PKC regulation of organic anion secretion in perfused S2 segments of rabbit proximal tubules

2000 ◽  
Vol 278 (1) ◽  
pp. F104-F109 ◽  
Author(s):  
Apichai Shuprisha ◽  
Ronald M. Lynch ◽  
Stephen H. Wright ◽  
William H. Dantzler
Keyword(s):  
Steady State ◽  
Organic Anion ◽  
Physiological Role ◽  
Coupling Reaction ◽  
Peptide Hormone ◽  
Proximal Tubules ◽  
Epifluorescence Microscopy ◽  
Bathing Medium ◽  
Renal Proximal Tubules ◽  
Anion Secretion

To examine the role of protein kinase C (PKC) in organic anion (OA) secretion, we used epifluorescence microscopy to study steady-state transepithelial secretion of 1 μM fluorescein (FL) by isolated perfused S2 segments of rabbit renal proximal tubules. Addition of 100 nM phorbol 12-myristate 13-acetate (PMA), a known PKC activator, to the bathing medium decreased steady-state secretion of FL by ∼30% after 25 min. This inhibition was irreversible and, indeed, increased to ∼40% at 25 min following removal of PMA [10 μM 1,2-dioctanoyl- sn-glycerol (DOG) produced a comparable inhibition]. The inhibition produced by PMA was blocked when 100 nM of either staurosporine (ST) or bisindolylmaleimide I (BIM), both known PKC inhibitors, was added to the bath for a 20-min preexposure followed by the addition of PMA. ST or BIM alone had no significant effect on FL secretion, suggesting that the basal FL secretion rate was not under influence of PKC. Addition of 1 μM of either the peptide hormone bradykinin (BK) or the α1-receptor agonist phenylephrine (PE), both of which stimulate PKC via a ligand-receptor-PKC coupling reaction, to the bath also inhibited FL secretion by ∼22 and ∼27%, respectively. However, the inhibition was completely reversible after removal of BK or PE. Pretreatment of tubules with 100 nM BIM eliminated the inhibition of FL secretion produced by exposure to PE. We conclude that PKC negatively regulates the net secretion of OAs in rabbit renal proximal tubules. The data indicate that BK or catecholamines can play a physiological role in regulating OA secretion via PKC activation.

Indirect coupling of organic anion secretion to sodium in teleost (Paralichthys lethostigma) renal tubules

1991 ◽  
Vol 261 (6) ◽  
pp. R1470-R1477 ◽  
Author(s):  
D. S. Miller ◽  
J. B. Pritchard
Keyword(s):  
Steady State ◽  
Organic Anion ◽  
Basolateral Membrane ◽  
Epifluorescence Microscopy ◽  
Renal Tubules ◽  
Bathing Medium ◽  
Anion Secretion ◽  
Southern Flounder ◽  
Indirect Coupling ◽  
Tubular Lumen

Recent findings in both rat and crab indicate that renal accumulation of p-aminohippurate (PAH) across the basolateral membrane can be coupled indirectly to the Na gradient through PAH-glutarate exchange and Na/glutarate cotransport. However, the role of this mechanism in net transepithelial PAH secretion was not examined. Therefore, proximal tubules from Southern flounder kidney were used to assess both the presence of indirect coupling in the fish and its relationship to net secretion. [14C]glutarate uptake by proximal tubular masses was concentrative, Na dependent, and Li inhibitable. Glutarate efflux from preloaded masses was stimulated by addition of PAH to the medium. Thus flounder tubules exhibited both Na/glutarate uptake and glutarate-PAH exchange. Furthermore, steady-state [3H]PAH accumulation was increased 50% by 10-50 microM glutarate, and this increase was abolished by Li, indicating indirect coupling of PAH entry to Na. To determine whether indirect coupling affected net secretion as well as tissue accumulation, the steady-state accumulation of an anionic dye, fluorescein (FL), was measured in individual renal tubules by use of epifluorescence microscopy and video-image analysis. FL accumulated in tubules to levels that were 20-40 times higher than the medium. In most fish, luminal fluorescence was measurably higher than cellular, and uptake in both compartments was markedly reduced by PAH and Li. Moreover, FL accumulation in cells and lumina was increased by 70-100% when 50 microM glutarate was added to the bathing medium. Thus glutarate not only stimulated uphill FL entry into the cells, but also stimulated active secretion into the tubular lumen.(ABSTRACT TRUNCATED AT 250 WORDS)

P-glycoprotein-mediated secretion of a fluorescent cyclosporin analogue by teleost renal proximal tubules

1995 ◽  
Vol 268 (1) ◽  
pp. F46-F52 ◽  
Author(s):  
U. Schramm ◽  
G. Fricker ◽  
R. Wenger ◽  
D. S. Miller
Keyword(s):  
Steady State ◽  
Fluorescence Intensity ◽  
Fundulus Heteroclitus ◽  
Organic Anion ◽  
Proximal Tubules ◽  
Metabolic Inhibitors ◽  
Epifluorescence Microscopy ◽  
Transport Systems ◽  
Simple Diffusion ◽  
P Glycoprotein

The transport of a fluorescent cyclosporin analogue was measured in killifish (Fundulus heteroclitus) proximal tubules by means of epifluorescence microscopy and digital image analysis. Renal cells rapidly accumulated the cyclosporin analogue from the medium and attained steady state within 60 min; luminal fluorescence increased over the first 60-90 min. At steady state, luminal fluorescence intensity was two to three times higher than cellular. Cellular fluorescence intensity was a linear function of medium substrate concentration and was not affected by any treatment used. In contrast, luminal fluorescence exhibited a saturable component as the medium concentration of the cyclosporin was increased. Secretion into the lumen was blocked by metabolic inhibitors, vanadate, other cyclosporins, such as cyclosporin A and cyclosporin G, and substrates for P-glycoprotein (verapamil, vinblastine, and quinine) but not by substrates for the renal organic anion or organic cation transport systems, such as p-aminohippurate or tetraethylammonium. The data are consistent with the fluorescent cyclosporin analogue entering proximal tubule cells by simple diffusion and then being pumped into the tubular lumen by P-glycoprotein.

Real-time assessment of α-ketoglutarate effect on organic anion secretion in perfused rabbit proximal tubules

1999 ◽  
Vol 277 (4) ◽  
pp. F513-F523 ◽  
Author(s):  
Apichai Shuprisha ◽  
Ronald M. Lynch ◽  
Stephen H. Wright ◽  
William H. Dantzler
Keyword(s):  
Steady State ◽  
Real Time ◽  
Organic Anion ◽  
Selective Inhibition ◽  
Tubular Secretion ◽  
Proximal Tubules ◽  
Renal Tubular Secretion ◽  
Anion Secretion ◽  
Renal Tubular ◽  
Time Assessment

To determine the quantitative roles of the basolateral and luminal Na+-dicarboxylate (Na-DC) cotransporters in establishing and maintaining the α-ketoglutarate (αKG) gradient required for renal tubular secretion of organic anions, we measured net steady-state transepithelial secretion of fluorescein (FL) in real time in isolated, perfused S2 segments of rabbit renal proximal tubules. Net “basal” FL secretion in the absence of exogenous αKG had a K t of ∼4 μM and a maximal transepithelial secretion rate ( J max) of ∼380 fmol ⋅ min−1 ⋅ mm−1(where K t is the FL concentration that produces one-half the J max). It could be almost completely inhibited by basolateral p-aminohippurate (PAH). Selective inhibition of the basolateral Na-DC cotransporter indicated that recycling via this transporter of αKG that had been exchanged for FL supports ∼25% of the “basal” FL secretion. Physiological αKG concentrations of 10 μM in the bath or 50 μM in the perfusate stimulated net secretion of FL by ∼30 or ∼20%, respectively. These data indicate that the basolateral Na-DC cotransporter supports ∼42% of the net FL secretion. The luminal and basolateral effects of physiological concentrations of αKG were additive, indicating that the combined function of the luminal and basolateral Na-DC cotransporters can support ∼50% of the net FL secretion. This apparently occurs by their establishing and maintaining ∼50% of the outwardly directed αKG gradient that is responsible for driving basolateral FL/αKG exchange. The remaining ∼50% would be maintained by metabolic production of αKG in the cells.

Transport of PAH, urate, TEA, and fluid by isolated perfused and nonperfused avian renal proximal tubules

1994 ◽  
Vol 266 (4) ◽  
pp. R1085-R1094 ◽  
Author(s):  
O. H. Brokl ◽  
E. J. Braun ◽  
W. H. Dantzler
Keyword(s):  
Steady State ◽  
Basolateral Membrane ◽  
Proximal Tubules ◽  
Intracellular Concentration ◽  
Electrochemical Gradient ◽  
Bathing Medium ◽  
Renal Proximal Tubules ◽  
Electrical Gradient ◽  
High Concentrations ◽  
First Time

Transport of organic anions [p-aminohippurate (PAH) and urate] and organic cations [tetraethylammonium (TEA)] and reabsorption of fluid were studied for the first time in individual renal proximal tubules isolated from avian kidneys. In isolated nonperfused tubules, PAH and urate uptake occurred against electrochemical gradients, whereas TEA uptake appeared to result from the electrical gradient. Radiolabeled PAH uptake and radiolabeled urate uptake were inhibited to an equal extent by high concentrations of unlabeled PAH and probenecid, suggesting that they might share the same transport system. However, the rate of uptake of radiolabeled PAH was significantly stimulated by preloading with alpha-ketoglutarate (alpha-KG), suggesting PAH/alpha-KG countertransport as in mammals and reptiles, whereas uptake of radiolabeled urate was not clearly stimulated. In isolated perfused tubules, net fluid reabsorption averaged approximately 2 nl.min-1.mm-1 and was inhibited by ouabain with or without bicarbonate in the perfusate and bathing medium. In these perfused tubules, the unidirectional bath-to-lumen fluxes of PAH and urate exceeded the unidirectional lumen-to-bath fluxes, indicating net secretion of both compounds. During the bath-to-lumen fluxes the uptake across the basolateral membrane was against an electrochemical gradient for both compounds. However, for PAH the steady-state intracellular concentration was about half that observed in nonperfused tubules, as generally expected during net secretion, whereas for urate the steady-state intracellular concentration was about twice that observed in nonperfused tubules, suggesting stimulation of uptake during net secretion. During the PAH lumen-to-bath flux, the steady-state intracellular concentration was significantly above that in the perfusate, suggesting that this flux involved transport into the cells from the lumen against an electrochemical gradient. However, during the urate lumen-to-bath flux, there was no urate in the cells, suggesting that this flux, as in reptiles, occurred by a paracellular route.

Inhibition of initial transport rate of basolateral organic anion carrier in renal PT by BK and phenylephrine

1999 ◽  
Vol 277 (2) ◽  
pp. F251-F256 ◽  
Author(s):  
Michael Gekle ◽  
Sigrid Mildenberger ◽  
Christoph Sauvant ◽  
Dallas Bednarczyk ◽  
Stephen H. Wright ◽  
...  
Keyword(s):  
Direct Action ◽  
Organic Anion ◽  
Specific Inhibitor ◽  
Peptide Hormone ◽  
Organic Anion Transporter ◽  
Epifluorescence Microscopy ◽  
Anion Transporter ◽  
Initial Uptake Rate ◽  
Continuous Presence

The effect of ligands for phospholipase C-coupled receptors and of protein kinase C (PKC) stimulation with phorbol ester [phorbol 12-myristate 13-acetate (PMA)] or 1,2-dioctanoyl- sn-glycerol on the activity of the basolateral organic anion transporter (OAT) in S2 segments of single, nonperfused rabbit proximal tubules (PT) was measured with the use of fluorescein and epifluorescence microscopy. The initial uptake rate (25 s, OAT activity) was measured in real time by using conditions similar to those found in vivo. Stimulation of PKC with PMA or 1,2-dioctanoyl- sn-glycerol led to an inhibition of OAT activity, which could be prevented by 10−7 mol/l of the PKC-specific inhibitor bisindolylmaleimide. The α1-receptor agonist phenylephrine as well as the peptide hormone bradykinin induced a reversible decrease of OAT activity, which was prevented by bisindolylmaleimide. The observed effect was not due to a decrease in the concentration of the counterion α-ketoglutarate or to impaired α-ketoglutarate recycling, because it was unchanged in the continuous presence of α-ketoglutarate or methyl succinate. We conclude that physiological stimuli can inhibit the activity of OAT in rabbit PT via PKC. The effect is not mediated by alterations in counterion availability but by a direct action on the OAT.

Daunomycin secretion by killfish renal proximal tubules

1995 ◽  
Vol 269 (2) ◽  
pp. R370-R379 ◽  
Author(s):  
D. S. Miller
Keyword(s):  
Organic Cation ◽  
Basolateral Membrane ◽  
Organic Cation Transporter ◽  
Proximal Tubules ◽  
Epifluorescence Microscopy ◽  
Buffer Solution ◽  
Simple Diffusion ◽  
Renal Proximal Tubules ◽  
Cellular Accumulation ◽  
Tubular Lumen

Epifluorescence microscopy and video-image analysis were used to measure the uptake of the fluorescent anthracycline daunomycin by intact killifish renal proximal tubules. When tubules were incubated in medium containing 2-5 microM daunomycin, the drug accumulated in the cells and the tubular lumen. At steady state, luminal fluorescence was two to three times greater than cellular fluorescence. Luminal accumulation of daunomycin was reduced when tubules were exposed to the multidrug-resistance (MDR) transporter modifiers verapamil and cyclosporin A (CSA), but not tetraethylammonium (TEA), a model substrate for the renal organic cation transport system. NaCN and vanadate reduced luminal drug accumulation. In contrast, cellular daunomycin accumulation was not affected by verapamil, CSA, TEA, or vanadate and was only slightly reduced by NaCN. When the pH of the buffer solution was decreased from 8.25 to 7.25, luminal, but not cellular, accumulation of daunomycin was again reduced by CSA; however, TEA now reduced cellular and luminal accumulation. These findings are consistent with daunomycin being actively secreted in killifish proximal tubule by two mechanisms. At pH 8.25, daunomycin crossed the basolateral membrane by simple diffusion and was secreted into the tubular lumen by the MDR transporter. At pH 7.25, daunomycin was transported across the basolateral membrane by simple diffusion and carrier-mediated uptake on the organic cation transporter and was secreted into the lumen by the MDR transporter and the organic cation/H+ exchanger.

Interaction of the metal chelator DMPS with OAT1 and OAT3 in intact isolated rabbit renal proximal tubules

2004 ◽  
Vol 286 (1) ◽  
pp. F68-F76 ◽  
Author(s):  
A. Lungkaphin ◽  
V. Chatsudthipong ◽  
K. K. Evans ◽  
C. E. Groves ◽  
S. H. Wright ◽  
...  
Keyword(s):  
Heavy Metals ◽  
Heavy Metal ◽  
Urinary Excretion ◽  
Basolateral Membrane ◽  
Proximal Tubules ◽  
Bathing Medium ◽  
Renal Proximal Tubules ◽  
Estrone Sulfate ◽  
Metal Chelator ◽  
Prevalent Form

2,3-Dimercapto-1-propanesulfonic acid (DMPS) is used clinically to increase urinary excretion of heavy metals, including mercury and arsenic. We used single S2 segments and suspensions of rabbit renal proximal tubules (RPT) to test the interaction of this anionic heavy metal chelator with basolateral transporters OAT1 and OAT3. RTPCR revealed expression of both transporters in single S2 segments. [3H]PAH and 3H-labeled estrone sulfate ([3H]ES) were used as specific substrates for rbOAT1 and rbOAT3, respectively. PAH and ES were transported into nonperfused single RPT segments with Kt values of 67 ± 20 and 3.4 ± 1.2 μM, respectively, and into tubule suspensions with Kt values of 58 ± 17 and 7.7 ± 2.1 μM, respectively. Reduced DMPS (DMPSH) inhibited uptake of both substrates into single tubule segments with Kapp values of 405 ± 49 μM (for [3H]PAH) and 320 ± 66 μM (for [3H]ES). Oxidized DMPS (DMPSS), the prevalent form in the blood, also inhibited uptakes of [3H]PAH ( Kapp of 766 ± 190 μM) and [3H]ES (696 ± 166 μM). Inward gradients of ES, DMPSH, and DMPSS trans-stimulated the 30-s efflux of preloaded [3H]ES across the basolateral membrane of RPT. Similarly, DMPSH, and PAH itself, trans-stimulated the 15-s efflux of [3H]PAH. In contrast, efflux of [3H]PAH was inhibited by the presence of DMPSS in the bathing medium. These data suggest that, whereas both OAT1 and OAT3 probably transport DMPSH, DMPSS transport may be limited to OAT3. This is the first evidence showing that both OAT1 and OAT3 can transport DMPS across the basolateral membrane of RPT.

scholarly journals The multidrug transporter MATE1 sequesters OCs within an intracellular compartment that has no influence on OC secretion in renal proximal tubules

2016 ◽  
Vol 310 (1) ◽  
pp. F57-F67 ◽  
Author(s):  
L. J. Martínez-Guerrero ◽  
K. K. Evans ◽  
W. H. Dantzler ◽  
S. H. Wright
Keyword(s):  
Cho Cells ◽  
Chinese Hamster ◽  
Proximal Tubules ◽  
Epifluorescence Microscopy ◽  
Apical Plasma Membrane ◽  
Multidrug Transporter ◽  
Renal Proximal Tubules ◽  
Intracellular Compartment ◽  
Parallel Pathway ◽  
Kinetics Of

Secretion of organic cations (OCs) across renal proximal tubules (RPTs) involves basolateral OC transporter (OCT)2-mediated uptake from the blood followed by apical multidrug and toxin extruder (MATE)1/2-mediated efflux into the tubule filtrate. Whereas OCT2 supports electrogenic OC uniport, MATE is an OC/H+ exchanger. As assessed by epifluorescence microscopy, cultured Chinese hamster ovary (CHO) cells that stably expressed human MATE1 accumulated the fluorescent OC N, N, N-trimethyl-2-[methyl(7-nitrobenzo[c][l,2,5]oxadiazol-4-yl)amino]ethanaminium (NBD-MTMA) in the cytoplasm and in a smaller, punctate compartment; accumulation in human OCT2-expressing cells was largely restricted to the cytoplasm. A second intracellular compartment was also evident in the multicompartmental kinetics of efflux of the prototypic OC [3H]1-methyl-4-phenylpyridinium (MPP) from MATE1-expressing CHO cells. Punctate accumulation of NBD-MTMA was markedly reduced by coexposure of MATE1-expressing cells with 5 μM bafilomycin (BAF), an inhibitor of V-type H+-ATPase, and accumulation of [3H]MPP and [3H]NBD-MTMA was reduced by >30% by coexposure with 5 μM BAF. BAF had no effect on the initial rate of MATE1-mediated uptake of NBD-MTMA, suggesting that the influence of BAF was a secondary effect involving inhibition of V-type H+-ATPase. The accumulation of [3H]MPP by isolated single nonperfused rabbit RPTs was also reduced >30% by coexposure to 5 μM BAF, suggesting that the native expression in RPTs of MATE protein within endosomes can increase steady-state OC accumulation. However, the rate of [3H]MPP secretion by isolated single perfused rabbit RPTs was not affected by 5 μM BAF, suggesting that vesicles loaded with OCs+ are not likely to recycle into the apical plasma membrane at a rate sufficient to provide a parallel pathway for OC secretion.

scholarly journals Specificity of basolateral organic anion exchanger in proximal tubule for cellular and extracellular solutes.

1992 ◽  
Vol 2 (7) ◽  
pp. 1192-1200 ◽  
Author(s):  
L P Sullivan ◽  
J J Grantham
Keyword(s):  
Organic Anion ◽  
Carbon Chain ◽  
Organic Anions ◽  
Anion Transport ◽  
Chain Fatty Acid ◽  
Proximal Tubules ◽  
Organic Anion Transport ◽  
Bathing Medium ◽  
Metabolic Intermediates ◽  
Serial Measurements

We used an epifluorimetric technique to study the mechanism of organic anion transport across the basolateral surface of isolated S2 segments of rabbit proximal tubules. Fluorescein influx and efflux rates across the basolateral surface of lumen-collapsed tubules were determined from serial measurements of cellular fluorescein content after its addition to or removal from the bathing medium. We examined the effect on fluorescein transport of monocarboxylic and dicarboxylic metabolic intermediates added to the bathing medium or preloaded into the cells. The presence of the monocarboxylates (octanoate, valerate, butyrate, propionate, and acetate) in the bathing medium inhibited fluorescein influx. The eight-carbon-chain fatty acid, octanoate, was nearly as potent an inhibitor as probenecid and was more potent than p-aminohippurate (PAH) (IC50 = 10, 6, and 141 microM, respectively); the shorter chain fatty acids were much less effective (IC50 greater than or equal to 1,000 microM). The dicarboxylates (succinate, adipate, and alpha-ketoglutarate) moderately inhibited fluorescein influx (IC50 = 505, 245, and 257 microM, respectively). To determine the effect of intracellular organic anions on fluorescein influx, tubules were preincubated with organic anions to load the cells; the compounds were then removed from the bath, and fluorescein influx was measured. Preincubation with monocarboxylates, octanoate, butyrate, acetate, and PAH had no effect. In contrast, the dicarboxylates alpha-ketoglutarate, glutarate, and adipate stimulated fluorescein influx by 58, 53, and 43%, respectively, but succinate had no effect. Fluorescein efflux was accelerated by medium octanoate, PAH, alpha-ketoglutarate, and succinate but not by acetate. Probenecid alone had no effect on fluorescein efflux, but it blocked the stimulation caused by medium PAH.(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.

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