Effects of Nucleobindin-2 on Tubular Cells and Mitochondria and Their Mechanisms

Background: We have reported that serum NUCB2 (Nucleobindin-2) is a potential biomarker of tubular tissue damage and prognostic factor in DKD. However, the mechanism by which NUCB2 improves renal tubular damage remains unclear. In this study, we investigated the effects of Nucleobindin-2 on tubular cells and mitochondria and the mechanisms underlying these effects. Methods: We analyzed the effects and mechanisms of NUCB2 on oxidative stress, apoptosis, and mitochondria under high glucose (HG) conditions using proximal tubular cells (HK2, RPTEC). Results: In oxidative stress cultured HG , NUCB2 reduced ROS production by 20% (Ample Red method), SOD2 expression was unchanged, HO-1 expression was decreased, and oxidative stress scavenging gene expression was not enhanced. Nrf2 expression was decreased both in the nucleus and cytoplasm, P22phox expression was decreased, poldip2 expression was increased, and p47phox expression was increased. This suggests that the expression balance of NOX2/NOX4 is altered, which may reduce oxidative stress production at the basal level for cellular maintenance. Cleaved caspase 3 expression was reduced by 40%, while Bax was decreased and Bcl2 was increased in a NUCB2 concentration-dependent manner, pERK was increased at HG concentration with NUCB2, suggesting that mitochondria were affected via the cRaf-ERK pathway. Analysis of mitochondrial dynamics revealed changes in mitochondrial fusion and fission-related proteins at the protein level and by electron microscopy. Furthermore, NUCB2 enhanced the LC3 1/2 ratio, suggesting that autophagy may also be involved. When we examined metabolism, piruvate kinase, GAPDH, and lactate were unchanged regardless of blood glucose, but PEPCK expression was increased, suggesting an enhancement of the glycogenic system. This may be due to the increased activity of the pentose pathway, which may have suppressed oxidative stress by producing NADPH. In addition, the expression of lipolysis (beta oxidation) and synthesis was enhanced by NUCB2 administration, regardless of blood glucose level. These results may indicate a correlation between lipid profiles and serum NUCB2 concentrations in humans. Conclusion: NUCB2 may reduce oxidative stress production, suppress apoptosis, and ameliorate mitochondrial damage at high glucose concentrations. NUCB2 may also have an effect on the metabolic system and lipid synthesis.