Therapeutic Effect of Lebanese Cannabis Oil Extract in the Management of Sodium Orthovanadate-Induced Nephrotoxicity in Rats

Abstract

Sodium orthovanadate is a non-selective protein tyrosine phosphatase inhibitor that can cause several types of kidney injury, including glomerulosclerosis, inflammation, and tubular damage. Cannabis is widely known for its medicinal use, and several studies have demonstrated its anti-diabetic and anti-inflammatory properties. The current study investigated the therapeutic effect of Lebanese cannabis oil extract (COE) against sodium orthovanadate-induced nephrotoxicity both in vitro and in vivo. Sprague Dawley male rats were intraperitoneally injected with 10 mg/kg sodium orthovanadate for 10 days followed by 5 mg/kg; 10 mg/kg; or 20 mg/kg intraperitoneal injection of cannabis oil extract, starting on day 4 until day 10. The body weight of the rats was monitored during the study, and clinical parameters, including serum urea, creatinine, and electrolytes, as well as kidney and heart pathology, were measured. Conditionally immortalized cultured rat podocytes were exposed to either sodium orthovanadate or selective phosphatase inhibitors, including DUSPi (DUSP1/6 inhibitor) and SF1670 (PTEN inhibitor), in the presence or absence of cannabis oil extract. MTS and an in vitro scratch assay were used to assess podocyte cell viability and migration, respectively. Western blot analysis was used to evaluate the phosphorylation levels of AKT and p38 MAPK. Rats injected with sodium orthovanadate displayed a marked reduction in body weight and an increase in serum creatinine and urea in comparison to the control non-treated group. All doses of COE caused a significant decrease in serum urea, with a significant decrease in serum creatinine observed at a dose of 20 mg/kg. Moreover, the COE treatment of rats injected with orthovanadate (20 mg/kg) showed a marked reduction in renal vascular dilatation, scattered foci of acute tubular necrosis, and numerous mitoses in tubular cells compared to the sodium orthovanadate-treated group. The cell viability assay revealed that COE reversed cytotoxicity induced by sodium orthovanadate and specific phosphatase inhibitors (DUSPi and SF1670) in rat podocytes. The in vitro scratch assay showed that COE partially restored the migratory capacity of podocytes incubated with DUSPi and SF1670. Time-course and dose-dependent experiments showed that COE (1 μg/mL) induced a significant increase in phospho-(S473)-AKT, along with a decrease in phospho (T180 + Y182) P38 levels. The current results demonstrated that Lebanese cannabis oil possesses important kidney protective effects against sodium orthovanadate-induced renal injury.

Keywords: AKT; cannabis oil extract; nephrotoxicity; p38; phosphatase inhibitors; podocytes; sodium orthovanadate.

Figure 1

Effect of DUSPi, SF1670, and Na₃VO₄ on podocyte cell viability. Rat podocytes were treated with indicated concentrations of DUSPi (A), SF1670 (B), and Na₃VO₄ (C) for 24 h. Cell viability was evaluated using Cell Titer 96 Aqueous Non-Radioactive Cell Proliferation Assay Kit. Data are expressed as mean ± SEM. Differences between groups were evaluated using one-way ANOVA followed by Bonferroni’s multiple comparison test. * p < 0.05; *** p < 0.001; **** p < 0.0001—significantly different from control.

Figure 2

Cannabis oil extract protects podocyte cells from phosphatase inhibitor-induced cytotoxicity. Immortalized rat podocytes were treated with (A) 10 μM DUSPi, (B) 2 μM SF1670, and (C) 1 mM Na₃VO₄ in the absence or presence of cannabis oil extract (COE) at the indicated concentrations for 24 h. Cell viability was evaluated via Cell Titer 96 Aqueous Non-Radioactive Cell Proliferation Assay. Data are expressed as mean ± SEM (n = 3). Differences between groups were evaluated using one-way ANOVA followed by Bonferroni’s multiple comparison test. #### p < 0.0001 vs. control; * p < 0.05; *** p < 0.001; **** p < 0.0001—significantly different from the DUSPi/SF1670 or Na₃VO₄-only treated group.

Figure 3

Podocyte cell migration. (A) Representative pictures show the migration of podocyte cells after induction of a scratch representing a wound. All the pictures were taken immediately after the scratch was induced (at zero hours) and 24 h after incision. Podocytes in the pictures were cultured in different conditions, as indicated. Pictures are taken at 10 times magnification. (B) The relative change in cell-free gap surfaces was measured at different time points after the creation of the wounding scratch and is expressed as fold change over zero time. Results are expressed as mean ± SEM of three independent experiments performed in triplicate. Statistical analysis was performed using one-way ANOVA followed by Bonferroni’s multiple comparison test. * p < 0.05; ** p < 0.01; **** p < 0.0001. Scale bar, 100 µM (condition Na₃VO₄/24 h).

Figure 4

Cannabis oil extract protects podocyte cells from sodium orthovanadate by inhibiting apoptosis and modulating AKT and p38 protein phosphorylation. (A) Cultured immortalized rat podocytes were treated with COE (1 μg/mL) for different durations of time (10–120 min), as indicated. Cell extracts (50 μg) were analyzed by Western blotting using anti-phospho-AKT1 (S473), anti-phospho-p38 (phospho T180 + Y182), Anti-AKT1 + AKT2 + AKT3 antibody, and anti p38 alpha/beta MAPK antibodies. The levels of phospho-AKT-(S473) and phospho-p38 were normalized to total AKT (B) and p38 MAPK (C), respectively, then to actin protein content, and the signal intensity was identified by densitometry. Data are expressed as mean ± SD (n = 3). Differences between groups were evaluated using one-way ANOVA followed by Bonferroni’s multiple comparison test. * p < 0.01; **** p < 0.0001 vs. control. (D) Cultured immortalized rat podocytes were treated with sodium orthovanadate Na₃VO₄ (1 mM) for 24 h in the presence or absence of COE (2 μg/mL). Cell extracts (50 μg) were analyzed by Western blotting using anti-PARP, anti-caspase-3, and anti-catalase antibodies. GAPDH was used as a loading control.

Figure 5

Average serum creatinine levels (mg/dL) (A); average serum urea levels (mg/dL) (B) in different groups of rats. Rats were intraperitoneally injected with 10 mg/kg sodium orthovanadate daily for 10 days. COE was injected daily from day 4 to day 10. Serum urea and creatinine levels were measured. Each column represents the mean ± SEM of seven animals. Statistical analysis was performed using two-way ANOVA followed by Bonferroni’s multiple comparison test. # p < 0.05; ## p < 0.01 vs. control; * p < 0.05; ** p < 0.01—significantly different from the sodium orthovanadate-only treated group.

Figure 6

Micrograph of renal sections of rats from different groups (H&E stained). (A) Control rats showing renal parenchyma with no specific changes (enlargement: ×200); (B–D) sodium orthovanadate-treated rats demonstrating (B) acute tubular necrosis (enlargement: ×400), (C) renal parenchyma with mild vascular congestion (enlargement: ×100) (scale bar 200 µM: (C)), and (D) numerous mitoses in tubular cells (enlargement: ×400). Cannabis-treated rats at different doses—(E) 5 mg/kg (enlargement: ×200), (F) 10 mg/kg (enlargement: ×400), and (G) 20 mg/kg (enlargement: ×200)—showing renal parenchyma with no specific changes (scale bar 100 µM: (F)).