Interaction of alpha-KG with basolateral organic anion transporter in isolated rabbit renal S3 proximal tubules

1995 ◽  
Vol 268 (6) ◽  
pp. F1109-F1116 ◽  
Author(s):  
S. Shpun ◽  
K. K. Evans ◽  
W. H. Dantzler
Keyword(s):  
Organic Anion ◽  
Basolateral Membrane ◽  
Proximal Tubules ◽  
Organic Anion Transporter ◽  
Anion Transporter ◽  
Significant Stimulation ◽  
S Uptake ◽  
S3 Segment ◽  
Uptake Data ◽  
Ethanesulfonic Acid

To understand the basolateral p-aminohippurate (PAH) transporter in the S3 segment of rabbit proximal tubules and its relationship to the transporter in the S2 segment, we measured the 30-s uptake and efflux of PAH across the basolateral membrane of single isolated S3 segments at 37 degrees C in bicarbonate-buffered media. Kinetic analysis of uptake data revealed a concentration of PAH at one-half Jmax of approximately 107 microM (same as in the S2 segment) but a Jmax of 600 fmol.min-1.nl-1 (one-tenth that of S2 segment). The coefficient for efflux across the basolateral membrane was also only one-sixth to one-tenth of that in the S2 segment. These data suggest that the basolateral PAH transporter is the same in both segments but that there are fewer transporters in the S3 than in the S2 segment. However, the apparent inhibitor constant values for cis-inhibition by probenecid (approximately 29 microM in S3, approximately 15 microM in S2) and by alpha-ketoglutarate (alpha-KG) in the presence of LiCl (approximately 40 microM in S3, approximately 160 microM in S2) suggest that the transporters may not be identical in the two segments. In bicarbonate-buffered medium, preloading the tubules with 100 microM alpha-KG did not trans-stimulate PAH uptake across the basolateral membrane, whereas preloading with 1.0 microM alpha-KG caused a significant stimulation of 43%. However, in N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid-buffered medium, preloading the tubules with 100 microM alpha-KG caused a twofold increase in PAH uptake.(ABSTRACT TRUNCATED AT 250 WORDS)

Acute regulation of OAT3-mediated estrone sulfate transport in isolated rabbit renal proximal tubules

2004 ◽  
Vol 287 (5) ◽  
pp. F1021-F1029 ◽  
Author(s):  
S. Soodvilai ◽  
V. Chatsudthipong ◽  
K. K. Evans ◽  
S. H. Wright ◽  
W. H. Dantzler
Keyword(s):  
Organic Anion ◽  
Basolateral Membrane ◽  
Anion Transport ◽  
Proximal Tubules ◽  
Organic Anion Transporter ◽  
Dependent Manner ◽  
Organic Anion Transport ◽  
Renal Proximal Tubules ◽  
Estrone Sulfate ◽  
Anion Transporter

We investigated the regulation of organic anion transport driven by the organic anion transporter 3 (OAT3), a multispecific OAT localized at the basolateral membrane of the renal proximal tubule. PMA, a PKC activator, inhibited uptake of estrone sulfate (ES), a prototypic substrate for OAT3, in a dose- and time-dependent manner. This inhibition was reduced by 100 nM bisindoylmaleimide I (BIM), a specific PKC inhibitor. The α1-adrenergic receptor agonist phenylephrine also inhibited ES uptake, and this effect was reduced by BIM. These results suggest that PKC activation downregulates OAT3-mediated organic anion transport. In contrast, epidermal growth factor (EGF) increased ES uptake following activation of MAPK. Exposure to PGE2 or dibutyryl (db)-cAMP also enhanced ES uptake. Stimulation produced by PGE2 and db-cAMP was prevented by the PKA inhibitor H-89, indicating that this stimulation required PKA activation. In addition, inhibition of cyclooxygenase 1 (COX1) (but not COX2) inhibited ES uptake. Furthermore, the stimulatory effect of EGF was eliminated by inhibition of either COX1 or PKA. These data suggest that EGF stimulates ES uptake by a process in which MAPK activation results in increased PGE2 production that, in turn, activates PKA and subsequently stimulates ES uptake. Interestingly, EGF did not induce upregulation immediately following phenylephrine-induced downregulation; and phenylephrine did not induce downregulation immediately after EGF-induced upregulation. These data are the first to show the regulatory response of organic anion transport driven by OAT3 in intact renal proximal tubules.

scholarly journals Immunohistochemical Localization of Multispecific Renal Organic Anion Transporter 1 in Rat Kidney

1999 ◽  
Vol 10 (3) ◽  
pp. 464-471 ◽  
Author(s):  
AKIHIRO TOJO ◽  
TAKASHI SEKINE ◽  
NORIKO NAKAJIMA ◽  
MAKOTO HOSOYAMADA ◽  
YOSHIKATSU KANAI ◽  
...  
Keyword(s):  
Rat Kidney ◽  
Organic Anion ◽  
Basolateral Membrane ◽  
Organic Anions ◽  
Proximal Tubules ◽  
Organic Anion Transporter ◽  
Electron Microscopic ◽  
Renal Vasculature ◽  
Initial Portion ◽  
Anion Transporter

Abstract. Renal proximal convoluted tubules have an important role, i.e., to excrete organic anions, including numerous drugs and endogenous substances. Recently, multispecific organic anion transporter 1 (OAT1) was isolated from rat kidney. In this study, the cellular and subcellular localization of OAT1 in rat kidney was investigated. Kidneys from normal rats were perfused and fixed with periodate-lysine-paraformaldehyde solution and were then processed for immunohistochemical analysis using the labeled streptavidin-biotin method, preembedding horseradish peroxidase method, and immunogold method. Light microscopic examination revealed immunostaining for OAT1 in the middle portion of the proximal tubule (S2 segment), but not in the initial portion of the proximal convoluted tubule, next to the glomerulus. Nephron segments other than the S2 segment and the renal vasculature were not stained with antibody to OAT1. Electron-microscopic observation using a preembedding method revealed that OAT1 was exclusively expressed in the basolateral membrane of S2 segments of proximal tubules. The immunogold method showed no labeling for OAT1 in the cytoplasmic vesicles, suggesting that OAT1 may not move together with organic anions into the cells. These results are consistent with previous physiologic data showing that organic anions, including para-aminohippurate, are taken up by the basolateral Na+-independent organic anion/dicarboxylate exchanger and excreted at S2 segments. In conclusion, OAT1 was localized to the basolateral membrane of S2 segments of proximal tubules in rat kidneys.

Renal organic anion transporter 1 is maldistributed and diminishes in proximal tubule cells but increases in vasculature after ischemia and reperfusion

2008 ◽  
Vol 295 (6) ◽  
pp. F1807-F1816 ◽  
Author(s):  
Osun Kwon ◽  
Wei-Wei Wang ◽  
Shane Miller
Keyword(s):  
Proximal Tubule ◽  
Ischemia Reperfusion ◽  
Organic Anion ◽  
Basolateral Membrane ◽  
Kidney Injury ◽  
Organic Anion Transporter ◽  
Membrane Domain ◽  
Proximal Tubule Cells ◽  
Anion Transporter ◽  
Tubule Cells

Renal solute clearances are reduced in ischemic acute kidney injury. However, the mechanisms explaining how solute clearance is impaired have not been clarified. Recently, we reported that cadaveric renal allografts exhibit maldistribution of organic anion transporter 1 (OAT1) in proximal tubule cells after ischemia and reperfusion, resulting in impairment of PAH clearance. In the present study, we characterized renal OAT1 in detail after ischemia-reperfusion using a rat model. We analyzed renal OAT1 using confocal microscopy with a three-dimensional reconstruction of serial optical images, Western blot, and quantitative real-time RT-PCR. OAT1 was distributed to basolateral membranes of proximal tubule cells in controls. With ischemia, OAT1 decreased in basolateral membrane, especially in the lateral membrane domain, and appeared diffusely in cytoplasm. After reperfusion following 60-min ischemia, OAT1 often formed cytoplasmic aggregates. The staining for OAT1 started reappearing in lateral membrane domain 1 h after reperfusion. The basolateral membrane staining was relatively well discernable at 240 h of reperfusion. Of note, a distinct increase in OAT1 expression was noted in vasculature early after ischemia and after reperfusion. The total amount of OAT1 protein expression in the kidney diminished after ischemia-reperfusion in a duration-dependent manner until 72 h, when they began to recover. However, even at 240 h, the amount of OAT1 did not reach control levels. The kidney tissues tended to show a remarkable but transient increase in mRNA expression for OAT1 at 5 min of ischemia. Our findings may provide insights of renal OAT1 in its cellular localization and response during ischemic acute kidney injury and recovery from it.

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

2012 ◽  
Vol 302 (10) ◽  
pp. F1293-F1299 ◽  
Author(s):  
Volker Vallon ◽  
Satish A. Eraly ◽  
Satish Ramachandra Rao ◽  
Maria Gerasimova ◽  
Michael Rose ◽  
...  
Keyword(s):  
Organic Anion ◽  
Basolateral Membrane ◽  
Tubular Secretion ◽  
Organic Anion Transporter ◽  
Xenopus Laevis Oocytes ◽  
Mean Values ◽  
Anion Transporters ◽  
Anion Transporter ◽  
Neutral Compounds ◽  
Quantitative Contribution

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.

Liver X receptor activation downregulates organic anion transporter 1 (OAT1) in the renal proximal tubule

2012 ◽  
Vol 302 (5) ◽  
pp. F552-F560 ◽  
Author(s):  
Suticha Kittayaruksakul ◽  
Sunhapas Soodvilai ◽  
Nithi Asavapanumas ◽  
Chatchai Muanprasat ◽  
Varanuj Chatsudthipong
Keyword(s):  
Proximal Tubule ◽  
Organic Anion ◽  
Basolateral Membrane ◽  
Receptor Activation ◽  
Inhibitory Effect ◽  
Renal Proximal Tubule ◽  
Organic Anion Transporter ◽  
Anion Transporter ◽  
X Receptors ◽  
Lxr Agonists

Liver X receptors (LXRs) play an important role in the regulation of cholesterol by regulating several transporters. In this study, we investigated the role of LXRs in the regulation of human organic anion transporter 1 (hOAT1), a major transporter localized in the basolateral membrane of the renal proximal tubule. Exposure of renal S2 cells expressing hOAT1 to LXR agonists (TO901317 and GW3965) and their endogenous ligand [22(R)-hydroxycholesterol] led to the inhibition of hOAT1-mediated [14C]PAH uptake. This inhibition was abolished by coincubation of the above agonists with 22(S)-hydroxycholesterol, an LXR antagonist. Moreover, it was found that the effect of LXR agonists was not mediated by changes in intracellular cholesterol levels. Interestingly, the inhibitory effect of LXRs was enhanced in the presence of 9- cis retinoic acid, a retinoic X receptor agonist. Kinetic analysis revealed that LXR activation decreased the maximum rate of PAH transport ( Jmax) but had no effect on the affinity of the transporter ( Kt). This result correlated well with data from Western blot analysis, which showed the decrease in hOAT1 expression following LXR activation. Similarly, TO901317 inhibited [14C]PAH uptake by the renal cortical slices as well as decreasing mOAT1 protein expression in mouse kidney. Our findings indicated for the first time that hOAT1 was downregulated by LXR activation in the renal proximal tubule.

Transport of a fluorescent cAMP analog in teleost proximal tubules

2007 ◽  
Vol 293 (6) ◽  
pp. R2382-R2389 ◽  
Author(s):  
Valeska Reichel ◽  
Rosalinde Masereeuw ◽  
Jeroen J. M. W. van den Heuvel ◽  
David S. Miller ◽  
Gert Fricker
Keyword(s):  
Fundulus Heteroclitus ◽  
Organic Anion ◽  
Membrane Vesicles ◽  
Proximal Tubules ◽  
Organic Anion Transporter ◽  
Renal Tubules ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Camp Analog ◽  
Anion Transporter

Previous studies have shown that killifish ( Fundulus heteroclitus) renal proximal tubules express a luminal membrane transporter that is functionally and immunologically analogous to the mammalian multidrug resistance-associated protein isoform 2 (Mrp2, ABCC2). Here we used confocal microscopy to investigate in killifish tubules the transport of a fluorescent cAMP analog (fluo-cAMP), a putative substrate for Mrp2 and Mrp4 (ABCC4). Steady-state luminal accumulation of fluo-cAMP was concentrative, specific, and metabolism-dependent, but not reduced by high K+ medium or ouabain. Transport was not affected by p-aminohippurate (organic anion transporter inhibitor) or p-glycoprotein inhibitor (PSC833), but cell-to-lumen transport was reduced in a concentration-dependent manner by Mrp inhibitor MK571, leukotriene C4 (LTC4), azidothymidine (AZT), cAMP, and adefovir; the latter two compounds are Mrp4 substrates. Although MK571 and LTC4 reduced transport of the Mrp2 substrate fluorescein-methotrexate (FL-MTX), neither cAMP, adefovir, nor AZT affected FL-MTX transport. Fluo-cAMP transport was not reduced when tubules were exposed to endothelin-1, Na nitroprusside (an nitric oxide generator) or phorbol ester (PKC activator), all of which signal substantial reductions in cell-to-lumen FL-MTX transport. Fluo-cAMP transport was reduced by forskolin, and this reduction was blocked by the PKA inhibitor H-89. Finally, in membrane vesicles from Spodoptera frugiperda (Sf9) cells containing human MRP4, ATP-dependent and specific uptake of fluo-cAMP could be demonstrated. Thus, based on inhibitor specificity and regulatory signaling, cell-to-lumen transport of fluo-cAMP in killifish renal tubules is mediated by a transporter distinct from Mrp2, presumably a teleost form of Mrp4.

scholarly journals The MRP4/ABCC4 Gene Encodes a Novel Apical Organic Anion Transporter in Human Kidney Proximal Tubules: Putative Efflux Pump for Urinary cAMP and cGMP

2002 ◽  
Vol 13 (3) ◽  
pp. 595-603 ◽  
Author(s):  
Rémon A. M. H. van Aubel ◽  
Pascal H. E. Smeets ◽  
Janny G. P. Peters ◽  
René J. M. Bindels ◽  
Frans G. M. Russel
Keyword(s):  
Efflux Pump ◽  
Organic Anion ◽  
Membrane Vesicles ◽  
Human Kidney ◽  
Proximal Tubules ◽  
Organic Anion Transporter ◽  
Anion Transporter ◽  
Dependent Transport ◽  
Camp And Cgmp ◽  
Kidney Proximal Tubules

ABSTRACT. The cyclic nucleotides cAMP and cGMP play key roles in cellular signaling and the extracellular regulation of fluid balance. In the kidney, cAMP is excreted across the apical proximal tubular membrane into urine, where it reduces phosphate reabsorption through a dipyridamole-sensitive mechanism that is not fully understood. It has long been known that this cAMP efflux pathway is dependent on ATP and is inhibited by probenecid. However, its identity and whether cGMP shares the same transporter have not been established. Here the expression, localization, and functional properties of human multidrug resistance protein 4 (MRP4) are reported. MRP4 is localized to the proximal tubule apical membrane of human kidney, and membrane vesicles from Sf9 cells expressing human MRP4 exhibit ATP-dependent transport of [3H]cAMP and [3H]cGMP. Both probenecid and dipyridamole are potent MRP4 inhibitors. ATP-dependent [3H]methotrexate and [3H]estradiol-17β-d-glucuronide transport by MRP4 and interactions with the anionic conjugates S-(2,4-dinitrophenyl)-glutathione, N-acetyl-(2,4-dinitrophenyl)-cysteine, α-naphthyl-β-d-glucuronide, and p-nitrophenyl-β-d-glucuronide are also demonstrated. In kidneys of rats deficient in the apical anionic conjugate efflux pump Mrp2, Mrp4 expression is maintained at the same level. It is concluded that MRP4 is a novel apical organic anion transporter and the putative efflux pump for cAMP and cGMP in human kidney proximal tubules.

Short-term regulation of multidrug resistance-associated protein 3 in rat and human hepatocytes

2005 ◽  
Vol 288 (6) ◽  
pp. G1252-G1258 ◽  
Author(s):  
Priyamvada Chandra ◽  
Peijin Zhang ◽  
Kim L. R. Brouwer
Keyword(s):  
Multidrug Resistance ◽  
Organic Anion ◽  
Basolateral Membrane ◽  
Rat Hepatocytes ◽  
Human Hepatocytes ◽  
Organic Anion Transporter ◽  
Intracellular Camp ◽  
Short Term ◽  
Anion Transporter ◽  
Rat Livers

The short-term regulation of multidrug resistance-associated protein 3 (Mrp3/MRP3) by cAMP and PKC was investigated in sandwich-cultured rat and human hepatocytes and isolated perfused rat livers. The modulator glucagon (500 nM) and the phorbol ester PMA (0.1 μM) were utilized to increase intracellular cAMP and PKC levels, respectively. In glucagon-treated rat hepatocytes, efflux of the Mrp3 substrate 5-(6)-carboxy-2′,7′-dichlorofluorescein (CDF) increased ∼1.5-fold, even in hepatocytes treated with the organic anion transporter (Oatp) inhibitor sulfobromophthalein (BSP). Confocal microscopy revealed more concentrated Mrp3 fluorescence in the basolateral membrane (less diffuse staining pattern) with glucagon treatment. PMA had no effect on Mrp3 activity or localization in sandwich-cultured rat hepatocytes. Glucagon and PMA treatment in isolated perfused rat livers resulted in a threefold increase (14 ± 4.6 μl·min−1·g liver−1) and a fourfold decrease (1.3 ± 0.3 μl·min−1·g liver−1) in CDF basolateral clearance compared with control livers (4.7 ± 2.3 μl·min−1·g liver−1), whereas CDF biliary clearance was not statistically different. In sandwich-cultured human hepatocytes, glucagon treatment resulted in a 1.3-fold increase in CDF efflux and a concomitant increase in MRP3 fluorescence in the basolateral membrane. In summary, cAMP and PKC appear to be involved in the short-term regulation of Mrp3/MRP3, as demonstrated by alterations in activity and localization in rat and human hepatocytes.

Molecular Physiology of Renal p-Aminohippurate Secretion

Physiology ◽  
2001 ◽  
Vol 16 (3) ◽  
pp. 114-118 ◽  
Author(s):  
Gerhard Burckhardt ◽  
Andrew Bahn ◽  
Natascha A. Wolff
Keyword(s):  
Organic Anion ◽  
Organic Anions ◽  
Proximal Tubules ◽  
Organic Anion Transporter ◽  
Molecular Physiology ◽  
Renal Proximal Tubules ◽  
Anion Transporter ◽  
Tubule Lumen ◽  
Tubule Cells ◽  
Species Specific

Renal proximal tubules secrete various organic anions, including drugs and p-aminohippurate (PAH). Uptake of PAH from blood into tubule cells occurs by exchange with intracellular α-ketoglutarate and is mediated by the organic anion transporter 1. PAH exit into tubule lumen is species specific and may involve ATP-independent and -dependent transporters.

Characterization and identification of steroid sulfate transporters of human placenta

2003 ◽  
Vol 284 (2) ◽  
pp. E390-E398 ◽  
Author(s):  
Bernhard Ugele ◽  
Marie V. St-Pierre ◽  
Monika Pihusch ◽  
Andrew Bahn ◽  
Peer Hantschmann
Keyword(s):  
Organic Anion ◽  
Diffusion Constant ◽  
Physiological Role ◽  
Placental Tissue ◽  
Organic Anion Transporter ◽  
Uptake Mechanism ◽  
Estrone Sulfate ◽  
Anion Transporter ◽  
S Uptake ◽  
Steroid Sulfate

Human trophoblasts depend on the supply of external precursors, such as dehydroepiandrosterone-3-sulfate (DHEA-S) and 16α-OH-DHEA-S, for synthesis of estrogens. The aim of the present study was to characterize the uptake of DHEA-S by isolated mononucleated trophoblasts (MT) and to identify the involved transporter polypeptides. The kinetic analysis of DHEA-35S uptake by MT revealed a saturable uptake mechanism ( K m = 26 μM, V max = 428 pmol · mg protein−1 · min−1), which was superimposed by a nonsaturable uptake mechanism (diffusion constant = 1.2 μl · mg protein−1 · min−1). Uptake of [3H]DHEA-S by MT was Na+dependent and inhibited by sulfobromophthalein (BSP), steroid sulfates, and probenecid, but not by steroid glucuronides, unconjugated steroids, conjugated bile acids, ouabain, p-aminohippurate (PAH), and bumetanide. MT took up [35S]BSP, [3H]estrone-sulfate, but not 3H-labeled ouabain, estradiol-17β-glucuronide, taurocholate, and PAH. RT-PCR revealed that the organic anion-transporting polypeptides OATP-B, -D, -E, and the organic anion transporter OAT-4 are highly expressed, and that OATP-A, -C, -8, OAT-3, and Na+-taurocholate cotransporting polypeptide (NTCP) are not or are only lowly expressed in term placental tissue and freshly isolated and cultured trophoblasts. Immunohistochemistry of first- and third-trimester placenta detected OAT-4 on cytotrophoblast membranes and at the basal surface of the syncytiotrophoblast. Our results indicate that uptake of steroid sulfates by isolated MT is mediated by OATP-B and OAT-4 and suggest a physiological role of both carrier proteins in placental uptake of fetal-derived steroid sulfates.

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