Nicotinamide adenine dinucleotide (NAD) is a cofactor in redox reactions and an essential mediator of energy metabolism. The redox balance between NAD⁺ and NADH affects various diseases, cell differentiation, and aging, and in recent years there has been a growing need for measurement techniques with improved accuracy. However, NAD(H) measurements, representing both NAD⁺ and NADH, have been limited by the compound’s properties. We achieved highly sensitive simultaneous measurement of NAD⁺ and NADH under non-ion pairing, mobile phase conditions of water, or methanol containing 5 mM ammonium acetate. These were achieved using a simple pre-treatment and 7-min analysis time. Use of the stable isotope ¹³C₅-NAD⁺ as an internal standard enabled validation close to BMV criteria and demonstrated the robustness of NAD(H) determination. Measurements using this method showed that brain NAD(H) levels correlate strongly with plasma NAD(H) levels in the same mouse, indicating that NAD(H) concentrations in brain tissue are reflected in plasma. As NAD(H) is involved in various neurodegenerative diseases and cerebral ischemia, as well as brain diseases such as mitochondrial myopathies, monitoring changes in NADH levels in plasma after drug administration will be useful for development of future diagnostics and therapeutics.

Several genetic defects, including a mutation in myosin heavy chain 11 (Myh11), are reported to cause familial thoracic aortic aneurysm and dissection (FTAAD). We recently showed that mice lacking K1256 of Myh11 developed aortic dissection when stimulated with angiotensin II, despite the absence of major pathological phenotypic abnormalities prior to stimulation. In this study, we used a comprehensive, data-driven, unbiased, multi-omics approach to find underlying changes in transcription and metabolism that predispose the aorta to dissection in mice harboring the Myh11 K1256del mutation. Pathway analysis of transcriptomes showed that genes involved in membrane transport were downregulated in homozygous mutant (Myh11^ΔK/ΔK) aortas. Furthermore, expanding the analysis with metabolomics showed that two mechanisms that raise the cytosolic Ca²⁺ concentration—multiple calcium channel expression and ADP–ribose synthesis—were attenuated in Myh11^ΔK/ΔK aortas. We suggest that the impairment of the Ca²⁺ influx attenuates aortic contraction and that suboptimal contraction predisposes the aorta to dissection.

Emerging evidence has indicated a possible link between attenuation of contractility in aortic smooth muscle cells and pathogenesis of aortic dissection, as revealed through comprehensive, multi-omic analyses of familial thoracic aortic aneurysm and dissection models. While L-type voltage-gated calcium channels have been extensively investigated for their roles in smooth muscle contraction, more recent investigations have suggested that downregulation of T-type voltage-gated calcium channels, rather than their L-type counterparts, may be more closely associated with impaired contractility observed in vascular smooth muscle cells. This review provides a detailed examination of T-type voltage-gated calcium channels, highlighting their structure, electrophysiology, biophysics, expression patterns, functional roles, and potential mechanisms through which their downregulation may contribute to reduced contractile function. Furthermore, the application of multi-omic approaches in investigating calcium channels is discussed.

Tacrolimus (TAC)-induced chronic nephrotoxicity (TAC nephrotoxicity) has a detrimental effect on long-term kidney graft survival. However, the pathogenesis of TAC nephrotoxicity remains largely unknown. We explored it by focusing on metabolic changes in renal tissues. In this study, mice were separated into TAC and control groups (n = 5/group). TAC was administered to the TAC group (1 mg/kg/d for 28 days) subcutaneously. The control group was similarly treated with normal saline. Renal tissue metabolomes were evaluated. Renal fibrosis was observed only in the TAC group. Metabolomic analysis showed that carnitine and related metabolites were substantially lower in the TAC group than in the control group, presumably due to impaired biosynthesis and reabsorption. Low carnitine levels impair antioxidation in renal tissues and β-oxidation in mitochondria, which may lead to renal tissue damage. This metabolomic analysis revealed that carnitine deficiency in renal tissue appears to explain TAC nephrotoxicity.

Tacrolimus (TAC)-induced chronic nephrotoxicity (TAC nephrotoxicity) is a serious issue for long-term graft survival in kidney transplantation. However, the pathophysiology of TAC nephrotoxicity remains unclear. In this study, we analyzed whole blood samples from mice that developed TAC nephrotoxicity in order to discover its mechanism. Mice were divided into a TAC group and a control group (n = 5 per group). The TAC group received TAC subcutaneously (1 mg/kg/day for 28 days), while the control group received normal saline instead. After the administration period, whole blood was collected and metabolomic analysis was performed, revealing significant changes in 56 metabolites. The major metabolic changes were related to uremic toxins, vascular damage, and NAD⁺. NAD⁺ levels were significantly lower in the TAC group, and ADP-ribose, nicotinamide, and nicotinamide N-oxide, which are degradation products of NAD⁺, were significantly higher, suggesting impairment of the NAD⁺ salvage pathway. NAD⁺ deficiency suggests cellular aging and mitochondrial dysfunction, which may induce vascular damage and chronic kidney disease. Our study demonstrated a correlation between low NAD⁺ levels and the pathophysiology of TAC nephrotoxicity.

Nicotinamide adenine dinucleotide (NAD) is a critical cofactor in mitochondrial energy production. The NADH/NAD⁺ ratio, reflecting the balance between NADH (reduced) and NAD^{+ (}oxidized⁾, is a key marker for the severity of mitochondrial diseases. We recently developed a streamlined LC-MS/MS method for the precise measurement of NADH and NAD⁺. Utilizing this technique, we quantified NADH and NAD⁺ levels in fibroblasts derived from pediatric patients and in a Leigh syndrome mouse model in which mitochondrial respiratory chain complex I subunit Ndufs4 is knocked out (KO). In patient-derived fibroblasts, NAD⁺ levels did not differ significantly from those of healthy controls (p = 0.79); however, NADH levels were significantly elevated (p = 0.04), indicating increased NADH reductive stress. This increase, observed despite comparable total NAD(H) levels between the groups, was attributed to elevated NADH levels. Similarly, in the mouse model, NADH levels were significantly increased in the KO group (p = 0.002), further suggesting that NADH elevation drives reductive stress. This precise method for NADH measurement is expected to outperform conventional assays, such as those for lactate, providing a simpler and more reliable means of assessing disease progression.