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

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 [(3)H]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 [(3)H]MPP and [(3)H]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 [(3)H]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 [(3)H]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.

Urine concentrating mechanism in the inner medulla of the mammalian kidney: role of three-dimensional architecture.

The urine concentrating mechanism in the mammalian renal inner medulla (IM) is not understood, although it is generally considered to involve countercurrent flows in tubules and blood vessels. A possible role for the three-dimensional relationships of these tubules and vessels in the concentrating process is suggested by recent reconstructions from serial sections labelled with antibodies to tubular and vascular proteins and mathematical models based on these studies. The reconstructions revealed that the lower 60% of each descending thin limb (DTL) of Henle's loops lacks water channels (aquaporin-1) and osmotic water permeability and ascending thin limbs (ATLs) begin with a prebend segment of constant length. In the outer zone of the IM (i) clusters of coalescing collecting ducts (CDs) form organizing motif for loops of Henle and vasa recta; (ii) DTLs and descending vasa recta (DVR) are arrayed outside CD clusters, whereas ATLs and ascending vasa recta (AVR) are uniformly distributed inside and outside clusters; (iii) within CD clusters, interstitial nodal spaces are formed by a CD on one side, AVR on two sides, and an ATL on the fourth side. These spaces may function as mixing chambers for urea from CDs and NaCl from ATLs. In the inner zone of the IM, cluster organization disappears and half of Henle's loops have broad lateral bends wrapped around terminal CDs. Mathematical models based on these findings and involving solute mixing in the interstitial spaces can produce urine slightly more concentrated than that of a moderately antidiuretic rat but no higher.

Involvement of tyrosine kinase and PI3K in the regulation of OAT3-mediated estrone sulfate transport in isolated rabbit renal proximal tubules.

It was shown previously that OAT3 activity was differentially regulated by protein kinases including MAPK, PKA, and PKC. The present study investigated the short-term effect of tyrosine kinase and phosphatidylinositol 3-kinase (PI3K) on OAT3-mediated organic anion transport in S2 segments of renal proximal tubules. Genistein, a tyrosine kinase inhibitor, and wortmannin, a PI3K inhibitor, inhibited transport of estrone sulfate, a prototypic substrate for OAT3, in a dose-dependent manner. Previously, we showed that epidermal growth factor (EGF) stimulated OAT3 activity via the MAPK pathway. In the present study, we investigated whether EGF-stimulated OAT3 activity was dependent on tyrosine kinase and PI3K. We showed that EGF stimulation of OAT3 was reduced by inhibition of tyrosine kinase or PI3K, suggesting that they play a role in the stimulatory process. Inhibitory effects also indicated that tyrosine kinase and PI3K are involved in the MAPK pathway for EGF stimulation of OAT3 in intact renal proximal tubules, with PI3K acting upstream and tyrosine kinase acting downstream of mitogen-activated/extracellular signal-regulated kinase kinase activation.

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

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 alpha(1)-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 PGE(2) or dibutyryl (db)-cAMP also enhanced ES uptake. Stimulation produced by PGE(2) 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 PGE(2) 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.

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

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 microM, respectively, and into tubule suspensions with Kt values of 58 +/- 17 and 7.7 +/- 2.1 microM, respectively. Reduced DMPS (DMPSH) inhibited uptake of both substrates into single tubule segments with Kapp values of 405 +/- 49 microM (for [3H]PAH) and 320 +/- 66 microM (for [3H]ES). Oxidized DMPS (DMPSS), the prevalent form in the blood, also inhibited uptakes of [3H]PAH (Kapp of 766 +/- 190 microM) and [3H]ES (696 +/- 166 microM). 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.

Action of EGF and PGE2 on basolateral organic anion uptake in rabbit proximal renal tubules and hOAT1 expressed in human kidney epithelial cells.

We recently showed that, in a proximal tubule cell line (opossum kidney cells), epithelial growth factor (EGF) stimulates basolateral organic anion transport (OAT) via ERK1/2, arachidonic acid, phospholipase A2, and generation of prostaglandins. PGE2 binds the prostanoid receptor and, thus, activates adenylate cyclase and PKA, which stimulate basolateral organic anion uptake. In the present study, we investigated whether this regulatory cascade is also true 1) for ex vivo conditions in isolated renal proximal (S2) tubules from rabbit and 2) in a human renal epithelial cell line stably expressing human OAT1 (IHKE-hOAT1). EGF activated ERK1/2 in S2 tubules and IHKE-hOAT1, and, in both cases, inhibition of ERK activation (by U-0126) abolished this stimulation. In S2 tubules and IHKE-hOAT1, EGF led to an increase of organic anion uptake, which again was inhibited by U-0126. PGE2 stimulated basolateral organic anion uptake in rabbit S2 tubules and IHKE-hOAT1. EGF- and PGE2-mediated stimulation of organic anion uptake was abolished by inhibition of PKA in rabbit S2 tubules and IHKE-hOAT1, respectively. We conclude that 1) stimulation of basolateral organic anion uptake by EGF or PGE2 is a widespread (if not general) regulatory mechanism, 2) the signal transduction pathway involved seems to be general, 3) stimulation of basolateral organic anion uptake by EGF or PGE2 is also present under ex vivo conditions and, thus, is not a cell culture artifact, 4) activation of OAT1 is sufficient to explain the stimulatory effects of EGF and PGE2 in opossum kidney cells and rabbit S2 segments, and 5) stimulation of basolateral OAT1 by EGF or PGE2 is also important in humans and, thus, may have clinical implications.

Method for measuring luminal efflux of fluorescent organic compounds in isolated, perfused renal tubules.

To examine directly in real time the efflux of organic compounds [e. g., organic anions (OAs) such as fluorescein (FL)] across the luminal membrane of isolated, perfused renal tubules during net secretion, we devised an approach utilizing a recently developed epifluorescence microscopy system for continuous monitoring of fluorescence in the collected perfusate. To illustrate this approach, we measured the luminal efflux rate of FL in mineral oil-covered, isolated, perfused S2 segments of rabbit renal proximal tubules. The washout profile of FL showed a deviation from linearity at time 0 when plotted on a semilog scale, indicating that the luminal efflux of FL was a saturable process. We were able for the first time to determine the kinetic parameters of luminal efflux [FL concentration at one-half maximal FL efflux (K(t)(lumen)) of approximately 560 microM and maximal rate of FL efflux across the luminal membrane (J(max)(lumen)) of approximately 635 fmol. min(-1). mm(-1)]. From the present study, we conclude that the transport step for OAs across the luminal membrane of OAs is a carrier-mediated process. This approach will work to measure luminal transport in real time for any secreted organic compound that is sufficiently fluorescent to be measured with commonly available, highly sensitive optical equipment.

Intracellular pH in isolated rat renal papillary thin limbs of Henle's loop.

Intracellular pH (pHi) was measured in isolated, nonperfused and perfused rat papillary thin limbs of Henle's loops in N-2-hydroxyethylpiperazine-N'-2-ethansulfonic acid (HEPES)- or HEPES/bicarbonate-buffered medium at pH 7.4 using the pH-sensitive fluorescent dye 2',7'-bis(2-carboxyethyl)-5,6-carboxyfluorescein (BCECF). Resting pHi was about 6.7 in descending thin limbs (DTL) and about 6.9 in ascending thin limbs (ATL), even with a medium pH of 7.4. These values appeared to reflect the acid pH of the blood in the neighboring vasa recta found in vivo. The resting pHi did not differ whether or not the medium contained bicarbonate although the total buffering capacity of the tubule cells was increased in the presence of bicarbonate. In nonperfused DTL and ATL, pHi was further acidified following an NH4Cl pulse. The rate of recovery of pHi from this level to the resting pHi was reduced by Na+ removal from the bath in both DTL and ATL and by the addition of ethylisopropylamiloride (EIPA) to the bath in the presence of Na+ in DTL. The rate of recovery was not affected by Cl- removal from the bath or K+ (75 mM) or 4,4'-diisothiocyanostilbene-2,2'-disulfonate (DIDS) addition to the bath in either DTL or ATL. These results suggest that the common, amiloride-sensitive, basolateral Na+/H+ exchanger plays a role in the regulation of pHi in rat papillary DTL but that a different basolateral Na+/H+ exchanger or a luminal Na+/H+ exchanger is important in rat papillary ATL.

Cycloleucine fluxes during rat vasa recta and loop microinfusions in vivo and loop microperfusions in vitro.

Amino acids are apparently recycled between loops of Henle and vasa recta in rat papilla in vivo. To examine this process in the absence of metabolism, we performed continuous microinfusions of rat renal papillary ascending thin limbs (ATLs) and vasa recta in vivo, and microperflusions of isolated rat renal papillary descending thin limbs (DTLs) and ATLs in vitro using the nonmetabolizable, synthetic, neutral amino acid cycloleucine. Like naturally occurring amino acids, approximately = 25% of radiolabeled cycloleucine microinfused into ATLs in vivo was reabsorbed by a process that was not saturable or inhibitable. Also, like naturally occurring amino acids, approximately = 47% (relative to inulin) of radiolabeled cycloleucine microinfused into ascending vasa recta in vivo was transferred directly into ipsilateral tubular structures (probably DTLs) by a saturable and inhibitable process. In DTLs perfused in vitro, unidirectional bath-to-lumen fluxes (Jbl) tended to exceed unidirectional lumen-to-bath fluxes (Jlb), whereas in ATLs perfused in vitro Jlb tended to exceed Jbl, but the differences were not statistically significant. Moreover, none of the unidirectional fluxes was saturable or inhibitable, an observation compatible with apparent reabsorption from ATLs in vivo but incompatible with apparent movement from vasa recta to DTLs in vivo. These in vitro observations are like those made previously for the naturally occurring neutral amino acid L-alanine. The lack of saturation and inhibition, like the previous data on L-alanine, suggest that transepithelial movement of amino acids in thin limbs of Henle's loop may occur via a paracellular route and that regulation of amino acid movement in vivo may involve vasa recta, not DTLs. They also suggest that cycloleucine is a good nonmetabolizable surrogate for the study of neutral amino acid transport in the kidney.

Reabsorption of betaine in Henle's loops of rat kidney in vivo.

This study was designed 1) to localize and 2) to characterize betaine reabsorption from the tubular lumen in rat kidney in vivo, and 3) to test whether reabsorption is modulated by the diuretic state. [(14)C]betaine (+ [(3)H]inulin) was microperfused through the proximal convoluted tubule (PCT) and microinfused into late proximal (LP) and early distal (ED) tubules, long loops of Henle (LLH), and vasa recta of the rat in vivo et situ, and the fractional recovery of the (14)C label was determined end proximally (PCT) and in the final urine, respectively. [(14)C]betaine was not reabsorbed during ED microinfusion, whereas fractional reabsorption during LP microinfusion was 82% at 0.06 mM betaine and decreased gradually to 4.8% at 60 mM. L-Proline had lower Michaelis-Menten constant (K(m)) and sarcosine a higher K(m) than betaine. Chronic, but not acute, diuresis inhibited betaine reabsorption in Henle's loops. Fractional [(14)C]betaine reabsorption in PCT was much smaller than that during LP microinfusion. [(14)C]betaine (7.28 mM) microinfused 1) into LLH was reabsorbed to 30% and 2) into vasa recta appeared in the ipsilateral urine to a much higher extent than contralaterally. In both cases, no saturation was detected at 70 mM. We conclude that betaine is reabsorbed by mediated transport from descending limbs of short Henle's loops by a proline-preferring carrier in a diuresis-modulated manner. In the deep medulla, bidirectional blood/urine betaine transport exists.

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

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 microM 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 approximately 30% after 25 min. This inhibition was irreversible and, indeed, increased to approximately 40% at 25 min following removal of PMA [10 microM 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 microM of either the peptide hormone bradykinin (BK) or the alpha(1)-receptor agonist phenylephrine (PE), both of which stimulate PKC via a ligand-receptor-PKC coupling reaction, to the bath also inhibited FL secretion by approximately 22 and approximately 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.

Mixed descending- and ascending-type thin limbs of Henle's loop in mammalian renal inner medulla.

Previous studies have generally indicated that the entire descending (DTL) and ascending thin limbs (ATL) of Henle's loops in the mammalian inner medulla exhibit structurally and functionally distinct properties. In the present study, we found that about 50% of Munich-Wistar rat inner medullary thin limbs, lying at positions distinctly above the bend, had segments exhibiting structural characteristics of DTL located immediately adjacent to segments exhibiting structural characteristics of ATL. Multiple DTL-type and ATL-type segments of variable length existed along a single straight portion of these mixed tubules. Inner medullary thin limbs with repeating, sequential expression of DTL-type and ATL-type regions were also numerous in Sprague-Dawley rats, mice, and rabbits with no evidence of sexual dimorphism. RT-PCR of microdissected segments showed that the water channel aquaporin-1 (AQP1) and the urea transporter UT-A2 were expressed in pure DTL, but not in pure ATL, and in DTL-type, but not in ATL-type, regions of mixed-type thin limbs. Immunocytochemistry revealed expression of AQP1 in cells of pure DTL, but not pure ATL, and in cells of DTL-type, but not ATL-type, regions of mixed-type thin limbs. In contrast, the chloride channel ClC-K1 was expressed in pure ATL, but not pure DTL, and in ATL-type, but not DTL-type, regions of mixed-type thin limbs. Discontinuous axial expression of AQP1, UT-A2, and ClC-K1 along the straight portion of single thin limbs indicates that these nephrons possess a more heterogeneous structure than previously recognized.

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

To determine the quantitative roles of the basolateral and luminal Na(+)-dicarboxylate (Na-DC) cotransporters in establishing and maintaining the alpha-ketoglutarate (alphaKG) 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 alphaKG had a K(t) of approximately 4 microM and a maximal transepithelial secretion rate (J(max)) of approximately 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 alphaKG that had been exchanged for FL supports approximately 25% of the "basal" FL secretion. Physiological alphaKG concentrations of 10 microM in the bath or 50 microM in the perfusate stimulated net secretion of FL by approximately 30 or approximately 20%, respectively. These data indicate that the basolateral Na-DC cotransporter supports approximately 42% of the net FL secretion. The luminal and basolateral effects of physiological concentrations of alphaKG were additive, indicating that the combined function of the luminal and basolateral Na-DC cotransporters can support approximately 50% of the net FL secretion. This apparently occurs by their establishing and maintaining approximately 50% of the outwardly directed alphaKG gradient that is responsible for driving basolateral FL/alphaKG exchange. The remaining approximately 50% would be maintained by metabolic production of alphaKG in the cells.

Amino acid fluxes in rat thin limb segments of Henle's loop during in vitro microperfusion.

Amino acids are apparently recycled between loops of Henle and vasa recta in the rat papilla in vivo. To examine more closely papillary amino acid transport, we measured transepithelial fluxes of L-[(14)C]alanine and [(14)C]taurine in thin limbs of Henle's loops isolated from rat papilla and perfused in vitro. In descending thin limbs (DTL) in vitro, unidirectional bath-to-lumen fluxes tended to exceed unidirectional lumen-to-bath fluxes for both radiolabeled amino acids, although the difference was statistically significant only for taurine. In ascending thin limbs (ATL) in vitro, unidirectional lumen-to-bath fluxes tended to exceed unidirectional bath-to-lumen fluxes, although the difference was again statistically significant only for taurine. These results are compatible with apparent directional movements of amino acids in vivo. However, none of the unidirectional fluxes was saturable or inhibitable, an observation compatible with apparent reabsorption from the ATL in vivo but not compatible with apparent movement from vasa recta to DTL in vivo. There was no evidence of net active transepithelial transport when concentrations of radiolabeled amino acids were matched on both sides of perfused tubule segments. These data suggest that regulation of amino acid movement in vivo may involve the vasa recta, not the DTL of Henle's loops. The data also suggest that transepithelial movement of amino acids in thin limbs of Henle's loop may occur via a paracellular route.

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

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 alpha(1)-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 alpha-ketoglutarate or to impaired alpha-ketoglutarate recycling, because it was unchanged in the continuous presence of alpha-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.

Basolateral regulation of pHi in proximal tubules of avian loopless and long-looped nephrons in bicarbonate.

In isolated, nonperfused chicken proximal tubules from both loopless reptilian-type and long-looped mammalian-type nephrons, resting intracellular pH (pHi), measured with pH-sensitive fluorescent dye 2',7'-bis(2-carboxyethyl)-5,6-carboxyfluorescein (BCECF), was approximately 7.1 under control HCO3- conditions [20 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES)/5 mM HCO3(-)-buffered medium with pH 7.4 at 37 degrees C] and was reduced to approximately 6.8 in response to NH4Cl pulse. The rate of recovery of pHi (dpHi/dt) from this level to the resting level in proximal tubules from both nephron types was (1) significantly reduced by the removal of Na+ or both Na+ and Cl- from the bath, and (2) unaffected by the removal of Cl- from the bath or the presence of a high K+ concentration or Ba2+ in the bath. In proximal tubules from long-looped mammalian-type, but not loopless reptilian-type, nephrons, dpHi/dt was significantly reduced by the addition of either 5-(N-ethyl-N-isopropyl) amiloride (EIPA) or 4,4'-diisothiocyanostilbene-2,2'disulfonate (DIDS) to the bath. These data suggest that a Na+/H+ exchanger and most likely a Na(+)-dependent Cl-/HCO3- exchanger are involved in basolateral regulation of pHi in mammalian-type nephrons whereas none of the commonly identified basolateral acid-base transporters appear to be involved in regulation of pHi in reptilian-type nephrons.

Basolateral regulation of pHi in isolated snake renal proximal tubules in presence and absence of bicarbonate.

Intracellular pH (pHi) and its basolateral regulation were studied in isolated proximal-proximal and distal-proximal segments of garter snake (Thamnophis spp.) renal tubules with oil-filled lumens in HEPES-buffered and in HEPES-HCO-3-buffered media (pH 7.4 at 25 degrees C). pHi was measured with the pH-sensitive fluorescent dye 2',7'-bis(2-carboxyethyl)-5,6-carboxyfluorescein (BCECF) under resting conditions and in response to NH4Cl pulse. Resting pHi (approximately 7.1-7.2) and its response to and rate of recovery (dpHi/dt) from an NH4Cl pulse were not affected by the presence or absence of HCO-3 in either segment. Rate of recovery was depressed by Na+ removal in distal-proximal segments only and only in HEPES buffer. It was not affected by removal of Cl- or of both Na+ and Cl- or by reduction in membrane potential through addition of Ba2+ (5 mM) or high K+ (75 mM) in either segment in either HEPES or HEPES-HCO-3 buffer. The Na+/H+ exchange inhibitor ethylisopropylamiloride (EIPA) (100 microM) and the anion exchange inhibitor DIDS (100 microM) reduced dpHi/dt in the distal-proximal segments only and only in HEPES-HCO-3 buffer. The H+-ATPase inhibitor bafilomycin (1 microM), H+-K+-ATPase and K+/NH+4 exchange inhibitor Schering 28080 (10-100 microM), organic cation efflux inhibitor tetrapentylammonium (25 microM-20 mM), and K+ channel blocker tetraethylammonium (20 mM) had no effect on dpHi/dt in either segment. These data do not clearly support basolateral regulation of pHi in snake proximal renal tubules by commonly recognized Na+-dependent or Na+-independent acid or base transporters.

D-Serine is reabsorbed in rat renal pars recta.

D-Serine normally contributes up to 3% to total plasma serine and up to 23% in chronic renal failure. D-Serine is metabolized by tubular D-amino acid oxidase (D-AAO), and high D-serine plasma levels are nephrotoxic; both events are localized in the straight part of the proximal tubule. We therefore investigated if and how D-serine is reabsorbed there. We microinfused 14C-labeled D- or -L-serine + [3H]inulin into early proximal (EP), late proximal (LP), or early distal (ED) tubule sections of superficial nephrons and into long loops of Henle (LLH) of rats in vivo and in situ. The fractional reabsorption (FR) of the 14C label was determined from the 14C:3H ratio in the final urine. At 0.36 mM, FR of D-[14C]serine was 86% (EP), 90% (LP), and approximately 0 (ED, LLH). FR of D-serine could be saturated and inhibited by L-serine (and vice versa). D-methionine, but not D-glutamate or D-arginine, blocked FR of D-serine (LP). We conlude that filtered D-serine is able to enter the pars recta cells, thereby getting access to D-AAO. The uptake carrier has a very low stereospecificity and is, therefore, different from that in the proximal convolution. The colocalization of exclusive reabsorption and metabolism makes the pars recta the tubule site for the recycling of the carbon structure of D-amino acids and, at the same time, the target of D-serine nephrotoxicity.

Effects of inhibitors and substitutes for chloride in lumen on p-aminohippurate transport by isolated perfused rabbit renal proximal tubules.

The transport step for p-aminohippurate (PAH) from cell to lumen across the luminal membrane of rabbit proximal tubules has not been adequately defined. To examine this process more closely, we determined the effects of possible transport inhibitors and substitutes for chloride on PAH secretion in isolated perfused S2 segments of rabbit proximal tubules. The addition of 4-acetamido-4'-isothiocyano-2,2' disulfonic stilbene (10(-4) M) to the perfusate irreversibly inhibited PAH secretion, whereas the addition of probenecid (10(-4) M) to the perfusate reversibly inhibited PAH secretion. PAH secretion was unaffected by thiocyanate replacement of chloride in the luminal perfusate, reversibly inhibited by 15 to 20% by methyl sulfate replacement, and irreversibly inhibited by isethionate replacement. Because the luminal membrane is at least as permeable to thiocyanate as to chloride, less permeable to methyl sulfate, and much less permeable to isethionate, these data suggest that the PAH transport step from cells to lumen does not require chloride in the lumen but does require a highly permeant anion. During inhibition of PAH transport from cells to lumen, PAH uptake across the basolateral membrane was also reduced, suggesting some type of feedback inhibition. The data are compatible with PAH transport across the luminal membrane by an anion exchanger, a potential-driven uniporter, both carriers, or a carrier that can function in both modes.

Basolateral choline transport in isolated rabbit renal proximal tubules.

Choline can undergo both net secretion and net reabsorption by renal proximal tubules, but at physiological plasma levels net reabsorption occurs. During this process, choline enters the cells at the luminal side down an electrochemical gradient via a specific transporter with a high affinity for choline. It appeared likely that choline was then transported out of the cells against an electrochemical gradient at the basolateral membrane by countertransport for another organic cation. This possibility was examined by studying net transepithelial reabsorption and basolateral uptake and efflux of [14C]choline in isolated S2 segments of rabbit renal proximal tubules. Basolateral uptake, which was inhibited by other organic cations such as tetraethylammonium (TEA), appeared to occur by the standard organic cation transport pathway. However, the addition of TEA to the bathing medium not only failed to trans-stimulate net transepithelial reabsorption and basolateral efflux of [14C]choline but it actually inhibited transepithelial reabsorption by @60%. The results do not support the presence of a countertransport step for choline against an electrochemical gradient at the basolateral membrane. Instead, they suggest that choline crosses this membrane by some form of carrier-mediated diffusion even during the reabsorptive process.