Isolated Rabbit Renal Proximal Tubules Research Articles

In isolated rabbit renal proximal tubules, the antibiotic cephaloridine does not inhibit the mitochondrial uptake of succinate, a biomarker of its nephrotoxicity: A cellular metabolomic study with carbon 13 NMR

Cephaloridine is a model nephrotoxicant. In isolated rabbit renal mitochondria, cephaloridine (i) inhibits succinate uptake and (ii) reduces respiration. We have re‐examined this problem using isolated proximal tubules. Tubules were incubated in Krebs‐Henseleit buffer containing either 1,4‐13C‐succinate, or 2,3‐13C‐succinate or succinate + 13C‐bicarbonate without or with cephaloridine. Substrate removal and product formation were measured by enzymatic and carbon 13 NMR methods. Enzymatic fluxes were calculated by combining these results with an original mathematical model of renal succinate metabolism. Succinate was converted mainly into fumarate, malate, glucose and CO2. Cephaloridine induced an increase in the accumulation of fumarate, malate and a decrease in the production of glucose, CO2 and the level of ATP. Surprisingly, it did not change the removal of succinate. Fluxes through malate dehydrogenase, glucose 6‐phosphatase and citrate synthase were also diminished by cephaloridine.In conclusion, the absence of inhibition of mitochondrial uptake of succinate in intact renal proximal tubules strongly suggests that results obtained with isolated mitochondria do not necessarily apply to mitochondria in intact cells and, therefore, should be interpreted with great caution. These results also suggest that mitochondrial functions should be studied in intact cells.

In isolated rabbit renal proximal tubules, the beta-lactam antibiotic cephaloridine does not inhibit the mitochondrial uptake of succinate, a biomarker of its nephrotoxicity: A cellular metabolomic study with carbon 13 NMR

In isolated rabbit renal proximal tubules, the beta-lactam antibiotic cephaloridine does not inhibit the mitochondrial uptake of succinate, a biomarker of its nephrotoxicity: A cellular metabolomic study with carbon 13 NMR

Activation of Protein Kinase Cζ Increases OAT1 (SLC22A6)- and OAT3 (SLC22A8)-mediated Transport

Organic anion transporters (OATs) play a pivotal role in the clearance of small organic anions by the kidney, yet little is known about how their activity is regulated. A yeast two-hybrid assay was used to identify putative OAT3-associated proteins in the kidney. Atypical protein kinase Czeta (PKCzeta) was shown to bind to OAT3. Binding was confirmed in immunoprecipitation assays. The OAT3/PKCzeta interaction was investigated in rodent renal cortical slices from fasted animals. Insulin, an upstream activator of PKCzeta, increased both OAT3-mediated uptake of estrone sulfate (ES) and PKCzeta activity. Both effects were abolished by a PKCzeta-specific pseudosubstrate inhibitor. Increased ES transport was not observed in renal slices from OAT3-null mice. Transport of the shared OAT1/OAT3 substrate, rho-aminohippurate, behaved similarly, except that stimulation was reduced, not abolished, in the OAT3-null mice. This suggested that OAT1 activity was also modified by PKCzeta, subsequently confirmed using an OAT1-specific substrate, adefovir. Inhibition of PKCzeta also blocked the increase in ES uptake seen in response to epidermal growth factor and to activation of protein kinase A. Thus, PKCzeta acted downstream of the epidermal growth factor to protein kinase A signaling pathway. Activation of transport was accompanied by an increase in V(max) and was blocked by microtubule disruption, indicating that activation may result from trafficking of OAT3 into the plasma membrane. These data demonstrate that PKCzeta activation up-regulates OAT1 and OAT3 function, and that protein-protein interactions play a central role controlling these two important renal drug transporters.

Involvement of ezrin in cold storage preservation injury in renal proximal tubule epithelial cells: Possible structural and signaling roles

Renal transplantation is the treatment of choice for patients with end‐stage renal disease. Preservation injury is caused by ischemia/reperfusion and hypothermia during cold storage, which leads to early graft loss and delayed graft function. Advancements in organ preservation are limited by our understanding of basic root mechanisms that cause this injury. We hypothesize that sub‐lamellar cytoskeletal protein failure is involved and studied this in isolated rabbit renal proximal tubules and epithelial cells. Tubules cold stored in UW solution released ezrin from cytoskeletal actin attachments. Ezrin phosphorylation at T567 and Y353 decreased with progressive cold storage ischemia, which controls both ezrin binding to the cytoskeleton and survival signaling through Akt, respectively. Tubule epithelial cell death at reperfusion after cold storage ischemia was almost exclusively by apoptosis (annexin‐V FACS analysis). Transfection of primary renal epithelial cells with wild‐type mammalian ezrin cDNA increased survival and viability after cold storage ischemia and reperfusion and increased Akt phosphorylation (IHC). Conversely, inhibition of ezrin expression with specific siRNA duplexes decreased cell survival and viability. In conclusion, inactivation of ezrin during preservation may cause renal preservation injury by altering membrane structure and by reducing survival signaling.

Relative contribution of OAT1 and OAT3 transport activities in isolated perfused rabbit renal proximal tubules

The expression of both OAT1 and OAT3 along the isolated rabbit renal proximal tubule (RPT) was determined using RT-PCR. They were found to be very strong in S2 segment and weak in S1 and S3 segments. We further examined the relative transport activity of these transporters in isolated perfused rabbit RPT using [ 3H] para-aminohippurate ([ 3H]PAH), and estrone sulfate ([ 3H]ES) as specific substrates for rbOAT1 and rbOAT3, respectively. The transport activity of OAT1 was in the order S2 > S1 = S3 segments and that of OAT3 was in the order S1 = S2>>S3 segments. The addition of α-ketoglutarate (100 μM) in the bathing medium increased both OAT1 and OAT3 transport activities in all segments of proximal tubule. The kinetics of [ 3H]succinic acid transport, used to measure the activity of sodium dicarboxylate transporter 3 (NaDC3), were examined. The J max for succinic acid was in the order S2 > S3 and unmeasurable in the S1 segment. Our data indicate that both OAT1 and OAT3 play quantitatively significant roles in the renal transport of organic anions along the proximal tubule but predominately in S2 segment. The relative contribution of both transporters depends on their relative expression levels and may possibly be affected by the activity of NaDC3 in RPT.

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.

Interactions of Stevioside and Steviol with Renal Organic Anion Transporters in S2 Cells and Mouse Renal Cortical Slices

Our previous studies have shown that both stevioside and steviol inhibited transepithelial transport of para-aminohippurate (PAH) in isolated rabbit renal proximal tubules by interfering with organic anion transport system. The current study examined the direct interactions of stevioside and steviol with specific organic anion transporters. S2 cells expressing human organic anion transporters (hOAT1, hOAT2, hOAT3, and hOAT4) and an intact renal epithelium were used to determine the inhibitory effect of stevioside and steviol on organic anion transport. Stevioside at 0.5-1 mM showed no interaction with any OAT. In contrast, steviol markedly inhibited substrate uptake in all S2hOAT cells. Steviol had low IC50 for hOAT1 (11.4 microM) and hOAT3 (36.5 microM) similar to that of probenecid, whereas IC50 for hOAT2 (1000 microM) and hOAT4 (285 microM) was much higher. Results obtained in mouse renal cortical slices were very similar; that is, stevioside was without inhibitory effect and steviol was a potent inhibitor of PAH and estrone sulfate (ES) transport. Stevioside has no interaction with human or mouse OATs. In contrast, steviol interacts directly with human OATs, in particular, hOAT1 and hOAT3, with a potency approximating probenecid, suggesting that the inhibition of OAT-mediated transport by steviol could alter renal drug clearance.

Transport of the Natural Sweetener Stevioside and Its Aglycone Steviol by Human Organic Anion Transporter (hOAT1; SLC22A6) and hOAT3 (SLC22A8)

The natural sweetening agent stevioside and its aglycone metabolite, steviol, have been shown to inhibit transepithelial transport of para-aminohippurate (PAH) in isolated rabbit renal proximal tubules by interfering with basolateral entry. The aim of the present study was to determine which of the cloned basolateral organic anion transporters were involved in the renal transport of stevioside and steviol. This question was addressed in Xenopus laevis oocytes expressing human organic anion transporter 1 (hOAT1), 3 (hOAT3), and winter flounder OAT (fOat1). The parent compound, stevioside, had no inhibitory effect on either PAH (hOAT1) or ES (estrone sulfate; hOAT3) uptake. In contrast, steviol showed significant, dose-dependent inhibition of PAH and ES uptake in hOAT1- or hOAT3-expressing oocytes, respectively. The IC(50) of steviol for hOAT1-mediated PAH transport was 11.1 microM compared with 62.6 microM for hOAT3-mediated ES uptake. The Michaelis-Menten inhibition constants (K(i)) for steviol transport mediated by hOAT1 and hOAT3 were 2.0 +/- 0.3 and 5.4 +/- 2.0 microM, respectively. Trans-stimulation of PAH efflux by steviol was assessed to determine whether steviol itself was transported by hOAT1 or hOAT3. A low concentration of 1 microM steviol increased the efflux of [(3)H]PAH (trans-stimulated) via both hOAT1 and hOAT3. In addition, it was shown by electrophysiology that steviol entry induced inward current in fOat1-expressing oocytes. In conclusion, stevioside had no interaction with either hOAT1 or hOAT3, whereas hOAT1, hOAT3, and fOat1 were all shown to be capable of steviol transport and thus, can play a role in its renal transport and excretion.

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.

A pathophysiologic study of the kidney tubule to optimize organ preservation solutions

Tissue damage at the time of organ transplantation has a negative impact on the subsequent success of the procedure, both in the immediate and longer term. Hypothermia is the principal element used to prolong organ viability ex vivo, but paradoxically also induces cellular edema through inhibition of energy-dependent adenosine triphosphatases (ATPases). This induces an electrolyte imbalance that leads to fluid influx and cell swelling. It is important, therefore, that improvements are made in the preservation of ischemic organs to reduce this injury. This study has applied a novel in vitro system to model cold and warm ischemic-induced renal tubule swelling that characterizes tissue damage in ischemia/reperfusion injury. Biochemical blockade of ATPases in this system using strophanthidin modeled the effects of energy depletion and induced cell swelling. By measuring such tubule swelling and changes to tubular cell volume in isolated rabbit renal proximal tubules, an analysis was made that defined the basis on which an optimal preservation solution may be developed. The data show that our model could reproduce ischemically induced cell swelling and characterized the response at the cellular level of tubules to different components of preservation solutions. The data indicate that an isosmolar, phosphate-buffered, sucrose solution prevented tubule swelling more effectively than Euro-Collins, hyperosmolar citrate, or University of Wisconsin solutions that are in routine clinical use. Future developments in organ preservation may significantly improve transplant outcomes. Our novel analysis forms the basis of future whole-organ studies that ultimately may allow us to propose an optimum platform for improved preservation solutions.

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.

The Anaplerotic Substrate Alanine Stimulates Acetate Incorporation into Glutamate and Glutamine in Rabbit Kidney Tubules: A 13C NMR STUDY

Although acetate, the main circulating volatile fatty acid in humans and animals, is metabolized at high rates by the renal tissue, little is known about the precise fate of its carbons and about the regulation of its renal metabolism. Therefore, we studied the metabolism of variously labeled [(13)C]acetate and [(14)C]acetate molecules and its regulation by alanine, which is also readily metabolized by the kidney, in isolated rabbit renal proximal tubules. With acetate as the sole substrate, 72% of the C-1 and 49% of the C-2 of acetate were released as CO(2); with acetate plus alanine, the corresponding values were decreased to 49 and 25%. The only other important products formed from the acetate carbons were glutamine, and to a smaller extent, glutamate. By combining (13)C NMR and radioactive and enzymatic measurements with a novel model of acetate metabolism, fluxes through the enzymes involved were calculated. Thanks to its anaplerotic effect, alanine caused a stimulation of acetate removal and a large increase in fluxes through pyruvate carboxylase, citrate synthase, and the enzymes involved in glutamate and glutamine synthesis but not in flux through alpha-ketoglutarate dehydrogenase. We conclude that the anaplerotic substrate alanine not only accelerates the disposal of acetate but also prevents the wasting of the latter compound as CO(2).

NBD-TMA: a novel fluorescent substrate of the peritubular organic cation transporter of renal proximal tubules

Traditionally, the measurement of transport activity has employed radiolabeled compounds. The resulting experimental procedures do not measure transport in real time and are limited in temporal and spatial resolution. The use of epifluorescence microscopy provides the ability to measure transport activity in real time with high temporal and spatial resolution. Using epifluorescence microscopy we characterized the transport of the fluorescent organic cation, [2-(4-nitro-2,1,3-benzoxadiazol-7-yl)aminoethyl]trimethylammonium (NBD-TMA+, MW 266). NBD-TMA+ has structural characteristics common to other secreted organic cations and is fluorescent (lambda(ex)=458 nm; lambda(em)=530 nm). The excitation and emission spectra are insensitive to changes in [Cl-] and minimally sensitive to pH in the physiologically relevant range (pH 5.0-7.4). A microscope equipped with a photon-detection system was used to measure accumulation of NBD-TMA+ by isolated rabbit renal proximal tubules. Accumulation of NBD-TMA+ by proximal tubules was time dependent and saturable (Michaelis-Menten constant Km 12 microM). Proximal tubule accumulation of NBD-TMA+ was inhibited by the organic cations tetraethylammonium (TEA+) (apparent inhibitory constant K(app)TEA 134 microM), cimetidine, and N1-methylnicotinamide (NMN). Our experimental results provide strong evidence that NBD-TMA+ is transported by one or more of the basolateral organic cation transporters involved in the renal secretion of this chemical class of compound. This fluorescent substrate provides a sensitive means of investigating organic cation transport.

Acetate stimulates flux through the tricarboxylic acid cycle in rabbit renal proximal tubules synthesizing glutamine from alanine: a 13C NMR study

Although glutamine synthesis has a major role in the control of acid-base balance and ammonia detoxification in the kidney of herbivorous species, very little is known about the regulation of this process. We therefore studied the influence of acetate, which is readily metabolized by the kidney and whose metabolism is accompanied by the production of bicarbonate, on glutamine synthesis from variously labelled [13C]alanine and [14C]alanine molecules in isolated rabbit renal proximal tubules. With alanine as sole exogenous substrate, glutamine and, to a smaller extent, glutamate and CO2, were the only significant products of the metabolism of this amino acid, which was removed at high rates. Absolute fluxes through the enzymes involved in alanine conversion into glutamine were assessed by using a novel model describing the corresponding reactions in conjunction with the 13C NMR, and to a smaller extent, the radioactive and enzymic data. The presence of acetate (5 mM) led to a large stimulation of fluxes through citrate synthase and α-oxoglutarate dehydrogenase. These effects were accompanied by increases in the removal of alanine, in the accumulation of glutamate and in flux through the anaplerotic enzyme pyruvate carboxylase. Acetate did not alter fluxes through glutamate dehydrogenase and glutamine synthetase; as a result, acetate did not change the accumulation of ammonia, which was negligible under both experimental conditions. We conclude that acetate, which seems to be an important energy-provider to the rabbit renal proximal tubule, simultaneously traps as glutamate the extra nitrogen removed as alanine, thus preventing the release of additional ammonia by the glutamate dehydrogenase reaction.

Acetate stimulates flux through the tricarboxylic acid cycle in rabbit renal proximal tubules synthesizing glutamine from alanine: a 13C NMR study

Although glutamine synthesis has a major role in the control of acid-base balance and ammonia detoxification in the kidney of herbivorous species, very little is known about the regulation of this process. We therefore studied the influence of acetate, which is readily metabolized by the kidney and whose metabolism is accompanied by the production of bicarbonate, on glutamine synthesis from variously labelled [(13)C]alanine and [(14)C]alanine molecules in isolated rabbit renal proximal tubules. With alanine as sole exogenous substrate, glutamine and, to a smaller extent, glutamate and CO(2), were the only significant products of the metabolism of this amino acid, which was removed at high rates. Absolute fluxes through the enzymes involved in alanine conversion into glutamine were assessed by using a novel model describing the corresponding reactions in conjunction with the (13)C NMR, and to a smaller extent, the radioactive and enzymic data. The presence of acetate (5 mM) led to a large stimulation of fluxes through citrate synthase and alpha-oxoglutarate dehydrogenase. These effects were accompanied by increases in the removal of alanine, in the accumulation of glutamate and in flux through the anaplerotic enzyme pyruvate carboxylase. Acetate did not alter fluxes through glutamate dehydrogenase and glutamine synthetase; as a result, acetate did not change the accumulation of ammonia, which was negligible under both experimental conditions. We conclude that acetate, which seems to be an important energy-provider to the rabbit renal proximal tubule, simultaneously traps as glutamate the extra nitrogen removed as alanine, thus preventing the release of additional ammonia by the glutamate dehydrogenase reaction.

Toxicity And Transport Of Three Synthesized Mercury-Thiol-Complexes In Isolated Rabbit Renal Proximal Tubule Suspensions

ABSTRACTPrevious work has suggested that endogenous sulfhydryls, such as glutathione (GSH) and cysteine, are involved in the uptake and toxicity of HgCh2- To study this possibility, uptake and toxicity of synthesized Hg(SG)2, Hg(cysteinylglycine)2 [Hg(CYS-GLY)2] and Hg(CYS)2 were investigated in rabbit renal proximal tubule suspensions (RPT). The intracellular K+ was used as a toxicity indicator, and the mercury content in the tubules was measured by proton induced x-ray emission analysis. The toxicity rank order of the three synthesized mercury-thiol-complexes from the highest to the lowest was: Hg(CYS)2 > Hg(CYS-GLY)2 > Hg(SG)2- However, no significant difference among the mercury contents in the tubules exposed to these synthesized mercury-thiol-complexes was detected. Acivicin (0.25mM), an inhibitor of γ -glutamyltranspeptidase (GGT), decreased the toxicity of Hg(SG)2 in a manner that did not decrease the uptake of mercury in the tubules. This suggests that the toxicity of Hg(SG)2 requires processing to Hg(CYS-GLY)2 or Hg(CYS)2, while Hg(SG)2 may be taken up by the tubules via Na+-dependent GSH transporter since 10 mM acivicin, an inhibitor of this transporter dramatically decreased the uptake of Hg(SG)2. Organic anion transporter plays a minor role, if any, in the toxicity and uptake of Hg(SG)2 and Hg(CYS)2 since p-aminohippuric acid (PAH), an inhibitor of organic anion transporter, did not have significant effect on their uptake and toxicity. L-phenylalanine, an inhibitor of the neutral amino acid decreased the uptake of mercury, but to a lesser extent. This suggested that neutral amino acid transporter seemed to play a role, in part, in the toxicity and uptake of synthesized Hg(CYS)2- In summary, the data suggested that basolateral transport is important for the toxicity of the three synthesized mercury-thiol-complexes, and a variety of mechanisms are involved in the toxicity and uptake of these complexes in isolated rabbit RPT.

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.

Mechanism of anion permeation in the basolateral membrane of isolated rabbit renal proximal tubule S3 segment.

Conventional and double-barreled microelectrodes were used to examine the anion selectivity of Cl- conductance in the basolateral membrane of rabbit renal proximal tubule S3 segment. The permeability sequence determined by anion replacements in the presence of K+ channel blocker quinine was SCN- > I- > Br- > Cl- > gluconate in both nonperfused and luminally perfused tubules. The anion-selective microelectrodes with higher sensitivity to I- enabled us to measure intracellular I- activities. With these electrodes, we could compare the conductive movements of Cl- and I- in response to the increase in bath K+ concentrations and confirmed that the conductance sequence was also I- > Cl-. Although the basolateral potential changes generated by Cl- and Br- currents were stimulated by adenosine 3',5'-cyclic monophosphate (cAMP), the potential changes by SCN- and I- currents were somewhat inhibited by cAMP. In addition, the conductive uptake of I- was, in contrast to that of Cl-, inhibited by cAMP These results are consistent with the existence of at least two distinct anion conductances in this membrane, which are differently regulated by cAMP.

Mechanism of [Ca2+]i increase by extracellular ATP in isolated rabbit renal proximal tubules.

The effects of extracellular ATP on cytosolic Ca2+ concentrations ([Ca2+]i) and cell membrane potentials (Vb) of rabbit renal proximal tubules were investigated using fura 2 and microelectrodes. ATP transiently increased [Ca2+]i without an apparent sustained phase, and the maximum effect was obtained at 10 microM. ADP, adenosine 5'-O-(3-thiotriphosphate), and 2-methylthioadenosine 5'-triphosphate were equally effective as ATP, whereas UTP, adenosine, and alpha beta-methyleneadenosine 5'-triphosphate were far less effective. The [Ca2+]i responses to ATP were strongly inhibited by reactive blue 2, a P2-purinergic receptor antagonist. The removal of extracellular Ca2+ as well as the addition of thapsigargin also markedly attenuated the responses to ATP. In addition, ATP had virtually no effect on Vb, except for the occasional small depolarization by 300 microM ATP. These results indicate that extracellular ATP increases [Ca2+]i through a P2-purinergic receptor and that this effect of ATP is dependent on both extracellular and intracellular Ca2+.

Stable Measurements of Intracellular Calcium Concentrations with Fura-2 in Isolated Rabbit Renal Proximal Tubules

The experiments were done to determine the optimal conditions for measurement of intracellular Ca2+ concentrations ([Ca2+]i) with a fluorescence dye fura-2 in isolated