Uraemic syndrome (also known as uremic syndrome) in patients with advanced chronic kidney disease involves the accumulation in plasma of small-molecule uraemic solutes and uraemic toxins (also known as uremic toxins), dysfunction of multiple organs and dysbiosis of the gut microbiota. As such, uraemic syndrome can be viewed as a disease of perturbed inter-organ and inter-organism (host-microbiota) communication. Multiple biological pathways are affected, including those controlled by solute carrier (SLC) and ATP-binding cassette (ABC) transporters and drug-metabolizing enzymes, many of which are also involved in drug absorption, distribution, metabolism and elimination (ADME). The remote sensing and signalling hypothesis identifies SLC and ABC transporter-mediated communication between organs and/or between the host and gut microbiota as key to the homeostasis of metabolites, antioxidants, signalling molecules, microbiota-derived products and dietary components in body tissues and fluid compartments. Thus, this hypothesis provides a useful perspective on the pathobiology of uraemic syndrome. Pathways considered central to drug ADME might be particularly important for the body's attempts to restore homeostasis, including the correction of disturbances due to kidney injury and the accumulation of uraemic solutes and toxins. This Review discusses how the remote sensing and signalling hypothesis helps to provide a systems-level understanding of aspects of uraemia that could lead to novel approaches to its treatment.

The organic anion transporters OAT1 (SLC22A6) and OAT3 (SLC22A8) have similar substrate specificity for drugs, but it is far from clear whether this holds for endogenous substrates. By analysis of more than 600 metabolites in the Oat3KO (Oat3 knockout) by LC/MS, we demonstrate OAT3 involvement in the movement of gut microbiome products, key metabolites, and signaling molecules, including those flowing through the gut-liver-kidney axis. Major pathways affected included those involved in metabolism of bile acids, flavonoids, nutrients, amino acids (including tryptophan-derivatives that are uremic toxins), and lipids. OAT3 is also critical in elimination of liver-derived phase II metabolites, particularly those undergoing glucuronidation. Analysis of physicochemical features revealed nine distinct metabolite groups; at least one member of most clusters has been previously validated in transport assays. In contrast to drugs interacting with the OATs, endogenous metabolites accumulating in the Oat1KO (Oat1 knockout) versus Oat3KO have distinct differences in their physicochemical properties; they are very different in size, number of rings, hydrophobicity, and molecular complexity. Consistent with the Remote Sensing and Signaling Hypothesis, the data support the importance of the OAT transporters in inter-organ and inter-organismal remote communication via transporter-mediated movement of key metabolites and signaling molecules (e.g. gut microbiome-to-intestine-to-blood-to-liver-to-kidney-to-urine). We discuss the possibility of an intimate connection between OATs and metabolite sensing and signaling pathways (e.g. bile acids). Furthermore, the metabolomics and pathway analysis support the view that OAT1 plays a greater role in kidney proximal tubule metabolism and OAT3 appears relatively more important in systemic metabolism, modulating levels of metabolites flowing through intestine, liver, and kidney.

Genes central to drug absorption, distribution, metabolism and elimination (ADME) also regulate numerous endogenous molecules. The Remote Sensing and Signaling Hypothesis argues that an ADME gene-centered network-including SLC and ABC "drug" transporters, "drug" metabolizing enzymes (DMEs), and regulatory genes-is essential for inter-organ communication via metabolites, signaling molecules, antioxidants, gut microbiome products, uremic solutes, and uremic toxins. By cross-tissue co-expression network analysis, the gut, liver, and kidney (GLK) formed highly connected tissue-specific clusters of SLC transporters, ABC transporters, and DMEs. SLC22, SLC25 and SLC35 families were network hubs, having more inter-organ and intra-organ connections than other families. Analysis of the GLK network revealed key physiological pathways (e.g., involving bile acids and uric acid). A search for additional genes interacting with the network identified HNF4α, HNF1α, and PXR. Knockout gene expression data confirmed ~60-70% of predictions of ADME gene regulation by these transcription factors. Using the GLK network and known ADME genes, we built a tentative gut-liver-kidney "remote sensing and signaling network" consisting of SLC and ABC transporters, as well as DMEs and regulatory proteins. Together with protein-protein interactions to prioritize likely functional connections, this network suggests how multi-specificity combines with oligo-specificity and mono-specificity to regulate homeostasis of numerous endogenous small molecules.

In vitro data indicates that the kidney proximal tubule (PT) transporters of uremic toxins and solutes (e.g., indoxyl sulfate, p-cresol sulfate, kynurenine, creatinine, urate) include two "drug" transporters of the organic anion transporter (OAT) family: OAT1 (SLC22A6, originally NKT) and OAT3 (SLC22A8). Here, we have examined new and prior metabolomics data from the Oat1KO and Oat3KO, as well as newly obtained metabolomics data from a "chemical double" knockout (Oat3KO plus probenecid). This gives a picture of the in vivo roles of OAT1 and OAT3 in the regulation of the uremic solutes and supports the centrality of these "drug" transporters in independently and synergistically regulating uremic metabolism. We demonstrate a key in vivo role for OAT1 and/or OAT3 in the handling of over 35 uremic toxins and solutes, including those derived from the gut microbiome (e.g., CMPF, phenylsulfate, indole-3-acetic acid). Although it is not clear whether trimethylamine-N-oxide (TMAO) is directly transported, the Oat3KO had elevated plasma levels of TMAO, which is associated with cardiovascular morbidity in chronic kidney disease (CKD). As described in the Remote Sensing and Signaling (RSS) Hypothesis, many of these molecules are involved in interorgan and interorganismal communication, suggesting that uremia is, at least in part, a disorder of RSS.

The hepatocyte nuclear factors, Hnf1a and Hnf4a, in addition to playing key roles in determining hepatocyte fate, have been implicated as candidate lineage-determining transcription factors in the kidney proximal tubule (PT) [Martovetsky et. al., (2012) Mol Pharmacol 84:808], implying an additional level of regulation that is potentially important in developmental and/or tissue-engineering contexts. Mouse embryonic fibroblasts (MEFs) transduced with Hnf1a and Hnf4a form tight junctions and express multiple PT drug transporters (e.g., Slc22a6/Oat1, Slc47a1/Mate1, Slc22a12/Urat1, Abcg2/Bcrp, Abcc2/Mrp2, Abcc4/Mrp4), nutrient transporters (e.g., Slc34a1/NaPi-2, Slco1a6), and tight junction proteins (occludin, claudin 6, ZO-1/Tjp1, ZO-2/Tjp2). In contrast, the coexpression (with Hnf1a and Hnf4a) of GATA binding protein 4 (Gata4), as well as the forkhead box transcription factors, Foxa2 and Foxa3, in MEFs not only downregulates PT markers but also leads to upregulation of several hepatocyte markers, including albumin, apolipoprotein, and transferrin. A similar result was obtained with primary mouse PT cells. Thus, the presence of Gata4 and Foxa2/Foxa3 appears to alter the effect of Hnf1a and Hnf4a by an as-yet unidentified mechanism, leading toward the generation of more hepatocyte-like cells as opposed to cells exhibiting PT characteristics. The different roles of Hnf4a in the kidney and liver was further supported by reanalysis of ChIP-seq data, which revealed Hnf4a colocalization in the kidney near PT-enriched genes compared with those genes enriched in the liver. These findings provide valuable insight, not only into the developmental, and perhaps organotypic, regulation of drug transporters, drug-metabolizing enzymes, and tight junctions, but also for regenerative medicine strategies aimed at restoring the function of the liver and/or kidney (acute kidney injury, AKI; chronic kidney disease, CKD).

Quantitative systems pharmacology (QSP) has emerged as a transformative science in drug discovery and development. It is now time to fully rethink the biological functions of drug metabolizing enzymes (DMEs) and transporters within the framework of QSP models. The large set of DME and transporter genes are generally considered from the perspective of the absorption, distribution, metabolism, and excretion (ADME) of drugs. However, there is a growing amount of data on the endogenous physiology of DMEs and transporters. Recent studies-including systems biology analyses of "omics" data as well as metabolomics studies-indicate that these enzymes and transporters, which are often among the most highly expressed genes in tissues like liver, kidney, and intestine, have coordinated roles in fundamental biological processes. Multispecific DMEs and transporters work together with oligospecific and monospecific ADME proteins in a large multiorgan remote sensing and signaling network. We use the Remote Sensing and Signaling Theory (RSST) to examine the roles of DMEs and transporters in intratissue, interorgan, and interorganismal communication via metabolites and signaling molecules. This RSST-based view is applicable to bile acids, uric acid, eicosanoids, fatty acids, uremic toxins, and gut microbiome products, among other small organic molecules of physiological interest. Rooting this broader perspective of DMEs and transporters within QSP may facilitate an improved understanding of fundamental biology, physiologically based pharmacokinetics, and the prediction of drug toxicities based upon the interplay of these ADME proteins with key pathways in metabolism and signaling. The RSST-based view should also enable more tailored pharmacotherapy in the setting of kidney disease, liver disease, metabolic syndrome, and diabetes. We further discuss the pharmaceutical and regulatory implications of this revised view through the lens of systems physiology.

Organic anion transporter 1 (OAT1/SLC22A6) is a drug transporter with numerous xenobiotic and endogenous substrates. The Remote Sensing and Signaling Theory suggests that drug transporters with compatible ligand preferences can play a role in "organ crosstalk," mediating overall organismal communication. Other drug transporters are well known to transport lipids, but surprisingly little is known about the role of OAT1 in lipid metabolism. To explore this subject, we constructed a genome-scale metabolic model using omics data from the Oat1 knockout mouse. The model implicated OAT1 in the regulation of many classes of lipids, including fatty acids, bile acids, and prostaglandins. Accordingly, serum metabolomics of Oat1 knockout mice revealed increased polyunsaturated fatty acids, diacylglycerols, and long-chain fatty acids and decreased ceramides and bile acids when compared with wildtype controls. Some aged knockout mice also displayed increased lipid droplets in the liver when compared with wildtype mice. Chemoinformatics and machine learning analyses of these altered lipids defined molecular properties that form the structural basis for lipid-transporter interactions, including the number of rings, positive charge/volume, and complexity of the lipids. Finally, we obtained targeted serum metabolomics data after short-term treatment of rodents with the OAT-inhibiting drug probenecid to identify potential drug-metabolite interactions. The treatment resulted in alterations in eicosanoids and fatty acids, further supporting our metabolic reconstruction predictions. Consistent with the Remote Sensing and Signaling Theory, the data support a role of OAT1 in systemic lipid metabolism.

The multispecific organic anion drug transporters OAT6 (SLC22A20) and OAT1 (SLC22A6) are expressed in nasal epithelial cells and both can bind odorants. Sequence analysis of OAT6 revealed an evolutionarily conserved 79-amino acid (AA) fragment present not only in OAT6 but also in other SLC22 transporters, such as the organic anion transporter (OAT), organic carnitine transporter (OCTN), and organic cation transporter (OCT) subfamilies. A similar fragment is also conserved in some odorant receptors (ORs) in both humans and rodents. This fragment is located in regions believed to be important for ligand/substrate preference and recognition in both classes of proteins, raising the possibility that it may be part of a potential common ligand/substrate recognition site in certain ORs and SLC22 transporters. In silico screening of an odorant database containing known OR ligands with a pharmacophore hypothesis (generated from a set of odorants known to bind OAT6 and/or OAT1), followed by in vitro uptake assays in transfected cells, identified OR ligands capable of inhibiting OAT6- and/or OAT1-mediated transport, albeit with different affinities. The conservation of the AA fragments between these two different classes of proteins, together with their coexpression in olfactory as well as other tissues, suggests the possibility that ORs and SLC22 transporters function in concert, and raises the question as to whether these transporters function in remote sensing and signaling and/or as transceptors.

The multispecific organic anion transporters, OAT1 (SLC22A6) and OAT3 (SLC22A8), the main kidney elimination pathways for many common drugs, are often considered to have largely-redundant roles. However, whereas examination of metabolomics data from Oat-knockout mice (Oat1 and Oat3KO) revealed considerable overlap, over a hundred metabolites were increased in the plasma of one or the other of these knockout mice. Many of these relatively unique metabolites are components of distinct biochemical and signaling pathways, including those involving amino acids, lipids, bile acids, and uremic toxins. Cheminformatics, together with a "logical" statistical and machine learning-based approach, identified a number of molecular features distinguishing these unique endogenous substrates. Compared with OAT1, OAT3 tends to interact with more complex substrates possessing more rings and chiral centers. An independent "brute force" approach, analyzing all possible combinations of molecular features, supported the logical approach. Together, the results suggest the potential molecular basis by which OAT1 and OAT3 modulate distinct metabolic and signaling pathways in vivo As suggested by the Remote Sensing and Signaling Theory, the analysis provides a potential mechanism by which "multispecific" kidney proximal tubule transporters exert distinct physiological effects. Furthermore, a strong metabolite-based machine-learning classifier was able to successfully predict unique OAT1 versus OAT3 drugs; this suggests the feasibility of drug design based on knockout metabolomics of drug transporters. The approach can be applied to other SLC and ATP-binding cassette drug transporters to define their nonredundant physiological roles and for analyzing the potential impact of drug-metabolite interactions.

The field of tissue engineering entered a new era with the development of human pluripotent stem cells (hPSCs), which are capable of unlimited expansion whilst retaining the potential to differentiate into all mature cell populations. However, these cells harbor significant risks, including tumor formation upon transplantation. One way to mitigate this risk is to develop expandable progenitor cell populations with restricted differentiation potential. Here, we used a cellular microarray technology to identify a defined and optimized culture condition that supports the derivation and propagation of a cell population with mesodermal properties. This cell population, referred to as intermediate mesodermal progenitor (IMP) cells, is capable of unlimited expansion, lacks tumor formation potential, and, upon appropriate stimulation, readily acquires properties of a sub-population of kidney cells. Interestingly, IMP cells fail to differentiate into other mesodermally-derived tissues, including blood and heart, suggesting that these cells are restricted to an intermediate mesodermal fate.