Green olive leaf extract (OLE) provides cytoprotection in renal cells exposed to low doses of cadmium

Abstract

Cadmium (Cd) is a heavy and highly toxic metal that contaminates air, food and water. Cadmium accumulates in several organs altering normal functions. The kidney is the major organ at risk of damage from chronic exposure to cadmium as a contaminant in food and water. This study aims to investigate the beneficial effects of OLE in renal collecting duct MCD4 cells exposed to a low dose cadmium (1 μM). In MCD4 cells cadmium caused an increase in ROS production, as well as generation of lipid droplets and reduced cell viability. Moreover, cadmium exposure led to a remarkable increase in the frequency of micronuclei and DNA double-strand breaks, assessed using the alkaline comet assay. In addition, cadmium dramatically altered cell cytoskeleton architecture and caused S-glutathionylation of actin. Notably, all cadmium-induced cellular deregulations were prevented by co-treatment with OLE, possibly due to its antioxidant action and to the presence of bioactive phytocompounds. Indeed, OLE treatment attenuated Cd-induced actin S-glutathionylation, thereby stabilizing actin filaments. Taken together, these observations provide a novel insight into the biological action of OLE in renal cells and support the notion that OLE may serve as a potential adjuvant against cadmium-induced nephrotoxicity.

Fig 1. Cell viability of MCD4 cells.

Cells were left under basal condition or treated with OLE (0.001mg/ml; 0.01mg/ml; 0.1mg/ml) and were stained with crystal violet solution. Data are presented as means ± SEMs of 3 independent experiments.

Fig 2. ROS content.

ROS content was measured using dihydrorhodamine-123 fluorescence in MCD4 cells treated as described in Methods. As positive control, cells were treated with the oxidant tBHP. Data are shown as mean ± SEMs and analyzed by one-way ANOVA followed by followed by Tukey’s Multiple Comparison test. (#P<0.0001 vs CTR; *P<0.0001 vs tBHP).

Fig 3. ROS content.

ROS content was measured using dihydrorhodamine-123 fluorescence in MCD4 cells treated as described in the Methods section. As positive control, cells were treated with the oxidant tBHP. Data are shown as mean ± SEMs and analyzed by one-way ANOVA followed by followed by Tukey’s Multiple Comparison test. (#P<0.001 vs CTR; *P<0.001 vs tBHP).

Fig 4. Staining of F-actin in MCD4 cells.

(A) Cells were left under basal condition or treated with OLE (0.001mg/ml; 0.01mg/ml; 0.1mg/ml) in the presence or in the absence of the oxidant tBHP (2 mm) for 30 min. Specimens were incubated with Phalloidin-TRITC (400 μg/ml) for 45 min at room temperature to detect F-actin. (B) F-actin content was semi-quantified by actin polymerization assay. Confluent cells were treated as described under methods. After staining with TRITC-phalloidin, cells were extracted with cold methanol and the fluorescence absorbance of extracts was read (540/565 nm) in a RF-5301PC fluorimeter. The values obtained were compared using one-way Anova and Tukey’s multiple comparison test (*P<0.05 vs CTR; #P<0.01 vs tBHP).

Fig 5. Cell viability of MCD4 cells.

Cells were left under basal conditions or treated with OLE (0.01 mg/ml), CdCl₂ (0.1μM) or with OLE in the presence of CdCl₂. After treatment, cells were stained with crystal violet solution. Data are presented as means ± SEMs of 3 independent experiments (*P< 0.001 vs CTR).

Fig 6. Determination of micronuclei.

Nuclei were detected using DAPI staining. Percentage of micronucleated cells was enumerated according to DAPI staining. Cadmium treatment promotes micronuclei formation compared to untreated cells. Data are shown as mean ± SEMs and analyzed by one-way ANOVA followed by followed by Tukey’s Multiple Comparison test. (*P< 0.001 vs CTR).

Fig 7. Comet assay.

DNA damage was evaluated in MCD4 cells treated with OLE (0.01 mg/ml), CdCl₂ (0.1μM) or with OLE in the presence of CdCl2. Imaging analysis revealed that cadmium promotes tail formation. This effect was counteracted by co-treatment with OLE.

Fig 8. Detection of intracellular lipid droplets.

(A) Oil Red O staining for lipid droplet formation, the lipid droplets are stained dark red. (B) The number and the area of lipid droplets are shown by frequency distribution.

Fig 9. ROS content.

ROS content was measured using dihydrorhodamine-123 fluorescence in MCD4 cells treated as described in Methods. As positive control, cells were treated with tBHP. Data are shown as mean ± SEMs and analyzed by one-way ANOVA followed using Tukey’s Multiple Comparison test. (**P<0.01 vs CTR; ****P<0.0001 vs CTR).

Fig 10. F-actin visualization.

(A) Cells were left under basal condition (CTR) or treated with OLE (0.01 mg/ml), CdCl₂(0.1μM) or with OLE in the presence of CdCl₂. Specimens were incubated with Phalloidin-TRITC (400 μg/ml) for 45 min at room temperature to detect F-actin. (B) F-actin content was semi-quantified by actin polymerization assay. Confluent cells were treated as described in Methods. After staining with TRITC-phalloidin, cells were extracted with cold methanol and the fluorescence absorbance of extracts was read (540/565 nm) in an RF-5301PC fluorimeter. The values obtained were compared using one-way Anova and Tukey’s multiple comparison test (*P<0,05 vs CTR).

Fig 11. Pyrene-actin polymerization assay.

Cytosolic fractions, isolated from cells treated as described above, were incubated with pyrene actin and changes in fluorescence were measured over time. The results shown are representative of three independent experiments performed in triplicate.

Fig 12. Glutathionylation assay with BIOGEE.

Glutathionylated actin was detected as described in Methods. Densitometric analysis of the obtained bands revealed that actin S-glutathionylation significantly increased in cells treated with CdCl₂ with respect to CTR. The data (means ± SEMs) were analyzed by one-way ANOVA, followed by Tukey’s Multiple Comparison test.

Fig 13. Proposed model of the action of olive leaf extract in renal cells exposed to a low cadmium dose.

Details in the discussion.