Dynamics of D-amino acid oxidase ain kidney epithelial cells under amino acid starvation

Abstract

Abstract D-amino acid oxidase (DAO) is a flavoenzyme catalyzing the oxidation of D-amino acid (AA)s. In the kidney, its expression is detected in proximal tubules, and DAO is considered to play a role in the conversion of D-form AAs to α-keto acids. LLC-PK1 cells, a pig renal proximal tubule cell line, were used to elucidate the regulation of DAO protein synthesis and degradation. In this study, we showed that trypsinization of LLC-PK1 cells in culture system rapidly reduced the intracellular DAO protein level to ∼33.9% of that before treatment, even within 30 min. Furthermore, we observed that the DAO protein level was decreased when LLC-PK1 cells were subjected to AA starvation. To determine the degradation pathway, we treated the cells with chloroquine and MG132. DAO degradation was found to be inhibited by chloroquine, but not by MG132 treatment. We next examined whether or not DAO was degraded by autophagy. We found that AA starvation led to an increased accumulation of LC3-II, suggesting that DAO protein is degraded by autophagy due to AA starvation conditions. Furthermore, treatment with cycloheximide inhibited DAO protein degradation. Taken together, DAO protein is degraded by autophagy under starvation. The present study revealed the potential dynamics of DAO correlated with renal pathophysiology.