The impact of autophagy on the development of senescence in primary tubular epithelial cells

Abstract

Autophagy and senescence are 2 distinct pathways that are importantly involved in acute kidney injury and renal repair. Recent data indicate that the 2 processes might be interrelated. To investigate the potential link between autophagy and senescence in the kidney we isolated primary tubular epithelial cells (PTEC) from wild-type mice and monitored the occurrence of cellular senescence during autophagy activation and inhibition. We found that the process of cell isolation and transfer into culture was associated with a strong basal autophagic activation in PTEC. Specific inhibition of autophagy by silencing autophagy-related 5 (Atg5) counteracted the occurrence of senescence hallmarks under baseline conditions. Reduced senescent features were also observed in Atg5 silenced PTEC after γ-irradiation and during H-Ras induced oncogenic senescence, but the response was less uniform in these stress models. Senescence inhibition was paralleled by better preservation of a mature epithelial phenotype in PTEC. Interestingly, treatment with rapamycin, which acts as an activator of autophagy, also counteracted the occurrence of senescence features in PTEC. While we interpret the anti-senescent effect of rapamycin as an autophagy-independent effect of mTOR-inhibition, the more specific approach of Atg5 silencing indicates that overactivated autophagy can have pro-senescent effects in PTEC. These results highlight the complex interaction between cell culture dependent stress mechanisms, autophagy and senescence.

Keywords: Autophagy; chloroquine; p16INK4a; rapamycin; senescence; tubular epithelial cells.

Figure 1.

Experimental modification of baseline autophagy influences senescence marker expression and epithelial phenotype in renal primary tubular epithelial cells (PTEC). In vitro culturing of PTEC induces autophagy. (A) LC3 staining in mouse proximal tubular cells (mPT) cells and PTEC showing LC3 punctae (arrows) which are localized on the autophagosomal membrane during autophagy induction. (B) Representative LC3 immunoblots in PTEC as compared to mPT cells. (C) Electron microscopy image of PTEC with autophagic vacuoles representing baseline autophagy. (D) LC3 immunoblot in PTEC treated with increasing concentrations of rapamycin. (E) Immunocytochemistry for LC3 punctae upon autophagy induction with rapamycin. (F) Representative immunoblots for LC3 and p62 and (G) immunocytochemistry for LC3 aggregates and vacuole formation in PTEC upon autophagy inhibition with increasing concentrations of chloroquine. (H) Immunoblots for LC3, p62, p16^INK4a and p21 at day 12 in PTEC treated with rapamycin and chloroquine. (I) Immunoblots for Atg5, LC3, p16^INK4a, α-smooth muscle actin (α-SMA), E-cadherin (E-cadh.) and p21 in Atg5 or control siRNA treated PTEC harvested at indicated time points and passages (P0-P3). (J) Representative pictures of day 12 PTEC transfected with control or Atg5 siRNA in bright field and immunohistochemistry for epithelial marker ZO-1. Original magnification x 630 in A, E, and G and x 400 in J. Scale bar 1 µm in C. n = 3.

Figure 2.

Experimental modification of autophagy influences senescence marker expression and epithelial phenotype under pro-senescent stress conditions in renal primary tubular epithelial cells (PTEC). PTEC underwent established models of senescence induction using γ-irradiation (10 Gy) or retrovirus-mediated overexpression of constitutively active mutant H-Ras. In parallel, autophagy was suppressed by siRNA mediated knock down of Atg5. (A) Representative electron microscopy images from PTEC showing (a) presence of massive autophagy related structures (defined as early double membraned structures surrounding cellular content as well as later stages, where the inner membrane has already become indiscernible) at day 1 (b) a decline in autophagy at day 10 after γ-irradiation. (B) Immunoblots for Atg5, LC3, p16^INK4, α-smooth muscle actin (α-SMA), E-cadherin (E-cadh.) and p21 in γ-irradiated PTEC treated with Atg5 or control siRNA. (C, D) Quantification of γ-H2AX⁺/Ki67⁻ cells and BrdU incorporation in γ-irradiated PTEC treated with Atg5 or control siRNA. (E, F) Representative immunoblots for LC3, p62, p16^INK4a and p21 in γ-irradiated PTEC treated with rapamycin or chloroquine. (G) Representative electron microscopy images from PTEC showing presence of large autophagy related structures at day 6 after H-Ras transduction. (H) Immunoblots for H-Ras, p16^INK4a and p21 expression in Atg5 siRNA treated H-Ras transduced PTEC. (I, J) Quantification of γ-H2AX⁺/Ki67⁻ cells and BrdU incorporation in H-Ras transduced PTEC after Atg5 or control siRNA treatment. (K) Representative immunoblots for H-Ras, p16^INK4a and p21 upon rapamycin treatment in H-Ras transduced PTEC. Scale bar 1 µm in A and G. Data is presented as mean ± SEM. n = 7. *P < 0.05; **P < 0.005; ***P < 0.001.