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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. 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.

ORGANISM(S): Homo sapiens

PROVIDER: GSE151776 | GEO | 2020/10/22

REPOSITORIES: GEO

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Project description:Triptolide (TP), the major active component in Tripterygium wilfordii Hook. f. (Tripterygium Glycosides), contributes to treat rheumatoid arthritis and anticancer activities. However, the organ toxicity, especially nephrotoxicity and hepatotoxicity limit its clinical application. To fully understand the mechanism underlying the triptolide induced toxicity, iTRAQ based proteomics and targeted fatty acids analysis were performed. In the present study, mouse were treated with LD50 dose of triptolide, and plasma, liver and kidney tissues were harvested before drug administration (0 h) and after drug administration (0.5 h, 1 h, 2 h, 8 h, 16 h). The blood biochemical levels (ALT, AST, BUN and CRE) were used to evaluated the toxicity of triptolide. 2D-LC-MS/MS approach was used to compare different proteins among groups 0h, 1h, 2h and 8 h. Functional annotation of different proteins in liver between different groups reveals that the top 3 pathways influenced by the TP were acute phase response signaling, antigen presentation pathway and FXR/RXR activation, the molecular and cellular functions which was mainly effected were lipid metabolism and small molecule biochemistry. In kidney, the pathway including LPS/IL-1 Mediated Inhibition of RXR Function, EIF2 Signaling, acute phase response signaling, and LXR/RXR Activation were mainly affected. The proteomics data implicated that fatty acids (FAs) may involve in the organ toxicity induced by TP. Then targeted fatty acids analysis was carried out to determinate the concentration between the different groups (0.5 h, 1 h, 2 h, 8 h, 16 h) by HPLC-MRM. We found that fatty acids in liver (C17:0, C18:0, C18:2, C18:3, C20:0, C20:3, C20:4, C22:1, C22:2, C22:3, C22:4, C22:5, C22:6, C24:0, C24:1, C24:2, C24:3, C24:4, C24:5, C24:6) show significant difference among groups, while in kidney, FAs (C12:0, C12:1, C14:0, C14:1, C16:1, C16:2) show significant difference. P450 protein family show significant change in different group, protein CYP4A14 demonstrate change in both kidney and liver tissues. By combing proteomics and targeted FAs analysis, we deeply investigated the mechanism underlying the TP induced organ toxicity, and the results may provide deeply insight into prevention or intervention in the TP clinical usage and improving its safety.

Project description:The use of calcineurin inhibitor (CI) immunosuppressants has significantly improved the early allograft survival rate in organ transplantation. However, CI therapy has been associated with chronic nephrotoxicity, which limits their long-term utility. In order to understand the mechanisms of the toxicity, we analyzed the gene expression changes that underlie the development of CI immunosuppressant-mediated nephrotoxicity, in male Sprague-Dawley (SD) rats dosed daily with cyclosporine (CsA), FK506 or rapamycin (Rapa) for 1 to 28 days. We identified a group of genes, whose expression in rat kidney is quantitatively correlated with CI-induced kidney injury as observed in changes in blood urea nitrogen (BUN) levels and kidney histopathology. These genes include both up-regulated genes, such as Ren1 and Klks3, and down-regulated genes, such as Calb1, Egf, NCC, and kidney specific Wnk1 (KS-Wnk1). Using the down-regulated genes alone we successfully predicted CI immunosuppressant-mediated kidney injury in rats following 7 days of treatment. Among these genes are two mechanism-related genes, NCC and KS-Wnk1, both of which are involved in the sodium transport in the distal nephrons. The down-regulation of both genes at the mRNA and protein level in rat kidney following CI treatment was confirmed by quantitative RT-PCR and immunohistochemical staining, respectively. We hypothesize that decreased expression of NCC may cause reduced sodium chloride reabsorption in the distal tubules, and contribute to the prolonged activation of the Renin-Angiotensin-System (RAS), a demonstrated contributor to the development of CI-induced nephrotoxicity in both animal models and clinical settings. Therefore, NCC and KS-Wnk1 could potentially be used as biomarkers for early detection and prevention of CI-related nephrotoxicity in clinical practice. Male SD rats approximately 5-7 weeks old were maintained on certified rodent chow and dosed i.p. with olive oil, CsA (CAS # 59865-13-3, 2.5 or 25 mg/kg/day), FK506 (CAS # 104987-11-3, 0.6 or 6 mg/kg/day), or Rapa (CAS # 53123-88-9, 1 or 10 mg/kg/day) (LC Laboratories, Woburn, MA) for 1, 7, 14 or 28 consecutive days. Blood was collected at the terminal necropsy (24 hours after 1, 7, 14 or 28 days of treatment) for clinical chemistry analysis. Each kidney was cut in half (one cross section and the other longitudinal). One half of the left kidney and one half of the right kidney were used for histopathological evaluation and the remaining portions of the kidney were processed for gene expression analysis. Total RNA was isolated from rat kidney using QIAGEN RNeasy kits (QIAGEN Inc., Valencia, CA) following manufacture’s instruction. The rat genome array (RAT 230 2.0) GeneChip (Affymetrix, Santa Clara, CA) was used for gene expression profiling.

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Microphysiological System Modeling of Ochratoxin A-Associated Nephrotoxicity

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