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Chronic kidney disease (CKD) is a global health concern affecting millions worldwide. One of the critical challenges in CKD is the accumulation of uremic toxins such as p-cresol sulfate (pCS) and indoxyl sulfate (IS), which contribute to systemic damage and CKD progression. Understanding the transport mechanisms of these prominent toxins is essential for developing effective treatments. Here, we investigated whether pCS and IS are routed to the plasma membrane or to the cytosol by two key transporters, SLC22A11 and OAT1. To distinguish between cytosolic transport and plasma membrane insertion, we used a hyperosmolarity assay in which the accumulation of substrates into HEK-293 cells in isotonic and hypertonic buffers was measured in parallel using LC-MS/MS. Judging from the efficiency of transport (TE), pCS is a relevant substrate of SLC22A11 at 7.8 ± 1.4 µL min−1 mg protein−1 but not as good as estrone-3-sulfate; OAT1 translocates pCS less efficiently. The TE of SLC22A11 for IS was similar to pCS. For OAT1, however, IS is an excellent substrate. With OAT1 and p-aminohippuric acid, our study revealed an influence of transporter abundance on the outcomes of the hyperosmolarity assay; very high transport activity confounded results. SLC22A11 was found to insert both pCS and IS into the plasma membrane, whereas OAT1 conveys these toxins to the cytosol. These disparate transport mechanisms bear profound ramifications for toxicity. Membrane insertion might promote membrane damage and microvesicle release. Our results underscore the imperative for detailed structural inquiries into the translocation of small molecules. [ABSTRACT FROM AUTHOR]

[Display omitted] Many drugs are largely hydrophobic molecules; a transporter might conceivably insert these into the plasma membrane. At least 18 transporters from diverse families have been reported to transport the model compound estrone sulfate alias estrone-3-sulfate (E3S). Out of these, we recently examined SLC22A11 (OAT4). We concluded from a comparison of E3S and uric acid transport that SLC22A11 does not translocate E3S into the cytosol, but into the plasma membrane. Here we present a hyperosmolarity alias hypertonicity assay to differentiate transport mechanisms. Human transporters were expressed heterologously in 293 cells. Solute uptake into intact cells was measured by LC-MS. Addition of mannitol or sucrose led to rapid cell shrinkage, but cell viability after 60 min in hyperosmolar buffer was not impaired. A decrease in substrate accumulation with increasing osmolarity as observed here for several substrates and the transporters SLC22A11, ETT (SLC22A4), OCT2 (SLC22A2), OAT3 (SLC22A8), and MATE1 (SLC47A1) suggests regular substrate translocation into the cytosol. An increase as observed for E3S transport by SLC22A11, OAT3, MATE1, SLC22A9, and SLC10A6 implies insertion into the membrane. In marked contrast to the other E3S transporters, the bile acid transporter SLC10A1 (NTCP, Na+ taurocholate co-transporting polypeptide) showed a decrease in the accumulation of E3S in hyperosmolar buffer; the same was observed with taurocholic acid. Indeed, our data from several functional assays strongly suggest that the transport mechanism is identical for both substrates. Apparently, a unique transport mechanism has been established for SLC10A1 by evolution that ensures the transport of amphipathic, detergent-like molecules into the cytosol. [ABSTRACT FROM AUTHOR]