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Microphysiological system modeling of ochratoxin A-associated nephrotoxicity.

ABSTRACT: Ochratoxin A (OTA) is one of the most abundant mycotoxin contaminants in food stuffs and possesses carcinogenic, nephrotoxic, teratogenic, and immunotoxic properties. Specifically, a major concern is severe nephrotoxicity, which is characterized by degeneration of epithelial cells of the proximal tubules and interstitial fibrosis. However, the mechanism of OTA toxicity, as well as the genetic risk factors contributing to its toxicity in humans has been elusive due to the lack of adequate models that fully recapitulate human kidney function in vitro. The present study attempts to evaluate dose-response relationships, identify the contribution of active transport proteins that govern the renal disposition of OTA, and determine the role of metabolism in the bioactivation and detoxification of OTA using a 3D human kidney proximal tubule microphysiological system (kidney MPS). We demonstrated that LC₅₀ values of OTA in kidney MPS culture (0.375-1.21 μM) were in agreement with clinically relevant toxic concentrations of OTA in urine. Surprisingly, no enhancement of kidney injury biomarkers was evident in the effluent of the kidney MPS after OTA exposure despite significant toxicity observed by LIVE/DEAD staining. Instead, these biomarkers decreased in an OTA concentration-dependent manner. Furthermore, the effect of 1-aminobenzotriazole (ABT) and 6-(7-Nitro-2,1,3-benzoxadiazol-4-ylthio) hexanol (NBDHEX), pan-inhibitors of P450 and glutathione S-transferase (GST) enzymes, respectively, on OTA-induced toxicity in kidney MPS was examined. These studies revealed significant enhancement of OTA-induced toxicity by NBDHEX (3 μM) treatment, whereas ABT (1 mM) treatment decreased OTA-induced toxicity, suggesting roles for GSTs and P450 enzymes in the detoxification and bioactivation of OTA, respectively. Analysis of transcriptional changes using RNA-sequencing of kidney MPS treated with different concentrations of OTA revealed downregulation of several nuclear factor (erythroid derived-2)-like 2 (NRF2)-regulated genes by OTA treatment, including GSTs. The transcriptional repression of GSTs is likely playing a key role in OTA toxicity via attenuation of glutathione conjugation/detoxification. The sequential molecular events may explain the mechanism of toxicity associated with OTA. Additionally, OTA transport studies using kidney MPS in the presence and absence of probenecid (1 mM) suggested a role for organic anionic membrane transporter(s) in the kidney specific disposition of OTA. Our findings provide a clearer understanding of the mechanism of OTA-induced kidney injury, which may support changes in risk assessment, regulatory agency policies on allowable exposure levels, and determination of the role of genetic factors in populations at risk for OTA nephrotoxicity.

SUBMITTER: Imaoka T

PROVIDER: S-EPMC7560959 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Ochratoxin A (OTA) is one of the most abundant mycotoxin contaminants in food stuffs and possesses carcinogenic, nephrotoxic, teratogenic and immunotoxic properties. Especially, severe nephrotoxicity is of great concern, as characterized by degeneration of epithelial cells of the proximal tubules and interstitial fibrosis. However, its mechanism of toxicity, hazard identification as well as genetic risk factors contributing to OTA toxicity in humans has been elusive due to the lack of adequate models that fully recapitulate kidney function in vitro. The present study attempts to evaluate dose-response relationships, identify the contribution of active transport proteins that govern renal disposition of OTA, and determine the role of metabolism in bioactivation and detoxification of OTA using a 3D human kidney proximal tubule microphysiological system (kidney MPS). We demonstrated that IC50 values of OTA in kidney MPS culture (0.375 – 1.21 µM) were in good agreement with clinical toxic concentrations of OTA in urine. Surprisingly, no enhancement of kidney injury biomarkers was evident in the effluents of kidney MPS after OTA exposure despite significant toxicity observed by LIVE/DEAD staining, rather these biomarkers were decreased in OTA concentration-dependent manner. Furthermore, the effect of 1-aminobenzotriazole (ABT) and 6-(NBD-4-ylthio-) hexanol (NBDHEX), pan-inhibitor of P450 and GST enzymes, respectively, on the OTA-induced toxicity in kidney MPS was examined, which resulted in significant enhancement of OTA-induced toxicity by NBDHEX (3 µM) treatment whereas ABT (1 mM) treatment decreased OTA-induced toxicity, suggesting the roles of GSTs and P450 enzymes in the detoxification and bioactivation of OTA, respectively. Additionally, OTA transport studies using kidney MPS in the presence and absence of inhibitor of organic anion transporter(s), probenecid (1 mM), revealed the role of organic anionic membrane transporter(s) in the kidney specific disposition of OTA. Our findings provide a better understanding of the mechanism of OTA-induced kidney injury which may support changes in risk assessment, regulatory agency policies on allowable exposure levels and determination of genetic factors in high-risk populations against OTA nephrotoxicity.

Project description:Statins are used to lower cholesterol and prevent cardiovascular disease. Musculoskeletal side effects known as statin associated musculoskeletal symptoms (SAMS), are reported in up to 10% of statin users, necessitating statin therapy interruption and increasing cardiovascular disease risk. We tested the hypothesis that, when exposed to statins ex vivo, engineered human skeletal myobundles derived from individuals with (n = 10) or without (n = 14) SAMS and elevated creatine-kinase levels exhibit statin-dependent muscle defects. Myoblasts were derived from muscle biopsies of individuals (median age range of 62-64) with hyperlipidemia with (n = 10) or without (n = 14) SAMS. Myobundles formed from myoblasts were cultured with growth media for 4 days, low amino acid differentiation media for 4 days, then dosed with 0 and 5μM of statins for 5 days. Tetanus forces were subsequently measured. To model the change of tetanus forces among clinical covariates, a mixed effect model with fixed effects being donor type, statin concentration, statin type and their two way interactions (donor type*statin concentration and donor type* statin type) and the random effect being subject ID was applied. The results indicate that statin exposure significantly contributed to decrease in force (P<0.001) and the variability in data (R2C [R square conditional] = 0.62). We found no significant differences in force between myobundles from patients with/without SAMS, many of whom had chronic diseases. Immunofluorescence quantification revealed a positive correlation between the number of straited muscle fibers and tetanus force (R2 = 0.81,P = 0.015) and negative correlation between number of fragmented muscle fibers and tetanus force (R2 = 0.482,P = 0.051) with no differences between donors with or without SAMS. There is also a correlation between statin exposure and presence of striated fibers (R2 = 0.833, P = 0.047). In patient-derived myobundles, statin exposure results in myotoxicity disrupting SAA organization and reducing force. We were unable to identify differences in ex vivo statin myotoxicity in this system. The results suggest that it is unlikely that there is inherent susceptibility to or persistent effects of statin myopathy using patient-derived myobundles.

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Microphysiological system modeling of ochratoxin A-associated nephrotoxicity.

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