Generation of Knockout and Transgenic Zebrafish to Characterize Abcc4 Functions in Detoxification and Efflux of Lead

Lead (Pb) is one of the major heavy metals that are toxic to vertebrates and usually considered as environmental pollutants. ABCC4/MRP4 is an organic anion transporter that mediates cellular efflux of a wide range of exogenous and endogenous compounds such as cyclic nucleotides and anti-cancer drugs; however, it remains unclear whether ABCC4 and its orthologs function in the detoxification and excretion of toxic lead. In this study, we found that the transcriptional and translational expression of zebrafish abcc4 was significantly induced under lead exposure in developing zebrafish embryos and adult tissues. Overexpression of zebrafish Abcc4 markedly decreased the cytotoxicity and accumulation of lead in pig renal proximal tubule cell line (LLC-PK1 cells). To further understand the functions of zebrafish Abcc4 in lead detoxification, the clustered regularly interspaced palindromic repeats (CRISPR)/Cas9 system was used to create an abcc4−/− mutant zebrafish line. In comparison with the wild-type (WT) zebrafish, the abcc4−/− mutants showed a higher death rate and lead accumulation upon exposure to lead. Furthermore, a stable abcc4-transgenic zebrafish line was successfully generated, which exerted stronger ability to detoxify and excrete lead than WT zebrafish. These findings indicate that zebrafish Abcc4 plays a crucial role in lead detoxification and cellular efflux and could be used as a potential biomarker to monitor lead contamination in a water environment.

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Remote sensing and signaling in kidney proximal tubules stimulates gut microbiome-derived organic anion secretion

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1821809116 ◽

2019 ◽

Vol 116 (32) ◽

pp. 16105-16110 ◽

Cited By ~ 18

Author(s):

Jitske Jansen ◽

Katja Jansen ◽

Ellen Neven ◽

Ruben Poesen ◽

Amr Othman ◽

...

Keyword(s):

Proximal Tubule ◽

Gut Microbiome ◽

Organic Anion ◽

Organic Anion Transporter ◽

Indoxyl Sulfate ◽

Regulation Mechanism ◽

Uremic Toxin ◽

Proximal Tubule Cell ◽

Anion Secretion ◽

Anion Transporter

Membrane transporters and receptors are responsible for balancing nutrient and metabolite levels to aid body homeostasis. Here, we report that proximal tubule cells in kidneys sense elevated endogenous, gut microbiome-derived, metabolite levels through EGF receptors and downstream signaling to induce their secretion by up-regulating the organic anion transporter-1 (OAT1). Remote metabolite sensing and signaling was observed in kidneys from healthy volunteers and rats in vivo, leading to induced OAT1 expression and increased removal of indoxyl sulfate, a prototypical microbiome-derived metabolite and uremic toxin. Using 2D and 3D human proximal tubule cell models, we show that indoxyl sulfate induces OAT1 via AhR and EGFR signaling, controlled by miR-223. Concomitantly produced reactive oxygen species (ROS) control OAT1 activity and are balanced by the glutathione pathway, as confirmed by cellular metabolomic profiling. Collectively, we demonstrate remote metabolite sensing and signaling as an effective OAT1 regulation mechanism to maintain plasma metabolite levels by controlling their secretion.

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