Sun-Young Ahn

Toward a Systems Level Understanding of Organic Anion and Other Multispecific Drug Transporters: A Remote Sensing and Signaling Hypothesis

Organic anion transporters (Oats) are located in the barrier epithelia of diverse organs, where they mediate the absorption and excretion of a wide range of metabolites, signaling molecules, and xenobiotics. Although their interactions with a broad group of substrates have been extensively studied and described, the primary physiological role of Oats remains elusive. The presence of overlapping substrate specificities among the different Oat isoforms, together with recent metabolomic data from the Oat1, Oat3, and renal-specific transporter (RST/URAT1) knockout mice, suggests a possible role in remote signaling wherein substrates excreted through one Oat isoform in one organ are taken up by another Oat isoform located in a different organ, thereby mediating communication between different organ systems, or even between different organisms. Here we further develop this “remote sensing and signaling hypothesis” and suggest how the regulation of SLC22 subfamily members (including those of the organic cation, organic carnitine, and unknown substrate transporter subfamilies) can be better understood by considering the organisms broader need to communicate between epithelial and other tissues by simultaneous regulation of transport of metabolites, signaling molecules, drugs, and toxins. This systems biology perspective of remote signaling (sensing) could help reconcile an enormous array of tissue-specific data for various SLC22 family genes and, possibly, other multispecific transporters, such as those of the organic anion transporting polypeptide (OATP, SLC21) and multidrug resistance-associated protein (MRP) families.

Organic Anion Transporter 3 Contributes to the Regulation of Blood Pressure

Renal organic anion transporters (OAT) are known to mediate the excretion of many drugs, but their function in normal physiology is not well understood. In this study, mice lacking organic anion transporter 3 (Oat3) had a 10 to 15% lower BP than wild-type mice, raising the possibility that Oat3 transports an endogenous regulator of BP. The aldosterone response to a low-salt diet was blunted in Oat3-null mice, but baseline aldosterone concentration was higher in these mice, suggesting that aldosterone dysregulation does not fully explain the lower BP in the basal state; therefore, both targeted and global metabolomic analyses of plasma and urine were performed, and several potential endogenous substrates of Oat3 were found to accumulate in the plasma of Oat3-null mice. One of these substrates, thymidine, was transported by Oat3 expressed in vitro. In vivo, thymidine, as well as two of the most potent Oat3 inhibitors that were characterized, reduced BP by 10 to 15%; therefore, Oat3 seems to regulate BP, and Oat3 inhibitors might be therapeutically useful antihypertensive agents. Moreover, polymorphisms in human OAT3 might contribute to the genetic variation in susceptibility to hypertension.

Interaction of Organic Cations with Organic Anion Transporters

Studies of the organic anion transporters (Oats) have focused mainly on their interactions with organic anionic substrates. However, as suggested when Oat1 was originally identified as NKT (Lopez-Nieto, C. E., You, G., Bush, K. T., Barros, E. J., Beier, D. R., and Nigam, S. K. (1997) J. Biol. Chem. 272, 6471–6478), since the Oats share close homology with organic cation transporters (Octs), it is possible that Oats interact with cations as well. We now show that mouse Oat1 (mOat1) and mOat3 and, to a lesser degree, mOat6 bind a number of “prototypical” Oct substrates, including 1-methyl-4-phenylpyridinium. In addition to oocyte expression assays, we have tested binding of organic cations to Oat1 and Oat3 in ex vivo assays by analyzing interactions in kidney organ cultures deficient in Oat1 and Oat3. We also demonstrate that mOat3 transports organic cations such as 1-methyl-4-phenylpyridinium and cimetidine. A pharmacophore based on the binding affinities of the tested organic cations for Oat3 was generated. Using this pharmacophore, we screened a chemical library and were able to identify novel cationic compounds that bound to Oat1 and Oat3. These compounds bound Oat3 with an affinity higher than the highest affinity compounds in the original set of prototypical Oct substrates. Thus, whereas Oat1, Oat3, and Oat6 appear to function largely in organic anion transport, they also bind and transport some organic cations. These findings could be of clinical significance, since drugs and metabolites that under normal physiological conditions do not bind to the Oats may undergo changes in charge and become Oat substrates during pathologic conditions wherein significant variations in body fluid pH occur.

Analysis of Three-dimensional Systems for Developing and Mature Kidneys Clarifies the Role of OAT1 and OAT3 in Antiviral Handling

The organic anion transporters OAT1 (SLC22A6, originally identified by us as NKT) and OAT3 (SLC22A8) are critical for handling many toxins, metabolites, and drugs, including antivirals (Truong, D. M., Kaler, G., Khandelwal, A., Swaan, P. W., and Nigam, S. K. (2008) J. Biol. Chem. 283, 8654–8663). Although microinjected Xenopus oocytes and/or transfected cells indicate overlapping specificities, the individual contributions of these transporters in the three-dimensional context of the tissues in which they normally function remain unclear. Here, handling of HIV antivirals (stavudine, tenofovir, lamivudine, acyclovir, and zidovudine) was analyzed with three-dimensional ex vivo functional assays using knock-out tissue. To investigate the contribution of OAT1 and OAT3 in various nephron segments, the OAT-selective fluorescent tracer substrates 5-carboxyfluorescein and 6-carboxyfluorescein were used. Although OAT1 function (uptake in oat3−/− tissue) was confined to portions of the cortex, consistent with a proximal tubular localization, OAT3 function (uptake in oat1−/− tissue) was apparent throughout the cortex, indicating localization in the distal as well as proximal nephron. This functional localization indicates a complex three-dimensional context, which needs to be considered for metabolites, toxins, and drugs (e.g. antivirals) handled by both transporters. These results also raise the possibility of functional differences in the relative importance of OAT1 and OAT3 in antiviral handling in developing and mature tissue. Because the HIV antivirals are used in pregnant women, the results may also help in understanding how these drugs are handled by developing organs.

Linkage of Organic Anion Transporter-1 to Metabolic Pathways through Integrated “Omics”-driven Network and Functional Analysis

The main kidney transporter of many commonly prescribed drugs (e.g. penicillins, diuretics, antivirals, methotrexate, and non-steroidal anti-inflammatory drugs) is organic anion transporter-1 (OAT1), originally identified as NKT (Lopez-Nieto, C. E., You, G., Bush, K. T., Barros, E. J., Beier, D. R., and Nigam, S. K. (1997) J. Biol. Chem. 272, 6471–6478). Targeted metabolomics in knockouts have shown that OAT1 mediates the secretion or reabsorption of many important metabolites, including intermediates in carbohydrate, fatty acid, and amino acid metabolism. This observation raises the possibility that OAT1 helps regulate broader metabolic activities. We therefore examined the potential roles of OAT1 in metabolic pathways using Recon 1, a functionally tested genome-scale reconstruction of human metabolism. A computational approach was used to analyze in vivo metabolomic as well as transcriptomic data from wild-type and OAT1 knock-out animals, resulting in the implication of several metabolic pathways, including the citric acid cycle, polyamine, and fatty acid metabolism. Validation by in vitro and ex vivo analysis using Xenopus oocyte, cell culture, and kidney tissue assays demonstrated interactions between OAT1 and key intermediates in these metabolic pathways, including previously unknown substrates, such as polyamines (e.g. spermine and spermidine). A genome-scale metabolic network reconstruction generated some experimentally supported predictions for metabolic pathways linked to OAT1-related transport. The data support the possibility that the SLC22 and other families of transporters, known to be expressed in many tissues and primarily known for drug and toxin clearance, are integral to a number of endogenous pathways and may be involved in a larger remote sensing and signaling system (Ahn, S. Y., and Nigam, S. K. (2009) Mol. Pharmacol. 76, 481–490, and Wu, W., Dnyanmote, A. V., and Nigam, S. K. (2011) Mol. Pharmacol. 79, 795–805). Drugs may alter metabolism by competing for OAT1 binding of metabolites.

Update on the molecular physiology of organic anion transporters.

Purpose of reviewOrganic anion transporters (OATs) mediate the renal absorption and excretion of a wide range of metabolites and xenobiotics. We discuss the recent advances that have been made in elucidating the binding and transport characteristics of OATs, new insights into their physiological role and regulation by various factors, and pharmacogenetics. Recent findingsOverlapping substrate specificity among the OATs is well established. However, recent findings have suggested distinct differences in the structural binding determinants among the OATs, which have important implications for understanding drug interactions and drug design. A potential role for OATs in blood pressure regulation and remote sensing has been reported. Meanwhile, factors regulating the expression of OATs continue to be identified and characterized. The effect of renal ischemia on OAT expression and function is currently being explored. Finally, recent studies identifying various OAT polymorphisms may facilitate prediction of individual drug response and toxicity. SummaryAs progress is made in unveiling the many functional aspects of the OATs, it is becoming clear that their significance is not only limited to a role in drug elimination from the body, but also extends to other vital physiological roles. Further delineation of the function and regulation of the OATs will uncover enormous potential clinical and pharmacological applications.

Scaffolding Proteins DLG1 and CASK Cooperate to Maintain the Nephron Progenitor Population during Kidney Development

DLG1 (discs-large homolog 1) and CASK (calcium/calmodulin-dependent serine protein kinase) interact at membrane-cytoskeleton interfaces and function as scaffolding proteins that link signaling molecules, receptors, and other scaffolding proteins at intercellular and synaptic junctions. Dlg1-null mice exhibit hydronephrosis, hydroureter, and occasionally hypoplastic kidneys, whereas Cask-null mice do not. To investigate whether DLG1 and CASK cooperate in the developing urogenital system, we generated mice deficient in both DLG1 and CASK either 1) globally, 2) in metanephric mesenchyme, or 3) in nephron progenitors. With each approach, Dlg1;Cask double-knockout (DKO) kidneys were severely hypoplastic and dysplastic and demonstrated rapid, premature depletion of nephron progenitors/stem cells. Several cellular and molecular defects were observed in the DKO kidneys, including reduced proliferation and increased apoptosis of cells in the nephrogenic zone and a progressive decrease in the number of cells expressing SIX2, a transcription factor essential for maintaining nephron progenitors. Fgf8 expression was reduced in early-stage DKO metanephric mesenchyme, accompanied by reduced levels of components of the Ras pathway, which is activated by fibroblast growth factor (FGF) signaling. Moreover, Dlg1(+/-);Cask(-/-) (het/null) kidneys were moderately hypoplastic and demonstrated impaired aggregation of SIX2-positive cells around the ureteric bud tips. Nephron progenitor-specific het/null mice survived with small kidneys but developed glomerulocystic kidney disease and renal failure. Taken together, these results suggest that DLG1 and CASK play critical cooperative roles in maintaining the nephron progenitor population, potentially via a mechanism involving effects on FGF signaling.

Acute Intermittent Porphyria A Diagnostic Challenge

Acute intermittent porphyria is a metabolic disorder rarely seen in prepubertal children. A delay in diagnosis of acute intermittent porphyria is common because of variable and nonspecific symptoms. We report an 8-year-old boy with right hemimegalencephaly and intractable seizures, who presented with dark-colored urine, hypertension, increasing lethargy, fluctuating seizures, and poor oral intake. He subsequently developed hyponatremia secondary to syndrome of inappropriate antidiuretic hormone secretion. His urinalysis was negative for red blood cells, and a random urine porphobilinogen level was elevated. Further biochemical and molecular testing confirmed the diagnosis of acute intermittent porphyria. His antiepileptic medications were discontinued and hemin administered, with dramatic clinical improvement. The diagnosis of acute intermittent porphyria was challenging because of his underlying neurologic condition. This case highlights the variable presentation of acute intermittent porphyria and emphasizes the importance of considering the diagnosis even in young patients with underlying neurologic conditions when they present with nonspecific neurovisceral symptoms or with unexplained neurologic deterioration.

DLG1 influences distal ureter maturation via a non-epithelial cell autonomous mechanism involving reduced retinoic acid signaling, Ret expression, and apoptosis.

The absence of Discs-large 1 (DLG1), the mouse ortholog of the Drosophila discs-large tumor suppressor, results in congenital hydronephrosis characterized by urinary tract abnormalities, reduced ureteric bud branching, and delayed disconnection of the ureter from the common nephric duct (CND). To define the specific cellular requirements for Dlg1 expression during urogenital development, we used a floxed Dlg1 allele and Pax2-Cre, Pax3-Cre, Six2-Cre, and HoxB7-Cre transgenes to generate cell type-restricted Dlg1 mutants. In addition, we used Ret(GFP) knockin and retinoic acid response element-lacZ transgenic mice to determine the effects of Dlg1 mutation on the respective morphogenetic signaling pathways. Mutation of Dlg1 in urothelium and collecting ducts (via HoxB7-Cre or Pax2-Cre) and in nephron precursors (via Pax2-Cre and Six2-Cre) resulted in no apparent abnormalities in ureteric bud branching or in distal ureter maturation, and no hydronephrosis. Mutation in nephrons, ureteric smooth muscle, and mesenchyme surrounding the lower urinary tract (via the Pax3-Cre transgene) resulted in congenital hydronephrosis accompanied by reduced branching, abnormal distal ureter maturation and insertion, and smooth muscle orientation defects, phenotypes very similar to those in Dlg1 null mice. Dlg1 null mice showed reduced Ret expression and apoptosis during ureter maturation and evidence of reduced retinoic acid signaling in the kidney. Taken together, these results suggest that Dlg1 expression in ureter and CND-associated mesenchymal cells is essential for ensuring distal ureter maturation by facilitating retinoic acid signaling, Ret expression, and apoptosis of the urothelium.