Main

Lead (Pb) is one of the most abundant toxic heavy metals in the environment which causes a broad range of biochemical, physiological and behavioral dysfunctions in human beings.1 Globally, Pb exposure is ubiquitous and routes of Pb exposure include inhalation of Pb-contaminated dust particles or aerosols, and ingestion of Pb-contaminated food or water.1, 2 The persistence of Pb in humans and its associated health risk is a matter of serious concern and a global issue. Pb is also a cumulative toxicant that is stored in the body for a long term, which exerts potent toxic effects on different tissues.3 Kidney is one of the most sensitive target organs for Pb toxicity, while the proximal tubule is the major site of Pb-induced renal injury.4 However, proximal tubular cells were less applied in the studies of Pb-induced nephrotoxicity. Primary cultures can better represent the live tissue than permanent cell lines, which are ideal for in vitro toxicity studies. Thus, primary rPT cells were established to elucidate low-level Pb-induced nephrotoxicity in this study.

Autophagy is a highly dynamic multi-step biological process which maintains cellular homeostasis via the degradation and recycling of damaged organelles, misfolded proteins, and long-lived macromolecules in lysosomes.5 Previous studies have demonstrated that basal autophagy is vital for normal proximal tubule function, and genetic or pharmacologic blockade of autophagy strongly enhanced acute kidney injury induced by cisplatin or ischemia-reperfusion.6, 7, 8, 9 Moreover, report by Lv et al.10 and Sui et al.11 showed that Pb promoted the autophagy in cultured osteoblasts and cardiofibroblasts, respectively. By contrast, our research group has recently found that 0.5 μM Pb treatment for 12 h blocked the autophagic flux in rPT cells,12 while the underlying molecular mechanism of impaired autophagic flux during Pb exposure remains to be elucidated.

Lysosomes are acidic organelles that contain various hydrolytic enzymes, which serve as cellular recycling centers for cargos received mainly from autophagy and endocytosis. Normal lysosomal degradation function is crucial to maintain cellular homeostasis and enable cell survival in the physiological state.13, 14 Due to its high hydrolase content, lysosomes are potentially harmful to the cell when damage occurs to the lysosomal membrane to induce lysosomal membrane permeabilization (LMP).14 LMP has been shown to cause the release of cathepsins and other hydrolases from the lysosomal lumen to the cytosol, which is a critical step in lysosome-mediated apoptosis.13 We have previously demonstrated that the apoptotic death was the chief mechanism in low-dose (0-1.0 μM) Pb-induced nephrotoxicity in rPT cells,15 which enable us to think whether LMP is involved in Pb-induced apoptosis in rPT cells. Based on the previous studies, this study will offer further evidences to elucidate the possible interaction mechanism between autophagy inhibition, impairment of lysosomal function and apoptosis in Pb-exposed rPT cells.

Results

Enhanced expression of autophagic marker LC3-II in Pb-exposed rPT cells

Immunoblot analysis of endogenous LC3-II has been widely used to reflect the progression of autophagy. Firstly, protein levels of LC3-II in rPT cells treated with Pb (0.25, 0.5 and 1 μM) for 3 h (Figure 1a), 6 h (Figure 1b) and 12 h (Figure 1c) was detected to investigate the effect of Pb exposure on autophagy, respectively. After 3 h treatment, only 1 μM Pb significantly elevated the LC3-II protein levels (Figure 1a). Significant differences were observed in the LC3-II protein levels after exposed to Pb (0.25, 0.5 and 1 μM) for 6 h (Figure 1b) and 12 h (Figure 1c), respectively; but 0.5 μM Pb resulted in a somewhat greater increase in LC3-II level than 1 μM Pb at these two time points. Likewise, there was a time-dependent enhancement of LC3-II protein level in 0.5 μM Pb-treated cells (Figure 1d). Thus, 0.5 μM Pb and 12 h exposure time were selected in subsequent experiments.

Figure 1
figure 1

Expression of the autophagy marker protein LC3-II in Pb-exposed rPT cells. Cells were incubated with increasing doses of Pb for 3 h (a), 6 h (b) and 12 h (c) to assess the protein levels of LC3-II. (d) Cells were treated with 0.5 μM Pb for different time periods to determine the protein levels of LC3-II. Upper panels: representative western blot images; lower panels: quantitative analysis of protein levels (mean±S.E.M., n=4). ns, not significant, *P<0.05, **P<0.01, as compared with control

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Autophagic flux was impaired by Pb treatment in rPT cells

Although the amount of LC3-II correlates with the number of autophagosomes, increased numbers of autophagosomes can be associated either with increased autophagosomes synthesis or decreased autophagosomes turnover.16 To distinguish these two possibilities, we assessed the autophagic flux. First, the effect of Pb exposure on the autophagosome formation in the presence of 3-MA or CQ (two well-defined autophagy inhibitors) was assessed using transient transfection method (Figures 2a and b). Co-incubation of Pb with 3-MA, which blocks the upstream steps of autophagy,20 It is also a concentration dependent meta-chromatic fluorescent dye. Upon the excitation by blue light, AO can be visualized as red fluorescence at high concentrations (in intact lysosomes) and green fluorescence at low concentrations (in the cytosol and the nucleus). Thus, AO relocation, from lysosomes to cytosol, and the decrease of granular (lysosomal) red fluorescence (dimer form) in combination with the increased diffuse (cytosolic) green fluorescence (monomer form) may imply the deterioration of lysosomal membrane stability with a decreased proton gradient, which permits the leakage of the lysosomal contents to cytosol.20 After incubated with 0.5 μM Pb (12 h) or 50 μM CQ (3 h) or EBSS medium (2 h), respectively, cells grown on coverslips in 24-well plates were loaded with 5 μg/ml AO at 37 °C for 30 min, rinsed twice with warm (37 °C) PBS and examined under confocal laser scanning microscope (TCS SPE, Leica, Germany) with excitation at 488 nm. Green fluorescence (emission peak between 530 and 550 nm) and red fluorescence (emission peak at about 650 nm) were simultaneously collected by two separate windows.

Lyso-Tracker Red staining

Cells were seeded on sterile coverslips placed in 24-well plates. After treatment with 0.5 μM Pb (12 h), EBSS medium (2 h) or 50 μM CQ (3 h), respectively, cells were incubated with 100 nM LTR (diluted in DMEM-F12 medium) for 30 min under ideal growth conditions (37 °C, 5% CO2) to label the lysosomes. Then slides were rapidly washed with warm PBS (37 °C) for three times, mounted as described above and observed under a laser scanning confocal microscope (TCS SPE, Leica, Germany).

Analysis of lysosomal degradation capacity

DQ-BSA-Green was used to determine the lysosomal degradation capacity. Cells grown on coverslips in 24-well plates were incubated with 10 μg/ml of DQ-BSA-Green for 12 h (37 °C, 5% CO2), washed twice with PBS to remove excess probe and refreshed the medium. Then cells were treated with 0.5 μM Pb (12 h), EBSS medium (2 h) or 50 μM CQ (3 h), respectively. Slides were mounted and observed under a laser scanning confocal microscope with excitation set at 488 nm. Degradation capacity was measured by the green fluorescence signal released due to the degradation of DQ-BSA-Green.

Analysis of apoptosis by morphological changes and flow cytometry

Cells were pretreated with 2 μM CA 074 or 20 μM Pep A for 1 h, followed by 0.5 μM Pb treatment for another 12 h to assess its effect on Pb-induced apoptosis, respectively. Firstly, harvested cells under the indicated treatments were stained with annexin V/PI to analyze the distribution of apoptotic cells using flow cytometer. Secondly, DAPI staining was applied to assess the morphological changes of treated cells and 200 cells were randomly selected to count the apoptotic cells within every batch of experiment, each one performed in triplicate. Both of these two methods have been extensively described in our previous study.15

Data presentation

Experiments were performed at least three times with similar results. Data are presented as the mean±S.E.M. of the indicated number of replicates. Statistical comparisons were made using one-way analysis of variance (ANOVA) (Scheffe’s F test) after ascertaining the homogeneity of variance between the treatments, and P<0.05 was regarded as significant.