L-NAME Administration Enhances Diabetic Kidney Disease Development in an STZ/NAD Rat Model.

One of the most important risk factors for developing chronic kidney disease (CKD) is diabetes. To assess the safety and efficacy of potential drug candidates, reliable animal models that mimic human diseases are crucial. However, a suitable model of diabetic kidney disease (DKD) is currently not available. The aim of this study is to develop a rat model of DKD by combining streptozotocin and nicotinamide (STZ/NAD) with oral N(ω)-Nitro-L-Arginine Methyl Ester (L-NAME) administration. Diabetes was induced in male Wistar rats by intravenous injection of 65 mg/kg STZ, 15 min after intraperitoneal injection of 230 mg/kg NAD. Rats were assigned to different groups receiving L-NAME (100 mg/kg/day) (STZ/NAD/L-NAME) or vehicle (STZ/NAD) for a period of 9 or 12 weeks by daily oral gavage. All rats developed hyperglycemia. Hyperfiltration was observed at the start of the study, whereas increased serum creatinine, albumin-to-creatinine ratio, and evolving hypofiltration were detected at the end of the study. Daily L-NAME administration caused a rapid rise in blood pressure. Histopathological evaluation revealed heterogeneous renal injury patterns, which were most severe in the STZ/NAD/L-NAME rats. L-NAME-induced NO-deficiency in STZ/NAD-induced diabetic rats leads to multiple characteristic features of human DKD and may represent a novel rat model of DKD.

Dihydronicotinamide riboside promotes cell-specific cytotoxicity by tipping the balance between metabolic regulation and oxidative stress.

Nicotinamide adenine dinucleotide (NAD+), the essential cofactor derived from vitamin B3, is both a coenzyme in redox enzymatic processes and substrate in non-redox events; processes that are intimately implicated in all essential bioenergetics. A decrease in intracellular NAD+ levels is known to cause multiple metabolic complications and age-related disorders. One NAD+ precursor is dihydronicotinamide riboside (NRH), which increases NAD+ levels more potently in both cultured cells and mice than current supplementation strategies with nicotinamide riboside (NR), nicotinamide mononucleotide (NMN) or vitamin B3 (nicotinamide and niacin). However, the consequences of extreme boosts in NAD+ levels are not fully understood. Here, we demonstrate the cell-specific effects of acute NRH exposure in mammalian cells. Hepatocellular carcinoma (HepG3) cells show dose-dependent cytotoxicity when supplemented with 100-1000 µM NRH. Cytotoxicity was not observed in human embryonic kidney (HEK293T) cells over the same dose range of NRH. PUMA and BAX mediate the cell-specific cytotoxicity of NRH in HepG3. When supplementing HepG3 with 100 µM NRH, a significant increase in ROS was observed concurrent with changes in the NAD(P)H and GSH/GSSG pools. NRH altered mitochondrial membrane potential, increased mitochondrial superoxide formation, and induced mitochondrial DNA damage in those cells. NRH also caused metabolic dysregulation, altering mitochondrial respiration. Altogether, we demonstrated the detrimental consequences of an extreme boost of the total NAD (NAD+ + NADH) pool through NRH supplementation in HepG3. The cell-specific effects are likely mediated through the different metabolic fate of NRH in these cells, which warrants further study in other systemic models.

Antioxidant Protection of NADPH-Depleted Oligodendrocyte Precursor Cells Is Dependent on Supply of Reduced Glutathione.

The pentose phosphate pathway is the main source of NADPH, which by reducing oxidized glutathione, contributes to antioxidant defenses. Although oxidative stress plays a major role in white matter injury, significance of NADPH for oligodendrocyte survival has not been yet investigated. It is reported here that the NADPH antimetabolite 6-amino-NADP (6AN) was cytotoxic to cultured adult rat spinal cord oligodendrocyte precursor cells (OPCs) as well as OPC-derived oligodendrocytes. The 6AN-induced necrosis was preceded by increased production of superoxide, NADPH depletion, and lower supply of reduced glutathione. Moreover, survival of NADPH-depleted OPCs was improved by the antioxidant drug trolox. Such cells were also protected by physiological concentrations of the neurosteroid dehydroepiandrosterone (10(-8) M). The protection by dehydroepiandrosterone was associated with restoration of reduced glutathione, but not NADPH, and was sensitive to inhibition of glutathione synthesis. A similar protective mechanism was engaged by the cAMP activator forskolin or the G protein-coupled estrogen receptor (GPER/GPR30) ligand G1. Finally, treatment with the glutathione precursor N-acetyl cysteine reduced cytotoxicity of 6AN. Taken together, NADPH is critical for survival of OPCs by supporting their antioxidant defenses. Consequently, injury-associated inhibition of the pentose phosphate pathway may be detrimental for the myelination or remyelination potential of the white matter. Conversely, steroid hormones and cAMP activators may promote survival of NADPH-deprived OPCs by increasing a NADPH-independent supply of reduced glutathione. Therefore, maintenance of glutathione homeostasis appears as a critical effector mechanism for OPC protection against NADPH depletion and preservation of the regenerative potential of the injured white matter.

NAD+-induced vasotoxicity in the pericyte-containing microvasculature of the rat retina: effect of diabetes.

PURPOSE: It was recently proposed that activation of P2X(7) purinoceptors may play a role in causing cell death in the pericyte-containing microvasculature of the diabetic retina. This hypothesis is supported by the observation that diabetes enhances lethal pore formation in retinal microvessels exposed to synthetic P2X(7) agonists. The goal of this study was to determine whether purinergic vasotoxicity can be triggered by the endogenous molecule nicotinamide adenosine dinucleotide (NAD(+)), which is a substrate for ecto-ribosylation reactions known to activate P2X(7) receptor/channels in other cell types. METHODS: Pericyte-containing retinal microvessels were isolated from normal and streptozotocin-injected rats. Trypan blue dye exclusion was used to assess cell viability, YO-PRO-1 uptake was used to identify cells with P2X(7)-induced pores, and ethenoadenosine antibodies were used to detect ecto-adenosine diphosphate (ADP)-ribosyltransferase (ART) activity. RESULTS: In freshly isolated retinal microvessels, it was found that extracellular NAD(+), but not its catabolites, caused cell death (half-maximal effective concentration [EC(50)] = 2 nM) by a mechanism involving the activation of P2X(7) purinoceptors and the formation of transmembrane pores. A series of experiments provided evidence that NAD(+), which is not a direct purinergic agonist, serves as a substrate for ecto-ribosylation reactions that subsequently trigger P2X(7)-dependent cell death in the retinal microvasculature. Soon after the onset of diabetes, the sensitivity of retinal microvessels to the vasotoxic effect of extracellular NAD(+) increased by approximately 100-fold. CONCLUSIONS: Purinergic vasotoxicity triggered by extracellular NAD(+) is a newly recognized mechanism that may contribute to the cell death observed in the pericyte-containing microvascular of the diabetic retina.

NAD metabolism and sirtuins: metabolic regulation of protein deacetylation in stress and toxicity.

Sirtuins are recently discovered NAD(+)-dependent deacetylases that remove acetyl groups from acetyllysine-modified proteins, thereby regulating the biological function of their targets. Sirtuins have been shown to increase organism and tissue survival in diverse organisms, ranging from yeast to mammals. Evidence indicates that NAD(+) metabolism and sirtuins contribute to mechanisms that influence cell survival under conditions of stress and toxicity. For example, recent work has shown that sirtuins and increased NAD(+) biosynthesis provide protection against neuron axonal degeneration initiated by genotoxicity or trauma. In light of their protective effects, sirtuins and NAD(+) metabolism could represent therapeutic targets for treatment of acute and chronic neurodegenerative conditions. Our work has focused on elucidating the enzymatic functions of sirtuins and quantifying perturbations of cellular NAD(+) metabolism. We have developed mass spectrometry methods to quantitate cellular NAD(+) and nicotinamide. These methods allow the quantitation of changes in the amounts of these metabolites in cells caused by chemical and genetic interventions. Characterization of the biochemical properties of sirtuins and investigations of NAD(+) metabolism are likely to provide new insights into mechanisms by which NAD(+) metabolism regulates sirtuin activities in cells. To develop new strategies to improve cell stress resistance, we have initiated proof of concept studies on pharmacological approaches that target sirtuins and NAD(+) metabolism, with the goal of enhancing cell protection against genotoxicity.

Safety of stabilized, orally absorbable, reduced nicotinamide adenine dinucleotide (NADH): a 26-week oral tablet administration of ENADA/NADH for chronic toxicity study in rats.

The safety of the stabilized, orally absorbable form of reduced nicotinamide adenine dinucleotide (NADH), known under the brand name ENADA, was investigated over a period of 26 weeks. Eighty healthy rats (40 males and 40 females) were divided into two groups. One tablet ENADA/NADH 5 mg per day was administered orally to one group while identical-looking white tablets not containing NADH (placebo) were given to the other group. The following parameters were statistically analyzed: body weight, body weight gain, food consumption, hematology, clinical chemistry, organ weight and organ histology. Clinical signs and mortality were recorded. There were no deaths associated with the study drug and no treatment-releated clinical signs. No differences in body weight between the placebo and the ENADA-treated males were observed. In the second half of the treatment period (weeks 13-26) females treated with NADH gained significantly (p < 0.05) more body weight than the controls. Food consumption in the treated males was similar to that in controls. From approximately week 15, the treated females consumed up to 10% more food than the controls. No differences were observed between the control and the treated groups in terms of hematology or clinical chemistry parameters. There was no apparent treatment-related effect on urine analysis parameters or on either the absolute or the relative organ weight. Furthermore, no macroscopic evidence of specific target organ toxicity associated with the test drug was observed. Histological findings in the treated rats were generally similar to those in control rats. A daily dose of 5 mg in a rat corresponds to a dose of 175 mg per day in a 70-kg human. This is 175 times the recommended daily dosage of 1 ENADA tablet per day. Hence ENADA/NADH 5 mg tablets can be generally regarded as safe.

Differential effects of NAD, nicotinamide and related compounds upon growth and nucleoside incorporation in human cells.

Two human melanoma cell lines, MM96 and MM127, were found to be highly sensitive to the toxicity of adenosine (D50 100-150 micrograms/ml) compared with other melanoma lines. HeLa cells and a lymphoblastoid line (D50 greater than 500 micrograms/ml). The MM127 line was also sensitive to NAD (D50 41 micrograms/ml) compared with the other lines (D50 greater than 400 micrograms/ml), and accumulated three-fold more NAD-derived isotopic label. Nicotinamide exhibited little toxicity in any cell type (D50 greater than 400 micrograms/ml); 25-100 micrograms/ml nicotinamide greatly increased the plating efficiency of melanoma cells and fibroblasts when low levels of foetal calf serum were used. The toxicity of DNA-damaging agents (alkylating agents and u.v.) in melanoma cells was not reduced in the presence of NAD, adenosine or nicotinamide. Studies of the effects of the latter compounds upon the incorporation of deoxynucleosides showed that: (a) melanoma cells have lower purine pools than fibroblasts; (b) [3H]deoxyguanosine incorporation was inhibited more than [3H]deoxyadenosine incorporation; (c) incorporation of [3H]deoxyadenosine and [3H]deoxyguanosine into RNA was inhibited by adenosine, thus providing a method for determination of guanine-specific DNA repair; and (d) NAD enhanced thymidine incorporation in intact melanoma cells but not in fibroblasts, in a pattern similar to the release from template restriction previously reported for permeabilised tumour cells.