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Ochratoxin A induces nephrotoxicity in vitro and in vivo via pyroptosis

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Abstract

Ochratoxin A (OTA), a prevalent nephrotoxic mycotoxin contaminant in food and feedstuff, has been reported to induce renal injury. To disclose the nephrotoxicity of continuous administration of OTA and to investigate potential mechanisms related to pyroptosis, male C57BL/6 mice were intraperitoneally injected with 1.0 and 2.0 mg/kg B.W. OTA every other day for 14 days. At 2.0 mg/kg B.W. OTA administration significantly increased histological injury and renal fibrosis molecules (α-SMA, Vimentin, TGF-β) and activated the NOD-like receptor protein 3 (NLRP3) inflammasome and induced pyroptosis compared with control. In the in vitro tests, Madin–Darby canine kidney (MDCK) epithelial cells were exposed to 0–4.0 μg/ml OTA for 24 h in serum-free medium. Data showed that OTA dose-dependently affected cell viability and significantly up-regulated renal fibrosis genes (α-SMA, Vimentin, TGF-β). 2.0 μg/ml OTA significantly induced NLRP3 inflammasome activation and caspase-1-dependent pyroptosis, increasing the expression and secretion of pro-inflammatory cytokines (IL-6, TNF-α) and pyroptosis-related genes (GSDMD, IL-1β, IL-18) in MDCK cells. These outcomes were significantly abrogated after inhibiting NLRP3 activation with inhibitor MCC950 and silencing NLRP3 with small interfering RNA (siRNA). Furthermore, knockdown of caspase-1 also ameliorated OTA-induced renal fibrosis via the inhibition of pyroptosis. Collectively, the chosen doses of OTA-triggered nephrotoxicity through NLRP3 inflammasome activation and caspase-1-dependent pyroptosis both in vitro and in vivo.

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Abbreviations

OTA:

Ochratoxin A

MDCK:

Madin–Darby canine kidney

CKD:

Chronic kidney disease

GSDMD:

Gasdermin D

LPS:

Lipopolysaccharide

NLRP3:

NOD-like receptor protein 3

PYD:

Pyrin domain

ASC:

Apoptosis-associated speck-like protein containing a C-terminal CARD

DMEM:

Dulbecco’s Modified Eagle’s Medium

FBS:

Fetal bovine serum

MTT:

3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide

LDH:

Lactate dehydrogenase

PBS:

Phosphate buffer saline

TBS:

Tris-buffered saline

IL-6:

Interleukin-6

TNF-α:

Tumor necrosis factor α

TEM:

Transmission electron microscopy

PVDF:

Polyvinylidene fluoride

SDS-PAGE:

Sodium salt–polyacrylamide gel electrophoresis

BCA:

Bicinchoninic acid

ECL:

Enhanced chemiluminescence

siRNA:

Small interfering RNA

ANOVA:

One-way analysis of variance

SEM:

Standard error mean

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Funding

This work was funded by the National Natural Science Foundation of China (31972745, 31811530300), the National Key R & D Program (2017YFD0501001), the Priority Academic Program Development of Jiangsu Higher Education Institutions (Jiangsu, China), MOE Joint International Research Laboratory of Animal Health and Food Safety, College of Veterinary Medicine, Nanjing Agricultural University and Project of National Center for International Research on Animal Gut Nutrition.

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  1. Hu Li and Xinru Mao have contributed equally to this work.

Authors and Affiliations

  1. College of Veterinary Medicine, Nanjing Agricultural University, Nanjing, 210095, China

    Hu Li, Xinru Mao, Kai Liu, Jiahao Sun, Benrui Li, Rahmani Mohammad Malyar, Dandan Liu, Cuiling Pan, Fang Gan, Yunhuan Liu, Kehe Huang & Xingxiang Chen

  2. Institute of Nutritional and Metabolic Disorders in Domestic Animals and Fowls, Nanjing Agricultural University, Nanjing, 210095, China

    Hu Li, Xinru Mao, Kai Liu, Jiahao Sun, Benrui Li, Rahmani Mohammad Malyar, Dandan Liu, Cuiling Pan, Fang Gan, Yunhuan Liu, Kehe Huang & Xingxiang Chen

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  1. Hu Li
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Contributions

HL and XM designed the research and wrote the paper; JS, BL and RMM assisted in performing the in vivo and in vitro experiments; DL, CP, FG and YL guided the experiments and image acquisition; KH and XC modified the syntax of the paper; XC assisted in designing the research and approved the final version to be submitted.

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Correspondence to Xingxiang Chen.

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Li, H., Mao, X., Liu, K. et al. Ochratoxin A induces nephrotoxicity in vitro and in vivo via pyroptosis. Arch Toxicol 95, 1489–1502 (2021). https://doi.org/10.1007/s00204-021-02993-6

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