zVAD-fmk prevents cisplatin-induced cleavage of autophagy proteins but impairs autophagic flux and worsens renal function

Abstract

Cisplatin injury to renal tubular epithelial cells (RTEC) is accompanied by autophagy and caspase activation. However, autophagy gradually decreases during the course of cisplatin injury. The role of autophagy and the mechanism of its decrease during cisplatin injury are not well understood. This study demonstrated that autophagy proteins beclin-1, Atg5, and Atg12 were cleaved and degraded during the course of cisplatin injury in RTEC and the kidney. zVAD-fmk, a widely used pancaspase inhibitor, blocked cleavage of autophagy proteins suggesting that zVAD-fmk would promote the autophagy pathway. Unexpectedly, zVAD-fmk blocked clearance of the autophagosomal cargo, indicating lysosomal dysfunction. zVAD-fmk markedly inhibited cisplatin-induced lysosomal cathepsin B and calpain activities and therefore impaired autophagic flux. In a mouse model of cisplatin nephrotoxicity, zVAD-fmk impaired autophagic flux by blocking autophagosomal clearance as revealed by accumulation of key autophagic substrates p62 and LC3-II. Furthermore, zVAD-fmk worsened cisplatin-induced renal dysfunction. Chloroquine, a lysomotropic agent that is known to impair autophagic flux, also exacerbated cisplatin-induced decline in renal function. These findings demonstrate that impaired autophagic flux induced by zVAD-fmk or a lysomotropic agent worsened renal function in cisplatin acute kidney injury (AKI) and support a protective role of autophagy in AKI. These studies also highlight that the widely used antiapoptotic agent zVAD-fmk may be contraindicated as a therapeutic agent for preserving renal function in AKI.

Fig. 1.

Cleavage of beclin-1, Atg5, and Atg12 in cisplatin (CP)-treated cells and effect of pancaspase inhibitor zVAD-fmk LLC-PK₁ cells were treated with 50 μM CP in the presence and absence of 20 μM zVAD-fmk for various time points as indicated. Cell lysates were subjected to Western blot analysis using antibodies to Atg5 (A), beclin-1 (B), and Atg12 (C). Actin was used as a loading control using a specific antibody to β-actin.

Fig. 2.

Effect of ZVAD-fmk on CP-induced autophagic flux. A: effect of zVAD-fmk on CP-induced LC3-II formation. Fluorescence staining of GFP-LC3 and formation of GFP-LC3-II in response to zVAD-fmk in CP-treated cells. LLC-PK₁ cells growing on coverslips at 70% confluency were transfected with GFP-LC3 plasmid. Following transfection as described in materials and methods, cells were treated with 50 μM CP in the presence and absence of 20 μM zVAD-fmk for various times as indicated. The appearance of CP-induced punctate staining dots indicates autophagosome-associated LC3-II. B: effect of zVAD-fmk on CP-induced LC3-II formation by Western blot analysis. LLC-PK₁ cells were treated with 50 μM CP in the presence and absence of 20 μM zVAD-fmk for various time periods as indicated. Conversion of LC3-I (18 kDa) to LC3-II (16 kDa) was determined in the cell lysates by Western blot using an antibody specific to LC3.

Fig. 3.

Effect of chloroquine (CHL) on zVAD-fmk-induced accumulation of LC3-II and p62 LLC-PK₁ cells were treated with 20 μM zVAD-fmk in the presence and absence of 20 μM chloroquine 1 h before the treatment with 50 μM CP for various time periods as indicated. Cell lysates were analyzed by Western blot for LC3-II and p62 using their specific antibodies. Actin was used as a loading control using a specific antibody to β-actin.

Fig. 4.

Effect of zVAD-fmk on CP-induced activation of calpain and cathepsin B. A: effect of zVAD-fmk on CP-induced activation of calpain. LLC-PK₁ cells were treated with 50 μM CP in the presence and absence of 20 μM zVAD-fmk for various times as indicated. Calpain activity in the cell lysates (50 μg of protein) was determined using N-succinyl-Leu-Leu-Val-Tyr-7-AMC fluorogenic substrate as described in materials and methods. B: effect of zVAD-fmk on CP-induced activation of cathepsin B. LLC-PK₁ cells were treated with 50 μM CP in the presence and absence of 20 μM zVAD-fmk for various times as indicated. Cathepsin B activity in the cell lysates (50 μg of protein) was determined using z-Arg-Arg-AMC fluorogenic substrate as described in materials and methods.

Fig. 5.

Dose-dependent effect of zVAD-fmk on CP-induced lactate dehydrogenase (LDH) release. Cells grown in 96-well plates in quadruplicates were treated at 70% confluency with either vehicle or various concentrations of zVAD-fmk 1 h before addition of CP (50 μM) and the LDH release assay was performed according to the instructions in the Cytotoxicity DetectionKitPlus Roche (Indianapolis, IN) as described in materials and methods.

Fig. 6.

Effect of overexpression of beclin-1 and Atg5 in CP-induced caspase activation and cell death. A: cells were transfected with beclin-1 and Atg5 plasmids as described in materials and methods and cell lysates were subjected to Western blots for beclin-1 and Atg5 protein expression. B: effect of beclin-1 or Atg5 overexpression on caspas-3/7 activation in response to CP injury. LLC-PK₁ cells were transfected with Atg5 or beclin-1 plasmids and treated with and without CP (50 μM) as indicated. Cell lysates were prepared and analyzed for caspase-3/7 activation as described in materials and methods. P < 0.01 compared with CP-treated cells. C: LLC-PK₁ cells were transfected with Atg5 or beclin-1 plasmids and treated with and without CP (50 μM) as indicated. The cells were stained with 0.5 μg/ml of 4′,6′-diamidino-2-phenylindole (DAPI) for 5 min, and the cells were washed twice in PBS. Coverslips were then mounted on slides using antifade mounting medium (Molecular Probes). Morphological changes of the nuclei were analyzed using a Zeiss deconvolution microscope.

Fig. 7.

Induction of autophagy and expression of autophagy proteins in kidney in response to CP injury. A: expression of autophagy proteins in CP nephrotoxicity. A mouse model of CP nephrotoxicity was developed as described in materials and methods. Mice were injected intraperitoneally with 20 mg/kg body wt CP in saline. Control mice were administered saline. Kidneys from mice were removed at 0 day [control (c)], 1 day (1d), 2 days (2d), 3 days (3d), and 4 days (4d). Tissue lysates from kidney cortices were prepared and 100-μg protein samples were subjected to Western blot analysis using antibody specific to LC3. B: induction of autophagy in kidney in CP nephrotoxicity. Formation of lipidated LC3 (LC3-II) from LC3-I was significantly increased in the kidneys from CP-treated mice. At 3 days following CP administration, the autophagosomes were maximally detected in the kidney cortex. The punctate fluorescent staining of autophagosomes was identified by immunostaining with antibody specific to LC3. Detection at ×20 and ×60 magnification is shown. C: cleavage of autophagy proteins during CP nephrotoxicity. C57BL/6 mice were treated with CP with or without zVAD-fmk as mentioned in materials and methods. Tissue lysates from kidney cortices at 0 day (c), 1 day, 2 days, 3 days, and 4 days were prepared and 100-μg protein samples were subjected to Western blot analysis using antibody specific to beclin-1.

Fig. 8.

zVAD-fmk and chloroquine impair autophagic flux in vivo. A: fluorescence staining of LC3-II in kidneys of mice treated with CP in the presence or absence of zVAD-fmk or chloroquine. C57BL/6 mice were treated with or without zVAD-fmk or chloroquine followed by CP as mentioned in materials and methods. Kidneys were harvested 3 days after treatment, formalin fixed, and paraffin embedded. Deparaffinized 5-μm sections were immunostained with polyclonal rabbit anti-LC3 followed with anti-rabbit Alexafluor-488-labeled secondary antibody and pictures were recorded on an Olympus BX51 fluorescence microscope at ×40 magnification. Kidney sections shown are as follows: control kidney section, kidney section of mouse treated with 20 mg/kg body wt CP after 3 days (3d CP), kidney section of mouse treated with 10 mg/kg body wt zVAD-fmk followed by 20 mg/kg body wt CP for 3 days (3d CP + zVAD-fmk), and kidney section of mouse treated with 50 mg·kg body wt⁻¹·day⁻¹ chloroquine + 20 mg/kg body wt CP for 3 days (3d CP + chloroquine). B: expression levels of LC3-II and p62 proteins in the presence and absence of ZVAD-fmk or chloroquine in CP nephrotoxicity. Mice were injected intraperitoneally with 20 mg/kg body wt CP in saline in presence and absence of 10 mg/kg body wt zVAD-fmk or 50 mg·kg body wt⁻¹·day⁻¹ chloroquine. Control mice were administered saline. Kidneys from mice were removed at 0 day (c), 1 day, 2 days, 3 days, and 4 days. Tissue lysates from kidney cortices were prepared and 100-μg protein samples were subjected to Western blot analysis using antibodies specific to LC3, p62, and actin. Actin detection is for the loading control. C: accumulation of p62 in the presence and absence of ZVAD-fmk or chloroquine in CP nephrotoxicity. Kidney sections from 3 days and following various treatments as shown were immunostained with p62 antibody as described in materials and methods. Green fluorescence shows accumulation of p62.

Fig. 9.

ZVAD-fmk and autophagy inhibitor chloroquine (Chlq) worsen CP-induced TdT-mediated dUTP nick-end labeling (TUNEL) staining. Mice were injected intraperitoneally with 20 mg/kg body wt CP in the presence and absence of 10 mg/kg body wt zVAD-fmk or 50 mg·kg body wt⁻¹·day⁻¹ chloroquine. Control mice were administered vehicle (10% DMSO in saline). Kidney sections were subjected to TUNEL staining according to the manufacturer's instructions using a kit from Roche Applied Science.

Fig. 10.

ZVAD-fmk and autophagy inhibitor chloroquine worsen CP-induced decline in renal function and histology. A: blood urea nitrogen (BUN) in response to CP, CP + chloroquine, and CP + zVAD-fmk. Mice were injected intraperitoneally with vehicle 10% DMSO in saline (n = 6), CP (1d and 3d period, n = 12 for each period), CP + zVAD-fmk (1d and 3d period, n = 13 for each period), CP + chloroquine (1d and 3d period, n = 9 for each period), zVAD alone for 3d (n = 6), or chloroquine alone for 3d (n = 6). Chloroquine (50 mg·kg body wt⁻¹·day⁻¹) or zVAD-fmk (10 mg/kg body wt) was administered intraperitoneally 1 h before the 20-mg/kg body wt CP injection. At the indicated times, BUN was determined. ***P < 0.001 and ****P < 0.0001 compared with control. B: serum creatinine was similarly determined using a diagnostic kit from International Bio-Analytical Industries (Boca Raton, FL). ***P < 0.001 compared with control. C: renal histology in response to vehicle saline (control) CP, zVAD-fmk alone (3d zVAD), chloroquine alone (3d Chlq), CP + zVAD-fmk (3d CP + zVAD), and CP + chloroquine (3d CP + Chlq). Kidney sections were stained with hematoxylin and eosin staining. D: histology score for kidney sections from mice treated with CP, CP + zVAD-fmk (3d CP + zVAD), and CP + chloroquine (3d CP + Chlq). The scores were determined in a blind manner as described in materials and methods. The results are expressed as means ± SE (n = 6).

Fig. 11.

Scheme depicting autophagic flux and common inhibitors of the autophagic pathway. Autphagic flux involves the dynamic process of autophagosome synthesis, delivery of the autophagic substrates to the lysosome, and degradation of the sequestered substrates by the lysosomal hydrolases. The “core” autophagic machinery that utilizes Atg proteins for the phagophore formation and its elongation to mature autophagosome formation work through the following functional protein complexes: 1) the ULK1/2 kinase complex is required for the induction of autophagy and involves at the site of autophagosome formation. Ulk1/2 is negatively regulated by mTORC1. 2) A class-III phosphatidylinositol 3-kinase complex is required for nucleation of the phagophore membrane. Beclin1 (Atg6) and Atg14 are part of this complex. 3) The elongation and expansion steps in autophagosome formation involve 2 conjugation systems that require ubiquitin-like proteins, Atg12 and Atg8/LC3-II. The Atg5-Atg12 conjugate subsequently associates noncovalently with Atg16 to form an Atg12-Atg5-Atg16 multimeric complex. The second conjugation step is the formation of LC3-II (Atg8-PE) by conjugation of LC3 with phosphatidylethanolamine (PE). Once the autophagosome is formed, most of the Atg proteins are dissociated which then allows fusion with the lysosome to form autolysosome. The sequestered contents and the inner membrane of the autolysosome are degraded by the lysosomal hydrolases. The autophagic flux is an indicator that the autophagy process has undergone to completion.