ consuming pathways NAD+ pathways. Each experiment was performed three times and data were subjected to bioinformatic analysis to unravel potential age-related effects on NAD metabolism. Participants/materials, setting, methods Mice were superovulated by intraperitoneal injection of PMSG followed by hCG 48h apart. MII oocytes were isolated by 0.3 mg/ml hyaluronidase. RNA was obtained from each sample by Arcturus PicoPure Kit, and reverse transcribed. Each cDNA was analysed in triplicate by employing a NAD Metabolism H41 Predesigned panel for use with SYBR® Green, containing 41 genes of the NAD pathway, 2 housekeeping genes and 6 control probes. Raw data were analysed by DataAssist software. Main results and the role of chance The comparison between aged and young oocytes were focused on genes showing an absolute fold change (FC) <0.7 or > 1.4, a present call in at least the 50% of experiments and a p-Value <0.05 (ANOVA). Excluding transcripts showing a concordant value < 80%, n.30 differentially expressed genes (DEGs) were found: n.26 transcripts down-expressed and n.4 genes up-regulated. Data obtained by Ingenuity Pathways Analysis (IPA) software (Ingenuity Systems) provide evidence that NAD+ biosynthesis in aged oocytes is severely compromised. Limitations, reasons for caution Our results on animal model must be taken with caution. Validation of NAD+ precursor or activators of NAD+ biosynthesis in vivo administration is required. Wider implications of the findings: Present results demonstrate that aging affect oocyte genes involved in the regulation of NAD+ availability and supports the hypothesis that modulation of NAD+ metabolism may be an “anti-aging” strategy. Overall, these data laid the foundation for a central role of NAD+ metabolism in the maintenance of oocyte competenc Trial registration number Not applicable

Analysis of the mechanism of Aldo-keto reductase dependent cis-platin resistance in HepG2 based on transcriptomic and NADH metabolic analysis

Aldo-keto oxidoreductase (AKR) inhibitors could reverse several cancer cells' resistance to Cis-platin, but their role in resistance remains unclear. Our RNA-seq results showed de novo NAD biosynthesis-related genes, and NAD(P)H-dependent oxidoreductases were significantly upregulated in Cis-platin-resistant HepG2 hepatic cancer cells (HepG2-RC cells) compared with HepG2 cells. Knockdown of AKR1Cs could increase Cis-platin sensitivity in HepG2-RC cells about two-fold. Interestingly, the AKR1C inhibitor meclofenamic acid could increase Cis-platin sensitivity of HepG2-RC cells about eight-fold, indicating that knockdown of AKR1Cs only partially reversed the resistance. Meanwhile, the amount of total NAD and the ratio of NADH/NAD+ were increased in HepG2-RC cells compared with HepG2 cells. The increased NADH could be explained as a directly operating antioxidant to scavenge radicals induced by Cis-platin. We report here that NADH, which is produced by NAD(P)H-dependent oxidoreductases, plays a key role in the AKR-associated Cis-platin resistance of HepG2 hepatic cancer cells.