Participation of autophagy in renal ischemia/reperfusion injury

https://doi.org/10.1016/j.bbrc.2008.01.059Get rights and content

Abstract

Renal ischemia–reperfusion (I/R) injury is inevitable in transplantation, and it results in renal tubular epithelial cells undergoing cell death. We observed an increase in autophagosomes in the tubular epithelial cells of I/R-injured mouse models, and in biopsy specimens from human transplanted kidney. However, it remains unclear whether autophagy functions as a protective pathway, or contributes to I/R-induced cell death. Here, we employed the human renal proximal tubular epithelial cell line HK-2 in order to explore the role of autophagy under hypoxia (1% O2) or activation of reactive oxygen species (500 μM H2O2). When compared to normoxic conditions, 48 h of hypoxia slightly increased LC3-labeled autophagic vacuoles and markedly increased LAMP2-labeled lysosomes. We observed similar changes in the mouse IR-injury model. We then assessed autophagic generation and degradation by inhibiting the downstream lysosomal degradation of autophagic vacuoles using lysosomal protease inhibitor. We found that autophagosomes increased markedly under hypoxia in the presence of lysosomal protease inhibitors, thus suggesting that hypoxia induces high turnover of autophagic generation and degradation. Furthermore, inhibition of autophagy significantly inhibited H2O2-induced cell death. In conclusion, high turnover of autophagy may lead to autophagic cell death during I/R injury.

Section snippets

Materials and methods

Allograft biopsy. Routine allograft biopsies were performed at one month after renal transplantation. All biopsy specimens were examined by light and electron microscopy. All patients gave written consent to undergo biopsy.

Cell Culture and in vitro I/R injury. Human renal proximal tubular epithelial cells (human kidney-2, HK-2; ATCC, Manassas, VA) were cultured in DMEM (Invitrogen Corp., Carlsbad, CA) with 10% fetal bovine serum under a 5% CO2 and 95% air atmosphere at 37 °C.

In order to mimic

Allograft biopsy specimens

We observed numerous PAS-positive vesicles in the cytoplasm of tubular epithelial cells from allograft biopsy specimens obtained from 1 month after renal transplantation (Fig. 1A), which were rarely observed in zero-hour biopsy specimens (data not shown). Electron microscopy showed vacuolar structures containing cytoplasm and that were clearly encircled by double membrane structures, resembling autophagosomes, in tubular epithelial cells, suggesting that some of these PAS-positive vesicles are

Discussion

Data presented in this study indicate that autophagosomes are present in human renal tubular epithelial cells from allograft biopsy specimens and in cultured human proximal tubular epithelial cells, where I/R injury may lead to autophagosomal cell death. This notion is supported by several lines of experimental evidence: (1) Electron microscopy from allograft biopsy specimens obtained at 1 month after renal transplantation showed increased autophagosomes in tubular epithelial cells; (2) In an

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