Through its genoprotective, mitochondrial bioenergetic modulation, and antioxidant effects, Fucoxanthin and its metabolite minimize Ochratoxin A-induced nephrotoxicity in HK-2 human kidney cells

Abstract

Background: Ochratoxin A (OTA) is a mycotoxin with reported multiorgan toxicity, especially kidney toxicity. Fucoxanthin (FX) and its hydrolyzed metabolite Fucoxanthinol (FXL) have reno-protective antioxidant and anti-inflammatory properties. This study evaluates the nephroprotective effects of FX and FLX on OTA-induced renal cytotoxicity using the HK-2 cell line.

Methods: Molecular docking was used to study the binding affinities with the main proteins of the studied pathways. Various in-vitro assays were used to test the hypothesis, including MTT, mitochondrial bioenergetics, oxidative stress, and apoptosis biomarkers.

Results: Docking revealed binding affinities of the tested chemicals with mitochondria, oxidative stress, and apoptosis. Data showed that OTA has a dose-dependent cytotoxic effect on HK-2 cells. Notably, FX and FXL improved cell viability. A significant deregulation of normal cellular pathways including genotoxicity (DNA damage percentage), mitochondrial bioenergetics disruption (PDH, α-KG, MCI and MCIII complexes activities, ATP levels and mitochondrial membrane potential), downregulation of some mitochondrial genes (ND1, ND5, CO-1 and ATP6/8) expression, mitophagy inhibition (PARK1 and parkin), Oxidative stress induction (ROS and TBARS), oxidative stress genes downregulation (HO-1 and Nrf2), antioxidant enzymatic activity reduction (ROS and CAT), and apoptotic mediator markers elevation (Caspases- 3, 8 and 9, and Bax/Bcl-2 ratio) were observed in OTA mono-treated cells compared to untreated control cells. All parameters were markedly normalized by combining FX or FLX with OTA, providing more protection in FXL co-treated samples.

Conclusion: Our results suggest that FX and FXL may be effective novel therapies for treating OTA-induced nephrotoxicity in vitro.

Keywords: Cellular bioenergetics; Fucoxanthin; Fucoxanthinol; Mitochondria; Nephrotoxicity; Ochratoxin A; Oxidative stress.

Fig. 1

The effects of OTA, FX, and FLX on the viability and DNA integrity of HK-2 cells after 24 h treatment. Cytotoxic effects of (A) OTA, (B) FX and FLX, and (C) combination of OTA with FX or FLX in various concentrations on HK-2 kidney cell line. (D) Effect of common inhibitors of cytotoxicity on HK-2 cells treated with 20 µM of OTA. The cell viability was determined after 24 h incubation by MTT assay. Percentage of comet assay parameters of DNA damage: tail moment (TM) (E), and tail DNA content (TD) (F) in OTA (20 µM) treated HK-2 cells, alone and in co-treatment with each of FX and FLX. Data are expressed as mean ± SD values from at least 3 independent experiments. Each treatment was applied to multiple wells and indicated a statistically significant difference between groups as *, **, and *** at p < 0.05, p < 0.01, and p < 0.001, respectively. Ochratoxin A, OTA; Fucoxanthin, FX; Fucoxanthinol, FXL

Fig. 2

The effects of FX and FLX treatment on the bioenergetics and mitophagy markers of OTA-induced nephrotoxicity in HK-2 cells after 24 h treatment. Bioenergetic parameters: cellular ATP (A), MMP (B), MCI (C), MCIII (D), α-KG (H) levels, and PDH activity (G). Mitophagy markers; Parkin (E) and PINK1 (F) protein levels. The results are demonstrated as mean ± SD values from at least 3 independent experiments. *, **, *** indicate significance between different groups at (p < 0.05), (p < 0.01), and (p < 0. 001), respectively. Ochratoxin A, OTA; Fucoxanthin, FX; Fucoxanthinol, FXL

Fig. 3

The effects of OTA (20 µM) treatment, alone and in co-administration with each of FX and FLX after 24 h treatment, on mRNA transcript levels of mitochondrial genes; ND1 (A), ND5 (B), CO1 (C), ATP 6/8 (D), and mitophagy coding genes; PINK1 (E) and Parkin (F). The gene expression was analysed by RT-qPCR, and the results are demonstrated as mean ± SD values from at least 3 independent experiments. *, **, *** indicate significance between different groups at (p < 0.05), (p < 0.01), and (p < 0. 001), respectively. Ochratoxin A, OTA; Fucoxanthin, FX; Fucoxanthinol, FXL

Fig. 4

The effects of OTA (20 µM) and its combination with FX and FLX for 24 h treatment on the intracellular ROS level, antioxidant profile, and oxidative stress markers in HK-2 cells after 24 h treatment. ROS levels (A), TBARS level (B), CAT activity (C), SOD activity, and mRNA transcript levels of oxidative stress genes; HO-1 (E) and Nrf2 (F). The gene expression was analysed by RT-qPCR. Results are displayed as mean ± SD values from at least 3 independent experiments. *, **, *** indicate significance between different groups at (p < 0.05), (p < 0.01), and (p < 0. 001), respectively. Ochratoxin A, OTA; Fucoxanthin, FX; Fucoxanthinol, FXL

Fig. 5

Apoptotic pathway mediators expression levels; caspase-3 (A), caspase-8 (B), caspase-9 (C), and Bax/Bcl2 ratio of mRNA transcript level (D), in control and different treated HK-2 cells with OTA (20 µM) and its combination with FX and FLX for 24 h. Results are displayed as mean ± SD values from at least 3 independent experiments. *, **, *** indicate significance between different groups at (p < 0.05), (p < 0.01), and (p < 0. 001), respectively. Ochratoxin A, OTA; Fucoxanthin, FX; Fucoxanthinol, FXL

Fig. 6

An illustrated graphic summarizes the renoprotective effects of FX and FLX on OTA-induced kidney cytotoxicity utilizing the HK-2 cell line during 24 h invitro. Data from various cytotoxicity mechanisms revealed that OTA had a dose-dependent cytotoxic effect on HK-2 cells after 24 h. FX and FXL increased cell viability and provided cytoprotection. OTA (20 µM) mono-treated cells exhibited significant improper regulation of normal cellular pathways, including genotoxicity (percentage of DNA damage), disruption of mitochondrial bioenergetics (activities of PDH, α-KG, MCI, and MCIII complexes, ATP levels, and mitochondrial membrane potential), downregulation of specific mitochondrial quality control genes (mRNA transcript levels of ND1, ND5, CO-1, and ATP6/8), inhibition of mitophagy (PARK1 and parkin), induction of oxidative stress (ROS and TBARS), downregulation of oxidative stress genes (HO-1 and Nrf2), reduction of antioxidant enzymatic activity (ROS and CAT), and elevation of apoptotic mediator markers (Caspases-3, 8, and 9, and BAX-BCL-2 ratio). Combining FX or FLX with OTA (20 µM) significantly standardized all parameters, demonstrating enhanced protection at higher dosages than lower ones