Journal: Scientific Reports

Article Title: Nicotinamide mononucleotide ameliorates adriamycin-induced renal damage by epigenetically suppressing the NMN/NAD consumers mediated by Twist2

doi: 10.1038/s41598-022-18147-2

Figure Lengend Snippet: Schematic diagram showing the NAD + metabolic pathway. Npt nicotinic acid phosphoribosyltransferase, Nmnat nicotinamide mononucleotide adenylyl transferase, NRK nicotinamide riboside kinase, 5′-NT 5′-nucleotidase, iNampt intracellular NAM phosphoribosyl transferase, Sirt1 Sirtuin1, Cyp2E1 Cytochrome P450 2E1, Nnmt nicotinamide N-Methyltransferase, Aox aldehyde oxidase, NaNM nicotinic acid mononucleotide, NAR nicotinic acid riboside, NAM nicotinamide, NMN nicotinamide mononucleotide, NR nicotinamide riboside, NAD nicotinamide adenine dinucleotide, NNO NAM N-oxide, MNA N1-methylniacinamide, 2py N1-methyl-2-pyridone-5-carboxamide, 4py N1-methyl-4-pyridone-3-carboxamide, PARP1 poly ADP-ribose polymerase 1.

Article Snippet: Briefly, paraffin sections (4 μm) were fixed in 3% formaldehyde and stained with the primary antibodies for Claudin-1 (Invitrogen, 51-9000, 1:50), Sirt1 (Sigma-Aldrich, 07-131, 1:100), Synaptopodin (Fitzgerald, 10R-S125A, undiluted), WT-1 (Santa Cruz, C-19, 1:200), Nampt (Bethyl Laboratories, A300-372A, 1:500), Nmnat1 (Proteintech, 11399-1AP, 1:500), Sirt3 (Cell Signaling, C73E3, 1:50), Sirt6 (LSBio, aa250-334, 1:2500), DNMT1 (Cell Signaling Technology, #5032, 1:100), PARP1 (Proteintech, 13371-1-AP, 1:200), Twist2 (Abcam, ab66031, 1:200), and H3K9me2 (Abcam; mAbcam 1220, 1:200).

Techniques:

Journal: Scientific Reports

Article Title: Nicotinamide mononucleotide ameliorates adriamycin-induced renal damage by epigenetically suppressing the NMN/NAD consumers mediated by Twist2

doi: 10.1038/s41598-022-18147-2

Figure Lengend Snippet: Effects of NMN treatment on NAD + metabolites and the salvage pathway. ( A ) Temporal changes in NAD + concentrations in the kidneys of mice in the Cont and ADR groups ( n = 6). ( B–D ) Renal tissue concentrations of NAD + metabolites, NAM ( B ), NMN ( C ), and NAD + ( D ) in the salvage pathway on day 28 in the Cont, ADR, and NMN500 groups ( n = 6). ( E ) Representative images of sections immunostained with Nampt, Nmnat1 in the kidneys of the Cont, ADR, and NMN500 groups (scale bar 50 µm). ( F–H ) Proportional staining areas for Nampt ( F ), Nmnat1 ( G ), and PARP1 ( H ) ( n = 20 sections/group). All data are shown as mean ± standard error of the mean. Statistical significance between each group is represented by a horizontal bar. * P < 0.05.

Article Snippet: Briefly, paraffin sections (4 μm) were fixed in 3% formaldehyde and stained with the primary antibodies for Claudin-1 (Invitrogen, 51-9000, 1:50), Sirt1 (Sigma-Aldrich, 07-131, 1:100), Synaptopodin (Fitzgerald, 10R-S125A, undiluted), WT-1 (Santa Cruz, C-19, 1:200), Nampt (Bethyl Laboratories, A300-372A, 1:500), Nmnat1 (Proteintech, 11399-1AP, 1:500), Sirt3 (Cell Signaling, C73E3, 1:50), Sirt6 (LSBio, aa250-334, 1:2500), DNMT1 (Cell Signaling Technology, #5032, 1:100), PARP1 (Proteintech, 13371-1-AP, 1:200), Twist2 (Abcam, ab66031, 1:200), and H3K9me2 (Abcam; mAbcam 1220, 1:200).

Techniques: Staining

Journal: Scientific Reports

Article Title: Nicotinamide mononucleotide ameliorates adriamycin-induced renal damage by epigenetically suppressing the NMN/NAD consumers mediated by Twist2

doi: 10.1038/s41598-022-18147-2

Figure Lengend Snippet: Nmnat1 epigenetic gene regulation by NMN and Dnmt1. ( A ) Promoter methylation downregulated Nmnat1 gene expression after NMN administration. The cells were treated with 5′-azacytidine (1 µM) for 4 days before incubation with NMN (100 µM) for 24 h. The Nmnat1 gene expression level was quantified by real-time polymerase chain reaction (PCR); n = 3 independent experiments. The positions of primers used for MSP. UMF unmethylated forward primer, UMR unmethylated reverse primer, MF methylated forward primer, MR methylated reverse primer. ( B–D ) The promoter methylation levels were examined by methylation-specific PCR (MSP). Methylation of the Nmnat1 promoter with or without NMN and a siRNA for Dnmt1 ( B ), Dnmt3a ( C ), and Dnmt3b ( D ). The upper panels show representative bands of MSP and the lower panels show the results of real-time MSP ( n = 3 independent experiments). ( E ) Schema depicting the epigenetic regulation of the expression of Nmnat1 by Dnmt1. In the FSGS state or after adriamycin treatment, the methylation level of the Nmnat1 promoter region was low. Thus, Twist2 could bind to the E-box sites and maintain the high expression level of Nmnat1 . In the presence of NMN, the methylation in this region is increased by Dnmt1. Consequently, the expression level of Nmnat1 is decreased because Twist2 cannot bind to the E-box sites. Statistical significance between each group is represented. * P < 0.05 by ANOVA with Tukey’s post hoc test. ( F ) Schematic model of NMN action in ADR-induced FSGS. In this study, we investigated the effect of a preemptive short-term NMN treatment on ADR-induced FSGS. This transient treatment reduced albuminuria immediately after treatment until 2 weeks after the treatment. We further demonstrated that NMN treatment retained the levels of NAD + in the kidney by suppressing the NMN consumer Nmnat1 and the NAD consumer PARP1 in the NAD + salvage pathway. Furthermore, NMN treatment increased Sirt1 expression and downregulated Claudin-1 expression, leading to the attenuation of the downregulation of Synaptopodin and the effacement of the podocyte foot processes. Therefore, this method could be a preventive strategy against FSGS.

Article Snippet: Briefly, paraffin sections (4 μm) were fixed in 3% formaldehyde and stained with the primary antibodies for Claudin-1 (Invitrogen, 51-9000, 1:50), Sirt1 (Sigma-Aldrich, 07-131, 1:100), Synaptopodin (Fitzgerald, 10R-S125A, undiluted), WT-1 (Santa Cruz, C-19, 1:200), Nampt (Bethyl Laboratories, A300-372A, 1:500), Nmnat1 (Proteintech, 11399-1AP, 1:500), Sirt3 (Cell Signaling, C73E3, 1:50), Sirt6 (LSBio, aa250-334, 1:2500), DNMT1 (Cell Signaling Technology, #5032, 1:100), PARP1 (Proteintech, 13371-1-AP, 1:200), Twist2 (Abcam, ab66031, 1:200), and H3K9me2 (Abcam; mAbcam 1220, 1:200).

Techniques: Methylation, Gene Expression, Incubation, Real-time Polymerase Chain Reaction, Expressing