Blockade of Organic Anion Transport in Humans After Treatment With the Drug Probenecid Leads to Major Metabolic Alterations in Plasma and Urine

Abstract

Probenecid is used to treat gout and hyperuricemia as well as increase plasma levels of antiviral drugs and antibiotics. In vivo, probenecid mainly inhibits the renal SLC22 organic anion transporters OAT1 (SLC22A6), OAT3 (SLC22A8), and URAT1 (SLC22A12). To understand the endogenous role of these transporters in humans, we administered probenecid to 20 healthy participants and metabolically profiled the plasma and urine before and after dosage. Hundreds of metabolites were significantly altered, indicating numerous drug-metabolite interactions. We focused on potential OAT1 substrates by identifying 97 metabolites that were significantly elevated in the plasma and decreased in the urine, indicating OAT-mediated clearance. These included signaling molecules, antioxidants, and gut microbiome products. In contrast, urate was the only metabolite significantly decreased in the plasma and elevated in the urine, consistent with an effect on renal reuptake by URAT1. Additional support comes from metabolomics analyses of our Oat1 and Oat3 knockout mice, where over 50% of the metabolites that were likely OAT substrates in humans were elevated in the serum of the mice. Fifteen of these compounds were elevated in both knockout mice, whereas six were exclusive to the Oat1 knockout and 4 to the Oat3 knockout. These may be endogenous biomarkers of OAT function. We also propose a probenecid stress test to evaluate kidney proximal tubule organic anion transport function in kidney disease. Consistent with the Remote Sensing and Signaling Theory, the profound changes in metabolite levels following probenecid treatment support the view that SLC22 transporters are hubs in the regulation of systemic human metabolism.

Figure 1:. Probenecid effect on the kidney.

Probenecid inhibits the function of URAT1 on the apical membrane of the proximal tubule. Unfiltered probenecid goes through the peritubular capillaries and inhibits the function of OAT1/OAT3.

Figure 2:. Probenecid treatment alters the plasma metabolome.

A) Probenecid levels in the plasma were significantly elevated 5 hours after oral dosage in all participants. B) Principal component analysis (PCA) reveals separation between pre and post treatment with probenecid plasma metabolomes. C) Hundreds of metabolites were significantly altered (elevated and decreased) following treatment with probenecid. D) Among the significantly elevated metabolites, 21 subpathways with at least 5 metabolites were enriched, including subpathways traditionally associated with OAT-mediated transport (Primary Bile Acid Metabolism, Phenylalanine Metabolism, Tyrosine Metabolism, Tryptophan Metabolism, etc.).

Figure 3:. Probenecid treatment alters the urine metabolome.

A) Probenecid levels in the urine were significantly elevated 5 hours after oral dosage in all participants. B) Principal component analysis reveals separation between pre and post treatment with probenecid urine metabolomes. C) Hundreds of metabolites were significantly altered following treatment with probenecid, with most being decreased. D) Among the significantly decreased metabolites, 23 subpathways with at least 5 metabolites were enriched, including subpathways traditionally associated with OAT-mediated transport (Secondary Bile Acid Metabolism, Tryptophan Metabolism, Phenylalanine Metabolism, etc.).

Figure 4:. Metabolites elevated in the plasma and decreased in the urine are likely OAT1/3 substrates.

Metabolites were further filtered by fold change criteria, with only plasma metabolites with fold changes over 1.25 and urine metabolites with fold changes under 0.80 included. Overall, 97 metabolites fit these criteria, with 40 having known chemical structures. 34 of these 40 compounds had a total negative charge, and many were also supported by existing in vitro data (Table S3).

Figure 5:. Presumed inhibition of urate reuptake transporters such as URAT1 led to a specific drug-metabolite interaction between probenecid and urate.

A) Urate was the only metabolite to be significantly decreased in the plasma (fold change < 0.8) and increased in the urine (fold change > 1.25) with more selective fold change criteria. B) The chemical structure for urate. C) Urate levels were significantly decreased in the plasma following treatment with probenecid (p-value: 1.20E-10, fold change: 0.606). D) Urate levels were significantly increased in the urine following treatment with probenecid (p-value: 0.008, fold change: 1.705).

Figure 6:. Multiple metabolites suggested to be OAT substrates are supported by in vivo Oat1 and Oat3 knockout mice.

Twenty-five metabolites elevated in the probenecid-treated human plasma, decreased in the probenecid-treated urine, and elevated in one or both knockout mice. Twenty of these metabolites had associated chemical structures. Fifteen (13 with chemical structures) were common to both knockout mice, while 6 (3 with chemical structures) were unique to the Oat1 knockout mice, and 4 were unique to the Oat3 knockout mice.