An Organic Anion Transporter 1 (OAT1)-centered Metabolic Network.

There has been a recent interest in the broader physiological importance of multispecific "drug" transporters of the SLC and ABC transporter families. Here, a novel multi-tiered systems biology approach was used to predict metabolites and signaling molecules potentially affected by the in vivo deletion of organic anion transporter 1 (Oat1, Slc22a6, originally NKT), a major kidney-expressed drug transporter. Validation of some predictions in wet-lab assays, together with re-evaluation of existing transport and knock-out metabolomics data, generated an experimentally validated, confidence ranked set of OAT1-interacting endogenous compounds enabling construction of an "OAT1-centered metabolic interaction network." Pathway and enrichment analysis indicated an important role for OAT1 in metabolism involving: the TCA cycle, tryptophan and other amino acids, fatty acids, prostaglandins, cyclic nucleotides, odorants, polyamines, and vitamins. The partly validated reconstructed network is also consistent with a major role for OAT1 in modulating metabolic and signaling pathways involving uric acid, gut microbiome products, and so-called uremic toxins accumulating in chronic kidney disease. Together, the findings are compatible with the hypothesized role of drug transporters in remote inter-organ and inter-organismal communication: The Remote Sensing and Signaling Hypothesis (Nigam, S. K. (2015) Nat. Rev. Drug Disc. 14, 29). The fact that OAT1 can affect many systemic biological pathways suggests that drug-metabolite interactions need to be considered beyond simple competition for the drug transporter itself and may explain aspects of drug-induced metabolic syndrome. Our approach should provide novel mechanistic insights into the role of OAT1 and other drug transporters implicated in metabolic diseases like gout, diabetes, and chronic kidney disease.

Changes in medication regimen complexity and the risk for 90-day hospital readmission and/or emergency department visits in U.S. Veterans with heart failure.

BACKGROUND: Heart failure (HF) hospitalization is associated with multiple medication modifications. These modifications often increase medication regimen complexity and may increase the risk of readmission and/or emergency department (ED) visit. OBJECTIVES: To determine the association between changes in medication regimen complexity (MRC) during hospitalization of patients with heart failure and the risk of readmission or ED visit at 90 days. Secondary objectives include examining the association between changes in MRC and time to readmission as well as the relationship between number of medications and MRC. METHODS: This was a retrospective cohort study that included U.S. Veterans hospitalized with heart failure. MRC was quantified using the medication regimen complexity index (MRCI). The change in MRCI was the difference between admission MRCI and discharge MRCI recorded during the index hospitalization. Demographic and clinical data were collected to characterize the study population. Patient data for up to one year after discharge was recorded to identify hospital readmissions and ED visits. RESULTS: A total of 174 patients were included in the analysis. Sixty-two patients (36%) were readmitted or had an ED visit at 90 days from the index hospitalization. The mean change (SD) in MRCI during the index hospitalization among the cohort was 4.7 (8.3). After multivariate logistic regression analysis, each unit increase in MRCI score was associated with a 4% lower odds of readmission or ED visit at 90 days but this finding was not statistically significant (OR 0.955; 95% CI 0.911-1.001). In the cox proportional hazard model, the median time to hospital readmission or ED visit was 214 days. Each unit increase in MRCI score was associated with a modest but non-significant increase in probability of survival from readmission or ED visit (HR 0.978; 95% CI 0.955, 1.001). CONCLUSION: Changes in medication regimen complexity that occur during hospitalization may also be associated with optimization of medical therapy and do not necessarily portend worse outcomes in patients with HF.

Transcriptome-based reconstructions from the murine knockout suggest involvement of the urate transporter, URAT1 (slc22a12), in novel metabolic pathways.

URAT1 (slc22a12) was identified as the transporter responsible for renal reabsorption of the medically important compound, uric acid. However, subsequent studies have indicated that other transporters make contributions to this process, and that URAT1 transports other organic anions besides urate (including several in common with the closely related multi-specific renal organic anion transporters, OAT1 (slc22a6) and OAT3 (slc22a8)). These findings raise the possibility that urate transport is not the sole physiological function of URAT1. We previously characterized mice null for the murine ortholog of URAT1 (mURAT1; previously cloned as RST), finding a relatively modest decrement in urate reabsorptive capacity. Nevertheless, there were shifts in the plasma and urinary concentrations of multiple small molecules, suggesting significant metabolic changes in the knockouts. Although these molecules remain unidentified, here we have computationally delineated the biochemical networks consistent with transcriptomic data from the null mice. These analyses suggest alterations in the handling of not only urate but also other putative URAT1 substrates comprising intermediates in nucleotide, carbohydrate, and steroid metabolism. Moreover, the analyses indicate changes in multiple other pathways, including those relating to the metabolism of glycosaminoglycans, methionine, and coenzyme A, possibly reflecting downstream effects of URAT1 loss. Taken together with the available substrate and metabolomic data for the other OATs, our findings suggest that the transport and biochemical functions of URAT1 overlap those of OAT1 and OAT3, and could contribute to our understanding of the relationship between uric acid and the various metabolic disorders to which it has been linked.

Striking differences between knockout and wild-type mice in global gene expression variability.

Microarray analyses of gene knockouts have traditionally focused on the identification of genes whose mean expression is different in knockout and wild-type mice. However, recent work suggests that changes in the variability of gene expression can have important phenotypic consequences as well. Here, in an unbiased sample of publicly available microarray data on gene expression in various knockouts, highly significant differences from wild-type (either increases or decreases) are noted in the gene expression coefficients of variation (CVs) of virtually every knockout considered. Examination of the distribution of gene-by-gene CV differences indicates that these findings are not attributable to a few outlier genes, but rather to broadly increased or decreased CV in the various knockouts over all the (tens of thousands of) transcripts assayed. These global differences in variability may reflect either authentic biological effects of the knockouts or merely experimental inconsistencies. However, regardless of the underlying explanation, the variability differences are of importance as they will influence both the statistical detection of gene expression changes and, potentially, the knockout phenotype itself.

Assessment of plasma C-reactive protein as a biomarker of posttraumatic stress disorder risk.

IMPORTANCE: Posttraumatic stress disorder (PTSD) has been associated in cross-sectional studies with peripheral inflammation. It is not known whether this observed association is the result of PTSD predisposing to inflammation (as sometimes postulated) or to inflammation predisposing to PTSD. OBJECTIVE: To determine whether plasma concentration of the inflammatory marker C-reactive protein (CRP) helps predict PTSD symptoms. DESIGN, SETTING, AND PARTICIPANTS: The Marine Resiliency Study, a prospective study of approximately 2600 war zone-deployed Marines, evaluated PTSD symptoms and various physiological and psychological parameters before deployment and at approximately 3 and 6 months following a 7-month deployment. Participants were recruited from 4 all-male infantry battalions imminently deploying to a war zone. Participation was requested of 2978 individuals; 2610 people (87.6%) consented and 2555 (85.8%) were included in the present analysis. Postdeployment data on combat-related trauma were included for 2208 participants (86.4% of the 2555 included) and on PTSD symptoms at 3 and 6 months after deployment for 1861 (72.8%) and 1617 (63.3%) participants, respectively. MAIN OUTCOMES AND MEASURES: Severity of PTSD symptoms 3 months after deployment assessed by the Clinician-Administered PTSD Scale (CAPS). RESULTS: We determined the effects of baseline plasma CRP concentration on postdeployment CAPS using zero-inflated negative binomial regression (ZINBR), a procedure designed for distributions, such as CAPS in this study, that have an excess of zeroes in addition to being positively skewed. Adjusting for the baseline CAPS score, trauma exposure, and other relevant covariates, we found baseline plasma CRP concentration to be a highly significant overall predictor of postdeployment CAPS scores (P = .002): each 10-fold increment in CRP concentration was associated with an odds ratio of nonzero outcome (presence vs absence of any PTSD symptoms) of 1.51 (95% CI, 1.15-1.97; P = .003) and a fold increase in outcome with a nonzero value (extent of symptoms when present) of 1.06 (95% CI, 0.99-1.14; P = .09). CONCLUSIONS: AND RELEVANCE A marker of peripheral inflammation, plasma CRP may be prospectively associated with PTSD symptom emergence, suggesting that inflammation may predispose to PTSD.

Multispecific drug transporter Slc22a8 (Oat3) regulates multiple metabolic and signaling pathways.

Multispecific drug transporters of the solute carrier and ATP-binding cassette families are highly conserved through evolution, but their true physiologic role remains unclear. Analyses of the organic anion transporter 3 (OAT3; encoded by Slc22a8/Oat3, originally Roct) knockout mouse have confirmed its critical role in the renal handling of common drugs (e.g., antibiotics, antivirals, diuretics) and toxins. Previous targeted metabolomics of the knockout of the closely related Oat1 have demonstrated a central metabolic role, but the same approach with Oat3 failed to reveal a similar set of endogenous substrates. Nevertheless, the Oat3 knockout is the only Oat described so far with a physiologically significant phenotype, suggesting the disturbance of metabolic or signaling pathways. Here we analyzed global gene expression in Oat3 knockout tissue, which implicated OAT3 in phase I and phase II metabolism (drug metabolizing enzymes or DMEs), as well as signaling pathways. Metabolic reconstruction with the recently developed "mouse Recon1" supported the involvement of Oat3 in the aforementioned pathways. Untargeted metabolomics were used to determine whether the predicted metabolic alterations could be confirmed. Many significant changes were observed; several metabolites were tested for direct interaction with mOAT3, whereas others were supported by published data. Oat3 thus appears critical for the handling of phase I (hydroxylation) and phase II (glucuronidation) metabolites. Oat3 also plays a role in bioenergetic pathways (e.g., the tricarboxylic acid cycle), as well as those involving vitamins (e.g., folate), steroids, prostaglandins, gut microbiome products, uremic toxins, cyclic nucleotides, amino acids, glycans, and possibly hyaluronic acid. The data seemingly consistent with the Remote Sensing and Signaling Hypothesis (Ahn and Nigam, 2009; Wu et al., 2011), also suggests that Oat3 is essential for the handling of dietary flavonoids and antioxidants.

Organic anion transport pathways in antiviral handling in choroid plexus in Oat1 (Slc22a6) and Oat3 (Slc22a8) deficient tissue.

Transporters in the choroid plexus (CP) regulate transport of numerous compounds of physiological and therapeutic interest between blood and CSF and thus likely play a key role in determining CNS levels of drugs, toxins and metabolites. Here, high CP expression was noted for the organic anion transporters, Oat1 (SLC22A6 or NKT) and Oat3 (SLC22A8) which are also the principal Oats in the renal proximal tubule, as well as SLC22A17, hypothesized to be involved in iron transport. Because Oat1 and Oat3 have overlapping substrate specificity, ex vivo preparations of CP from Oat1((-/-)) and Oat3((-/-)) mice were used to isolate the individual transport function of each, respectively. Tissue from either knockout mouse mediated the probenecid-inhibitable transport of the Oat substrate, 6-carboxyfluorescein (6CF), confirming the presence of Oat1 and Oat3 function. Because many antiviral medications are Oat substrates, including those crucial in the treatment of HIV infections, the interaction of the antivirals zidovudine, acyclovir, tenofovir, lamivudine, and stavudine, with Oat1 and Oat3 in CP, was investigated by determining the inhibition of 6CF uptake. All the antivirals tested manifested significant interaction with both Oat1 and Oat3, with the exception of stavudine which did not significantly affect Oat1 function. These results could have important implications for antiretroviral (and other drugs) penetration into or retention within the CNS, a major reservoir for virus during HIV infection. Apart from any effect at the blood brain barrier (BBB), designing specific inhibitors of Oat1 and Oat3 may be helpful in altering CNS drug levels by blocking organic anion transporters in the CP. The role of SLC22A17 in the CP deserves further exploration. The ability of Oats to regulate the movement of small molecules across the BBB, CP, proximal tubule and other tissues may also be important for their role in remote sensing and signaling [1,21]).

A role for the organic anion transporter OAT3 in renal creatinine secretion in mice.

Tubular secretion of the organic cation, creatinine, limits its value as a marker of glomerular filtration rate (GFR) but the molecular determinants of this pathway are unclear. The organic anion transporters, OAT1 and OAT3, are expressed on the basolateral membrane of the proximal tubule and transport organic anions but also neutral compounds and cations. Here, we demonstrate specific uptake of creatinine into mouse mOat1- and mOat3-microinjected Xenopus laevis oocytes at a concentration of 10 µM (i.e., similar to physiological plasma levels), which was inhibited by both probenecid and cimetidine, prototypical competitive inhibitors of organic anion and cation transporters, respectively. Renal creatinine clearance was consistently greater than inulin clearance (as a measure of GFR) in wild-type (WT) mice but not in mice lacking OAT1 (Oat1-/-) and OAT3 (Oat3-/-). WT mice presented renal creatinine net secretion (0.23 ± 0.03 µg/min) which represented 45 ± 6% of total renal creatinine excretion. Mean values for renal creatinine net secretion and renal creatinine secretion fraction were not different from zero in Oat1-/- (-0.03 ± 0.10 µg/min; -3 ± 18%) and Oat3-/- (0.01 ± 0.06 µg/min; -6 ± 19%), with greater variability in Oat1-/-. Expression of OAT3 protein in the renal membranes of Oat1-/- mice was reduced to ∼6% of WT levels, and that of OAT1 in Oat3-/- mice to ∼60%, possibly as a consequence of the genes for Oat1 and Oat3 having adjacent chromosomal locations. Plasma creatinine concentrations of Oat3-/- were elevated in clearance studies under anesthesia but not following brief isoflurane anesthesia, indicating that the former condition enhanced the quantitative contribution of OAT3 for renal creatinine secretion. The results are consistent with a contribution of OAT3 and possibly OAT1 to renal creatinine secretion in mice.

Syndrome of inappropriate antidiuretic hormone associated with moxifloxacin.

PURPOSE: A possible case of moxifloxacin-induced syndrome of inappropriate antidiuretic hormone (SIADH) is reported. SUMMARY: A 66-year-old Caucasian woman with stage II chronic obstructive pulmonary disease (COPD) arrived at the emergency department from an outpatient clinic complaining of worsening shortness of breath, headache, body aches, and dizziness. Five days before her arrival at the hospital, the patient was seen in an outpatient clinic with symptoms of COPD exacerbation and was given a corticosteroid taper of prednisone 40 mg daily and moxifloxacin 400 mg daily. The patient was hospitalized, and her serum sodium concentration was 110 meq/L. Moxifloxacin was continued on admission, and the patient was admitted to the intensive care unit for frequent neurologic examination, serial serum sodium measurements, and fluid restriction. Her laboratory test results were consistent with SIADH. Fluid restriction at 1 L/day initially corrected her serum sodium concentration to 119 meq/L, but increases in serum sodium plateaued by day 2 of admission (119-122 meq/L). Moxifloxacin was discontinued on hospital day 3. At discharge, on hospital day 5, her serum sodium concentration had increased to 131 meq/L. She was restarted on her home medications and followed up in an outpatient clinic one week later. After multiple etiologies were ruled out, drug-induced SIADH associated with moxifloxacin was the most likely diagnosis for this patient. Clinicians should be aware of this potential adverse drug effect when assessing patients with hyponatremia or SIADH. CONCLUSION: A 66-year-old woman developed severe hyponatremia after receiving moxifloxacin for five days for treatment of COPD exacerbation.

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.

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.

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 a large cluster of SLC22 transporter genes, including novel USTs, reveals species-specific amplification of subsets of family members.

When the organic anion transporter Oat1 was first identified as NKT (Lopez-Nieto CE, You G, Bush KT, Barros EJ, Beier DR, Nigam SK. J Biol Chem 272: 6471-6478, 1997), it was argued that it, together with Oct1, may be part of a larger subfamily (now known as SLC22) involved in organic ion and xenobiotic transport. The least studied among SLC22 transporters are the so-called unknown substrate transporters (USTs). Here, five novel genes located in a cluster on mouse chromosome 19, immediately between Slc22a8 (Oat3)/Slc22a6 (Oat1) and Slc22a19 (Oat5), were identified as homologs of human USTs. These genes display preferential expression in liver and kidney, and one gene, AB056422, has several splicing variants with differential tissue expression and embryonic expression. Along with Slc22a6, Slc22a8, and Slc22a19, these Usts define the largest known cluster of mammalian Slc22 genes. Given the established functions of Oats, these genes may also be involved in organic anion transport. Usts have characteristic motifs and share a signature residue in the possible active site of transmembrane domain 7, a conserved, positively charged, amino acid, Arg356, possibly a site for interaction with organic anions. In certain species, Oat1 and Oat3 appeared to be highly conserved, whereas the Ust part of this cluster appeared to undergo repeated species-specific amplification, suggesting strong environmental selection pressure, and perhaps providing an explanation for copy number variation in the human locus. One Ust amplification in mouse appears to be recent. This cluster may be coordinately regulated and under selective pressure in a species-specific manner.

Implications of the alternating access model for organic anion transporter kinetics.

Many transport proteins, including the clinically important organic anion transporters (OATs), appear to function via an "alternating access" mechanism. In analyzing the kinetics of these transporters, the terms K(m) and V(max) are often treated in the field as denoting, respectively, the affinity of the substrate for the transporter and the turnover (conformational switch) rate of the substrate-transporter complex. In fact, the expressions for both these parameters have very complex forms comprising multiple rate constants from conformational switch as well as association/dissociation steps in the cycling of the transporter and, therefore, do not have straightforward physical meanings. However, if the rapid equilibrium assumption is made (namely, that the association/dissociation steps occur far more rapidly than the conformational switch steps), these expressions become greatly simplified and their physical meaning clear, though still distinct from the conventional interpretations. V(max) will be a function of not just the rate of substrate-transporter complex turnover but also the rate of the "return" conformational switch and will vary largely with the slower of these two steps (the rate-limiting step). K(m) will be seen to be related to substrate affinity by a term that varies inversely with the substrate-transporter complex turnover rate, essentially because the greater this rate, the greater the extent to which transporters will be distributed in a conformation inaccessible to substrate. Here, an intuitive approach is presented to demonstrate these conclusions. The phenomena of trans-stimulation and trans-inhibition are discussed in the context of this analysis.

Organic anion transporter 3 inhibitors as potential novel antihypertensives.

Hypertension is an exceedingly common disease with potentially devastating complications. Unfortunately, existing treatments are often only partially effective, indicating the utility of the development of novel therapeutics. It has recently been discovered that loss of renal organic anion transporter 3 (Oat3) results in decreased blood pressure. This suggests (regardless of the underlying mechanisms, which are, as yet, unclear) that inhibition of Oat3 function might also decrease blood pressure, and, therefore, that inhibitors of Oat3 might serve as novel drugs in the treatment of hypertension. In support of this hypothesis, it has been found that at least some potent Oat3 inhibitors can lower blood pressure. These recent findings are discussed in the context of the alternating access model that has been proposed for organic anion transporter function, with particular attention to the little-appreciated phenomenon of trans-inhibition.

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.

Multiple organic anion transporters contribute to net renal excretion of uric acid.

Excretion of uric acid, a compound of considerable medical importance, is largely determined by the balance between renal secretion and reabsorption. The latter process has been suggested to be principally mediated by urate transporter 1 (URAT1; slc22a12), but the role of various putative urate transporters has been much debated. We have characterized urate handling in mice null for RST, the murine ortholog of URAT1, as well as in those null for the related organic anion transporters Oat1 and Oat3. Expression of mRNA of other putative urate transporters (UAT, MRP2, MRP4, Oatv1) was unaffected in the knockouts, as were general indexes of renal function (glomerular filtration rate, fractional excretion of fluid and electrolytes). While mass spectrometric analyses of urine and plasma revealed significantly diminished renal reabsorption of urate in RST-null mice, the bulk of reabsorption, surprisingly, was preserved. Oat1- and Oat3-null mice manifested decreased secretion rather than reabsorption, indicating that these related transporters transport urate in the "opposite" direction to RST. Moreover, metabolomic analyses revealed significant alteration in the concentration of several molecules in the plasma and urine of RST knockouts, some of which may represent additional substrates of RST. The results suggest that RST, Oat1, and Oat3 each contribute to urate handling, but, at least in mice, the bulk of reabsorption is mediated by a transporter(s) that remains to be identified. We discuss the data in the context of recent human genetic studies that suggest that the magnitude of the contribution of URAT1 to urate reabsorption might vary with ethnic background.

Overlapping in vitro and in vivo specificities of the organic anion transporters OAT1 and OAT3 for loop and thiazide diuretics.

Organic anion transporter (OAT) genes have been implicated in renal secretion of organic anions, but the individual in vivo contributions of OAT1 (first identified as NKT) and OAT3 remain unclear. Potential substrates include loop diuretics (e.g., furosemide) and thiazide diuretics (e.g., bendroflumethiazide), which reach their tubular sites of action mainly by proximal tubular secretion. Previous experiments in Oat1 knockout (-/-) mice revealed an almost complete loss of renal secretion of the prototypic organic anion p-aminohippurate (PAH) and a role of OAT1 in tubular secretion of furosemide (Eraly SA, Vallon V, Vaughn D, Gangoiti JA, Richter K, Nagle M, Monte JC, Rieg T, Truong DM, Long JM, Barshop BA, Kaler G, Nigam SK. J Biol Chem 281: 5072-5083, 2006). In this study we found that both furosemide and bendroflumethiazide inhibited mOat1- and mOat3-mediated uptake of a labeled tracer in Xenopus oocytes injected with cRNA, consistent with their being substrates for mouse OAT1 and OAT3. Experiments in Oat3(-/-) mice revealed intact renal secretion of PAH, but the dose-natriuresis curves for furosemide and bendroflumethiazide were shifted to the right and urinary furosemide excretion was impaired similar to the defect in Oat1(-/-) mice. Thus, whereas OAT1 (in contrast to OAT3) is the classic basolateral PAH transporter of the proximal tubule, both OAT1 and OAT3 contribute similarly to normal renal secretion of furosemide and bendroflumethiazide, and a lack of either one is not fully compensated by the other. Although microarray expression analysis in the kidneys of Oat1(-/-) and Oat3(-/-) mice revealed somewhat altered expression of a small number of transport-related genes, none were common to both knockout models. When searching for polymorphisms involved in human diuretic responsiveness, it may be necessary to consider both OAT1 and OAT3, among other genes.

Structural variation governs substrate specificity for organic anion transporter (OAT) homologs. Potential remote sensing by OAT family members.

Organic anion transporters (OATs, SLC22) interact with a remarkably diverse array of endogenous and exogenous organic anions. However, little is known about the structural features that determine their substrate selectivity. We examined the substrate binding preferences and transport function of olfactory organic anion transporter, Oat6, in comparison with the more broadly expressed transporter, Oat1 (first identified as NKT). In analyzing interactions of both transporters with over 40 structurally diverse organic anions, we find a correlation between organic anion potency (pKi) and hydrophobicity (logP) suggesting a hydrophobicity-driven association with transporter-binding sites, which appears particularly prominent for Oat6. On the other hand, organic anion binding selectivity between Oat6 and Oat1 is influenced by the anion mass and net charge. Smaller mono-anions manifest greater potency for Oat6 and di-anions for Oat1. Comparative molecular field analysis confirms these mechanistic insights and provides a model for predicting new OAT substrates. By comparative molecular field analysis, both hydrophobic and charged interactions contribute to Oat1 binding, although it is predominantly the former that contributes to Oat6 binding. Together, the data suggest that, although the three-dimensional structures of these two transporters may be very similar, the binding pockets exhibit crucial differences. Furthermore, for six radiolabeled substrates, we assessed transport efficacy (Vmax) for Oat6 and Oat1. Binding potency and transport efficacy had little correlation, suggesting that different molecular interactions are involved in substrate binding to the transporter and translocation across the membrane. Substrate specificity for a particular transporter may enable design of drugs for targeting to specific tissues (e.g. olfactory mucosa). We also discuss how these data suggest a possible mechanism for remote sensing between OATs in different tissue compartments (e.g. kidney, olfactory mucosa) via organic anions.

Olfactory mucosa-expressed organic anion transporter, Oat6, manifests high affinity interactions with odorant organic anions.

We have characterized the expression of organic anion transporter 6, Oat6 (slc22a20), in olfactory mucosa, as well as its interaction with several odorant organic anions. In situ hybridization reveals diffuse Oat6 expression throughout olfactory epithelium, yet olfactory neurons laser-capture microdissected from either the main olfactory epithelium (MOE) or the vomeronasal organ (VNO) did not express Oat6 mRNA. These data suggest that Oat6 is expressed in non-neuronal cells of olfactory tissue, such as epithelial and/or other supporting cells. We next investigated interaction of Oat6 with several small organic anions that have previously been identified as odortype components in mouse urine. We find that each of these compounds, propionate, 2- and 3-methylbutyrate, benzoate, heptanoate, and 2-ethylhexanoate, inhibits Oat6-mediated uptake of a labeled tracer, estrone sulfate, consistent with their being Oat6 substrates. Previously, we noted defects in the renal elimination of odortype and odortype-like molecules in Oat1 knockout mice. The finding that such molecules interact with Oat6 raises the possibility that odorants secreted into the urine through one OAT-mediated mechanism (Eraly et al., JBC 2006) are transported through the olfactory mucosa through another OAT-mediated mechanism. Oat6 might play a direct or indirect role in olfaction, such as modulation of the availability of odorant organic anions at the mucosal surface for presentation to olfactory neurons or facilitation of delivery to a distal site of chemosensation, among other possibilities that we discuss.