Eucalyptol inhibits diabetes‐associated renal fibrosis through blocking epithelial to mesenchymal transition in tubular epithelial cells under high glucose stimulation

Abstract

Renal tubulointerstitial fibrosis is an important event in the pathogenesis of diabetic nephropathy. Epithelial to mesenchymal transition (EMT) is a process by which epithelial cells acquire mesenchymal properties. This process contributes to the accumulation of matrix proteins in kidney. Under the pathologic conditions, renal tubular epithelial cells can undergo EMT characterized by loss of cell‐cell adhesion, apical‐basal polarity, followed by a shift in cytoskeletal dynamics toward front end‐back end polarity and increased cell migration. The current study investigated that eucalyptol, present in eucalyptus oil, inhibited renal tubular fibrogenesis stimulated by diabetic insult. Human renal proximal tubular epithelial cells (RPTEC) were incubated in media containing 5.5 mM glucose, 27.5 mM mannitol as an osmotic control or 33 mM glucose (high glucose) in the absence and presence of 1–20 μM eucalyptol for up to 72 h, there was no cytotoxicity observed from the MTT assay. By western blotting, we investigated that non‐toxic eucalyptol inhibited high glucose‐induced expression of the mesenchymal markers of N‐cadherin, whereas the induction of the epithelial marker E‐cadherin was enhanced from the sample in vitro. Also, we found those tendency in vivo. We raised db/con and db/db mice for 8 weeks. The db/db mice were supplemented with 10 mg/kg eucalyptol for 8 weeks. During 8 weeks, we recorded volume of daily water and food intake. And then we collected urine and blood sample. We measure urine volume, urinary albumin by using with enzyme linked‐immunosorbent assay in 24h urine sample and Hba1c level, plasma insulin concentration. As a result, db/db mice urine volume is about 8 times higher than control mice and db/db mice that treated with eucalyptol is less than half volume of db/db mice. Furthermore, the high urinary albumin amount is observed in db/db mice group than eucalyptol treated mice. Also, db/db mice Hba1c level is twice over as high as control mice. Likewise, Plasma insulin concentration is lowest in control mice and highest in db/db mice. Through the lysate of kidney, we investigated the EMT marker by western blotting. Under the treatment of eucalyptol, while the induction of N‐cadherin decreased, the expression of E‐cadherin increased. Therefore, we concluded that eucalyptol is effective in diabetic nephropathy especially in high glucose‐induced epithelial to mesenchymal transition. Furthermore, the collagen fiber accumulation in kidney tissue diminished under the eucalyptol treatment, it increased under the high glucose conditions by the Masson staining. Also, eucalyptol attenuated the CTGF induction and collagen production elevated by the presence of high glucose and reduced high glucose‐stimulated MT1‐MMP expression with congruent induction of TIMP‐2 in vitro. These results demonstrate that nontoxic eucalyptol abrogated renal tubular EMT‐mediated tubulointerstitial. Therefore, eucalyptol may be a potent renoprotective agent for the treatment of diabetes‐associated renal tubular fibrosis.