OS 36-03 E-BOX CpG HYPOMETHYLATION OF NMN-PRODUCING ENZYME NAMPT IN PROXIMAL TUBULES ENABLES CONSTANT SUPPLY OF NMN FROM TUBULES TO GLOMERULI, WHICH IS DISRUPTED IN DIABETIC NEPHROPATHY

Abstract

Nicotinamide phosphoribosyltransferase (Nampt) cooperates with a longevity gene, Sirt1, to exert the potentiation of stress resistance. We have reported that the high expression level of Nampt in PTs contributes to the sufficient supply of NMN to glomeruli. We also showed that downregulation of Nampt and NMN in PT in DN leads to glomerular damages (Nature Medicine 2013). However, the regulation mechanisms of Nampt expression remain unknown. We investigated how Nampt expression is consistently retained at high levels in PTs under normal conditions and how Nampt levels are decreased in DN. In murine models of DN, Sirt1 expression in PTs was decreased 8 weeks after DN onset. At 24 weeks, TGF-β production increased which directly lowered Nampt expression in PTs. Human renal biopsy samples showed the same patterns seen in mice. A promoter analysis identified that CpG islands reside within Nampt gene region in both humans and mice, which contain four enhancer (E-) box sites. AhR/ARNT binds to the distal two E-box sequences. Results from luciferase reporter assays and gel electrophoresis mobility shift assays demonstrated that the basal transcription of Nampt depends on AhR/ARNT binding to the E-box sites, identifying this as a core promoter. We found that TGF-β activated Dnmt1, leading to the hypermethylation of CpGs which blocked AhR/ARNT binding and decreased Nampt expression. Results from methylation-specific PCR and bisulfite sequencing using laser-microdissected PT regions assays showed that the methylation levels of E-box sites were very low in control non-DN mice but significantly higher in DN mice. Nampt's E-box is a core promoter with low CpG methylation easily bound by AhR/ARNT, which results in the high expression of Nampt providing sufficient NMN under normal conditions. The DN-induced proximal tubular TGF-β production and its concomitant hypermethylation of the E-box disrupt this balance.