Presentation Authors: Rei Unno*, Nagoya, Japan, Tsuyoshi Kawabata, Nagasaki, Japan, Hiroshi Takase, Teruaki Sugino, Yutaro Tanaka, Naoko Unno, Yasuhiro Fujii, Kazumi Taguchi, Shuzo Hamamoto, Ryosuke Ando, Atsushi Okada, Hiroyuki Kamiya, Takahiro Yasui, Nagoya, Japan
Introduction: We found that cellular damage caused by calcium oxalate (CaOx) was repaired by the induction of autophagy using renal tubule cultured cells and CaOx crystal model mice, and the failure of the repair mechanism caused kidney stones. In this study, we focused on selective autophagy and upstream regulatory factors mTORC1 and transcription factor TFEB and tried to elucidate the mechanism of calculus formation.
Methods: In vitro: We exposed renal tubular cells to calcium oxalate monohydrate (COM) crystals at a concentration of 20 Î¼g/cm2, and autophagy was examined by immunostaining and Western-blot. mTORC1 activity was examined by Western blotting of phosphorylated S6 kinase, and nuclear import of TFEB was examined by transfection of GFP-TFEB. In vivo: We used autophagy visualization (GFP-LC3) mice, and kidney crystal formation in mice was induced by daily intra-abdominal injection of 80 mg/kgâˆ’1 glyoxylic acid.We investigated autophagy by immunostaining and Western blotting. Finally, we examined the suppression effect of mTOR inhibitor, which can promote autophagy, on crystal formation.
Results: We found that autophagic activity was significantly decreased in mouse RTCs exposed to CaOx monohydrate crystals and in the kidneys of GFP-LC3 transgenic mice with CaOx nephrocalcinosis induced by glyoxylate. This caused accumulation of damaged intracellular organelles, such as mitochondria and lysosomes, the normal functioning of which is mediated by functional autophagy. An impairment of autophagy was also observed in the mucosa with plaques of CaOx kidney stone formers. We determined that the decrease in autophagy was caused by an upregulation of mTOR, which consequently resulted in the suppression of the upstream autophagy regulator transcription factor EB. Furthermore, we showed that an mTOR inhibitor could recover a decrease in autophagy and alleviate crystal-cell interactions and the formation of crystals associated with increased inflammatory responses.
Conclusions: We conclude that autophagy compromised by mTOR deregulation is a fundamental feature in the pathology of kidney stone formation, and propose that chemical inhibition of mTOR could be a prospective strategy for disease suppression.