Membrane Of Renal Proximal Tubules Research Articles

Protein Phosphatase 2A B56α During Development in the Spontaneously Hypertensive Rat

Mistargeting of the regulatory subunit of protein phosphatase 2A (PP2A), B56α is involved in the hyperphosphorylation and desensitization of the D1 dopamine receptor in renal proximal tubules of spontaneously hypertensive rats (SHRs). However, the renal expression of B56α before hypertension develops is not known. Therefore, we studied the expression of B56α and PP2A activity in the kidney during development in the SHR and its normotensive control, the Wistar‐Kyoto (WKY) rat. PP2A B56α was expressed in proximal and distal tubules with no differences in the pattern of expression in WKY and SHRs at any age. In brush border membranes of renal proximal tubules, PP2A B56α protein was greatest in the immature rats and decreased with development. However, PP2A activity did not change with age. PP2A B56α protein and PP2A activity were similar in WKY and SHRs except at 2 weeks when both PP2A B56α protein and PP2A activity were higher in SHRs than in WKY rats. The PP2A catalytic subunit co‐immunoprecipitated with the D1 receptor in renal proximal tubule cells. It is possible that the increased expression of PP2A B56α and increased basal PP2A activity in the young, especially in the SHRs, may serve as a compensatory mechanism in the increased phosphorylation and decreased renal D1 receptor function, including D1‐receptor mediated stimulation in renal proximal tubules of SHRs.

Identification of a Novel Voltage-driven Organic Anion Transporter Present at Apical Membrane of Renal Proximal Tubule

A novel transport protein with the properties of voltage-driven organic anion transport was isolated from pig kidney cortex by expression cloning in Xenopus laevis oocytes. A cDNA library was constructed from size-fractionated poly(A)+ RNA and screened for p-aminohippurate (PAH) transport in high potassium medium. A 1856-base pair cDNA encoding a 467-amino acid peptide designated as OATV1 (voltage-driven organic anion transporter 1) was isolated. The predicted amino acid sequence of OATV1 exhibited 60-65% identity to those of human, rat, rabbit, and mouse sodium-dependent phosphate cotransporter type 1 (NPT1), although OATV1 did not transport phosphate. The homology of this transporter to known members of the organic anion transporter family (OAT family) was about 25-30%. OATV1-mediated PAH transport was affected by the changes in membrane potential. The transport was Na+-independent and enhanced at high concentrations of extracellular potassium and low concentrations of extracellular chloride. Under the voltage clamp condition, extracellularly applied PAH induced outward currents in oocytes expressing OATV1. The current showed steep voltage dependence, consistent with the voltage-driven transport of PAH by OATV1. The PAH transport was inhibited by various organic anions but not by organic cations, indicating the multispecific nature of OATV1 for anionic compounds. This transport protein is localized at the apical membrane of renal proximal tubule, consistent with the proposed localization of a voltage-driven organic anion transporter. Therefore, it is proposed that OATV1 plays an important role to excrete drugs, xenobiotics, and their metabolites driven by membrane voltage through the apical membrane of the tubular epithelial cells into the urine.

The mouse sulfate anion transporter gene Sat1 (Slc26a1): cloning, tissue distribution, gene structure, functional characterization, and transcriptional regulation thyroid hormone.

Sulfate (SO(4)(2-)) is required for bone/cartilage formation and cellular metabolism. sat-1 is a SO(4)(2-) anion transporter expressed on basolateral membranes of renal proximal tubules, and is suggested to play an important role in maintaining SO(4)(2-) homeostasis. As a first step towards studying its tissue-specific expression, hormonal regulation, and in preparation for the generation of knockout mice, we have cloned and characterized the mouse sat-1 cDNA (msat-1), gene (sat1; Slc26a1) and promoter region. msat-1 encodes a 704 amino acid protein (75.4 kDa) with 12 putative transmembrane domains that induce SO(4)(2-) (also oxalate and chloride) transport in Xenopus oocytes. msat-1 mRNA was expressed in kidney, liver, cecum, calvaria, brain, heart, and skeletal muscle. Two distinct transcripts were expressed in kidney and liver due to alternative utilization of the first intron, corresponding to an internal portion of the 5'-untranslated region. The Sat1 gene (~6 kb) consists of 4 exons. Its promoter is ~52% G + C rich and contains a number of well-characterized cis-acting elements, including sequences resembling hormone responsive elements T(3)REs and VDREs. We demonstrate that Sat1 promoter driven basal transcription in OK cells was stimulated by tri-iodothyronine. Site-directed mutagenesis identified an imperfect T(3)RE at -454-bp in the Sat1 promoter to be responsible for this activity. This study represents the first characterization of the structure and regulation of the Sat1 gene encoding a SO(4)(2-)/chloride/oxalate anion transporter.

The organic cation transporters rOCT1 and hOCT2 are inhibited by cGMP.

The electrogenic cation transporters OCT1 and OCT2 in the basolateral membrane of renal proximal tubules mediate the first step during secretion of organic cations. Previously we demonstrated stimulation and change of selectivity for rat OCT1 (rOCT1) by protein kinase C. Here we investigated the effect of cGMP on cation transport by rOCT1 or human OCT2 (hOCT2) after expression in human embryonic kidney cells (HEK293) or oocytes of Xenopus laevis. In HEK293 cells, uptake was measured by microfluorimetry using the fluorescent cation 4-(4-(dimethyl-amino)styryl)-N-methylpyridinium iodide (ASP + ) as substrate, whereas uptake into Xenopus laevis oocytes was measured with radioactively labelled cations. In addition, ASP +-induced depolarizations of membrane voltages (Vm) were measured in HEK293 cells using the slow whole-cell patch-clamp method. Incubation of rOCT1-expressing HEK293 cells for 10 min with 100 mM 8-Br-cGMP reduced initial ASP + uptake by maximally 78% with an IC50 value of 24 +/- 16 mM. This effect was not abolished by the specific PKG inhibitor KT5823, indicating that a cGMP-dependent kinase is not involved. An inhibition of ASP + uptake by rOCT1 in HEK293 cells was also obtained when the cells were incubated for 10 min with 100 mM cGMP, whereas no effect was obtained when cGMP was given together with ASP +. ASP + (100 mM)-induced depolarizations of Vm were reduced in the presence of 8-Br-cGMP (100 mM) by 44 +/- 11% (n = 6). Since it could be demonstrated that [3H]cGMP is taken up by an endogeneous cyanine863-inhibitable transporter, the effect of cGMP is probably mediated from inside the cell. Uptake measurements with [14C]tetraethylammonium and [3H]2-methyl-4-phenylpyridinium in Xenopus laevis oocytes expressing rOCT1 performed in the absence and presence of 8-Br-cGMP showed that cGMP does not interact directly with the transporter. The data suggest that the inhibition mediated by cGMP observed in HEK293 cells occurs most likely via a mammalian cGMP-binding protein that interacts with OCT1-2 transporters.

Na+/H+ exchange activity and NHE-3 expression in renal tubules from the spontaneously hypertensive rat

The NHE-3 isoform of the Na+/H+ antiporter, in the apical membrane of renal proximal tubule, is responsible for the bulk transport of Na+ and fluid reabsorption. Studies have reported that apical NHE-3 translocates to internal pools, thereby facilitating natriuresis when blood pressure increases abruptly. The present study examined Na+/H+ exchange activity and NHE-3 expression in renal cortical tubules from the spontaneously hypertensive rat (SHR) and WKY rats before and after the development of hypertension. SHR 4 to 6 weeks of age were pre-hypertensive, 6 to 7 weeks old had mild hypertension, and 8 to 13 weeks old had severe hypertension. Renal proximal tubules (PTs) were isolated and purified by Percoll gradient centrifugation. NHE-3 protein and mRNA levels were determined by Western and Northern blots, respectively. Apical brush border membrane vesicles (BBMV) were prepared using the MgSO4 aggregation method and Na+/H+ exchange activity assessed using the acridine orange method. Na+/H+ exchange activity, determined as the rate of Na+-dependent intracellular pH (pHi) recovery assessed using BCECF after an acute acid load, was significantly greater in PTs from SHR than in WKY rats at all age groups (4 to 6 weeks, 0.30 +/- 0.04 vs. 0.24 +/- 0.02 pH U/30 sec, P < 0.05; 6 to 7 weeks, 0.42 +/- 0.07 vs. 0.29 +/- 0.05 pH U/30 sec, P < 0.05; and 8 to 13 weeks, 0.48 +/- 0.07 vs. 0.40 +/- 0.07 pH U/30 sec, P < 0.05). The Na+-dependent recovery in BBMV was also greater in SHR than WKY rats (1464 +/- 62 vs. 1042 +/- 79 fluorescence. U/5 sec, P < 0.001) and was unaffected by cariporide, a specific NHE-1 inhibitor. NHE-3 protein levels also were significantly higher in SHR than age-matched WKY rats at all stages during the development of hypertension (pre-hypertensive 1.8-fold; early onset hypertension twofold; established hypertension 1.5-fold; each P < 0.05). By contrast, NHE-3 mRNA levels were not different between SHR and WKY rats at each age group. Na+/H+ exchange activity and NHE-3 protein abundance in renal proximal tubules from the SHR are increased while NHE-3 mRNA is not. A post-transcriptional event(s) best explains the increase in NHE-3 protein expression since mRNA levels were not increased. The alterations in the SHR antedate the development of hypertension and fail to decrease as blood pressure increases with age in the SHR, which likely results in inappropriate renal sodium retention in the face of a chronic rise in blood pressure.

Molecular determinants for apical expression of the renal type IIa Na+/Pi-cotransporter.

Type IIa and IIb Na+/Pi-cotransporters have different patterns of expression in vivo: IIa is expressed in apical membranes of renal proximal tubules, and IIb in intestinal and lung epithelia. They are found in different subcellular locations when transfected in epithelial cells: IIa is apically expressed in renal proximal cells (OK), but mostly intracellularly in intestinal cells (CaCo2); IIb is apical in both cell types. To identify the domains responsible for the different expression of both cotransporters (in CaCo2), as well as those responsible for the apical expression of IIa (in OK), mutated cotransporters were fused to the Enhanced Green Fluorescent Protein (EGFP), and their expression analyzed by confocal microscopy. We conclude that the apical expression information for CaCo2 is contained within the C-terminal tail of IIb, but is not contained within IIa. From analysis of mutated IIa cotransporters we identified residues, within the C-terminal tail, involved in the apical expression of these cotransporters in OK cells: internal PR-residues and terminal TRL-residues. These signals are functional in OK but not in CaCo2-cells, supporting the concept that polarized targeting can be protein and cell specific.

Identification and characterization of human organic anion transporter 3 expressing predominantly in the kidney.

A cDNA encoding a multispecific organic anion transporter 3 (hOAT3) was isolated from a human kidney cDNA library. The hOAT3 cDNA consisted of 2179 base pairs that encoded a 543-amino-acid residue protein with 12 putative transmembrane domains. The deduced amino acid sequence of hOAT3 showed 36 to 51% identity to those of other members of the OAT family. Northern blot analysis revealed that hOAT3 mRNA is expressed in the kidney, brain, and skeletal muscle. When expressed in Xenopus laevis oocytes, hOAT3 mediated the transport of estrone sulfate (K(m) = 3.1 microM), p-aminohippurate (K(m) = 87.2 microM), methotrexate (K(m) = 10.9 microM), and cimetidine (K(m) = 57.4 microM) in a sodium-independent manner. hOAT3 also mediated the transport of dehydroepiandrosterone sulfate, ochratoxin A, PGE(2), estradiol glucuronide, taurocholate, glutarate, cAMP and uric acid. Estrone sulfate did not show any trans-stimulatory effects on either influx or efflux of [(3)H]estrone sulfate via hOAT3. hOAT3 interacted with chemically heterogeneous anionic compounds, such as nonsteroidal anti-inflammatory drugs, diuretics, sulfobromophthalein, penicillin G, bile salts and tetraethyl ammonium bromide. The hOAT3 protein was shown to be localized in the basolateral membrane of renal proximal tubules and the hOAT3 gene was determined to be located on the human chromosome 11q12-q13.3 by fluorescent in situ hybridization analysis. These results suggest an important role of hOAT3 in the excretion/detoxification of endogenous and exogenous organic anions in the kidney.

Effect of Acetaminophen on Na+, K+ ATPase and Alkaline Phosphatase on Plasma Membranes of Renal Proximal Tubules

In previous work we reported that 1 h after acetaminophen (APAP) administration, tubular function remained at control values, while 16 h later a significant deterioration of tubular function was observed. The aim of the present work was to study if APAP induces its renal toxic effects by altering the normal activity of key tubular plasma membrane enzymes. We analyzed the effects of a nephrotoxic dose of APAP (1000 mg/kg b.wt., i.p.) on the activities of the brush-border membrane (BBM) enzyme, alkaline phosphatase, and the basolateral membrane (BLM) enzyme Na+, K+ ATPase 1 h (APAP1h) and 16 h (APAP16h) after dosing. Na+, K+ ATPase abundance in homogenates and each membrane domain were analyzed by Western blot. Cortical adenosine 5′ triphosphate (ATP) content was also evaluated. At each time studied, APAP promoted a diminution of alkaline phosphatase in BBM. Na+, K+ ATPase activity in BLM showed a biphasic response to APAP. One hour after APAP administration it was significantly increased, but it was decreased 16 h after dosing. Na+, K+ ATPase protein abundance was elevated in homogenates, BLM, and BBM after 1 h of APAP dosing. After 16 h, Na+, K+ ATPase abundance was increased in homogenates, while in BLM it was decreased. No differences were observed in cortical ATP content in each time studied. Our present results could contribute to the understanding of the molecular basis of the previously reported time course alteration in the fractional excretion of sodium promoted by a nephrotoxic dose of APAP.

Cellular and molecular biology of the aquaporin water channels.

The high water permeability characteristic of mammalian red cell membranes is now known to be caused by the protein AQP1. This channel freely permits movement of water across the cell membrane, but it is not permeated by other small, uncharged molecules or charged solutes. AQP1 is a tetramer with each subunit containing an aqueous pore likened to an hourglass formed by obversely arranged tandem repeats. Cryoelectron microscopy of reconstituted AQP1 membrane crystals has revealed the three-dimensional structure at 3-6 A. AQP1 is distributed in apical and basolateral membranes of renal proximal tubules and descending thin limbs as well as capillary endothelia. Ten mammalian aquaporins have been identified in water-permeable tissues and fall into two groupings. Orthodox aquaporins are water-selective and include AQP2, a vasopressin-regulated water channel in renal collecting duct, in addition to AQP0, AQP4, and AQP5. Multifunctional aquaglyceroporins AQP3, AQP7, and AQP9 are permeated by water, glycerol, and some other solutes. Aquaporins are being defined in numerous other species including amphibia, insects, plants, and microbials. Members of the aquaporin family are implicated in numerous physiological processes as well as the pathophysiology of a wide range of clinical disorders.

Regulation of an inwardly rectifying ATP-sensitive K+ channel in the basolateral membrane of renal proximal tubule.

Functional coupling of Na+,K+-ATPase pump activity to a basolateral membrane (BLM) K+ conductance is crucial for sustaining transport in the proximal tubule. Apical sodium entry stimulates pump activity, lowering cytosolic [ATP], which in turn disinhibits ATP-sensitive K+ (KATP) channels. Opening of these KATP channels mediates hyperpolarization of the BLM that facilitates Na+ reabsorption and K+ recycling required for continued Na+,K+-ATPase pump turnover. Despite its physiological importance, little is known about the regulation of this channel. The present study focuses on the regulation of the BLM KATP channel by second messengers and protein kinases using membrane patches from dissociated, polarized Ambystoma proximal tubule cells. The channel is regulated by protein kinases A and C, but in opposing directions. The channel is activated by forskolin in cell-attached (c/a) patches, and by PKA in inside-out (i/o) membrane patches. However, phosphorylation by PKA is not sufficient to prevent channel rundown. In contrast, the channel is inhibited by phorbol ester in c/a patches, and PKC decreases channel activity (nPo) in i/o patches. The channel is pH sensitive, and lowering cytosolic pH reduces nPo. Increasing intracellular [Ca2+] ([Ca2+]i) in c/a patches decreases nPo, and this effect is direct since [Ca2+]i inhibits nPo with a Ki of approximately 170 nM in i/o patches. Membrane stretch and hypotonic swelling do not significantly affect channel behavior, but the channel appears to be regulated by the actin cytoskeleton. Finally, the activity of this BLM KATP channel is coupled to transcellular transport. In c/a patches, maneuvers that inhibit turnover of the Na+,K+-ATPase pump reduce nPo, presumably due to a rise in intracellular [ATP], although the associated cell depolarization cannot be ruled out as the possible cause. Conversely, stimulation of transport (and thus pump turnover) leads to increases in nPo, presumably due to a fall in intracellular [ATP]. These results show that the inwardly rectifying KATP channel in the BLM of the proximal tubule is a key element in the feedback system that links cellular metabolism with transport activity. We conclude that coupling of this KATP channel to the activity of the Na+,K+-ATPase pump is a mechanism by which steady state NaCl reabsorption in the proximal tubule may be maintained.

Properties of an inwardly rectifying ATP-sensitive K+ channel in the basolateral membrane of renal proximal tubule.

The potassium conductance of the basolateral membrane (BLM) of proximal tubule cells is a critical regulator of transport since it is the major determinant of the negative cell membrane potential and is necessary for pump-leak coupling to the Na+,K+-ATPase pump. Despite this pivotal physiological role, the properties of this conductance have been incompletely characterized, in part due to difficulty gaining access to the BLM. We have investigated the properties of this BLM K+ conductance in dissociated, polarized Ambystoma proximal tubule cells. Nearly all seals made on Ambystoma cells contained inward rectifier K+ channels (gammaslope, in = 24.5 +/- 0.6 pS, gammachord, out = 3.7 +/- 0.4 pS). The rectification is mediated in part by internal Mg2+. The open probability of the channel increases modestly with hyperpolarization. The inward conducting properties are described by a saturating binding-unbinding model. The channel conducts Tl+ and K+, but there is no significant conductance for Na+, Rb+, Cs+, Li+, NH4+, or Cl-. The channel is inhibited by barium and the sulfonylurea agent glibenclamide, but not by tetraethylammonium. Channel rundown typically occurs in the absence of ATP, but cytosolic addition of 0. 2 mM ATP (or any hydrolyzable nucleoside triphosphate) sustains channel activity indefinitely. Phosphorylation processes alone fail to sustain channel activity. Higher doses of ATP (or other nucleoside triphosphates) reversibly inhibit the channel. The K+ channel opener diazoxide opens the channel in the presence of 0.2 mM ATP, but does not alleviate the inhibition of millimolar doses of ATP. We conclude that this K+ channel is the major ATP-sensitive basolateral K+ conductance in the proximal tubule.

INCREASED SERINE-PHOSPHORYLATION OF THE D1A RECEPTOR IN RENAL PROXIMAL TUBULE CELLS IN SPONTANEOUS HYPERTENSION. • 2218

We have suggested that a defect in the coupling of the D1 receptor to G-protein/enzyme complex in renal proximal tubules is involved in the pathogenesis of spontaneous hypertension. The D1A receptor subtype may be involved since hypertension develops in mice mutant for the D1A receptor allele. However, we have found no mutation of the D1A receptor gene in the spontaneously hypertensive rat (SHR) or Dahl salt-sensitive rat suggesting that the D1A receptor defect may be due to post-translational modification. Since the renal D1 receptor resembles the phenomenon of desensitization, and desensitization occurs as a result of receptor phosphorylation, we studied the phosphorylation of the D1A receptor in renal proximal tubules from SHR and its normotensive control, the Wistar-Kyoto rat (WKY). Vehicle alone or fenoldopam, a D1 agonist, 1μg/kg/min, was infused for 5-10′ into the renal artery via the right suprarenal artery of anesthetized rats. Renal cortical homogenates (n=5) were then immunoprecipitated with anti-phosphoserine antibodies and immunoblotted with anti D1A antibodies. Basal serine-phosphorylated D1A receptor was greater in SHR (21.0±1.5 density units, DU) than in WKY(7.4±2.9 DU) (P<0.01 t-test); the D1 agonist increased serine-phosphorylated D1A receptor in WKY (55.0±2.8 DU) but not in SHR (17.0±6.3 DU) (P<0.01 t-test) (n=5/group). The“hyper” phosphorylation of the D1A receptor in the SHR was not the result of homologous desensitization because similar studies were obtained when the rats (n=3) were injected with carbidopa (40 μmol/kg) 24, 18, and 1 hr prior to study to decrease renal dopamine production. Moreover, the decreased ability of D1 agonist to stimulate adenylyl cyclase and phospholipase C (n=5-8) and the “hyper” phosphorylation (n=3) of the D1A receptor persist in immortalized proximal tubular cells from SHR (dopamine synthesis does not occur because of the absence of DOPA, the substrate for renal dopamine synthesis). The mechanism of “hyper” phosphorylation of the D1A receptor in renal proximal tubules from SHR is not known. However, we have recently found that protein phosphatase 2A activity in cytosol but not in membranes of renal proximal tubules is decreased in the SHR compared to the WKY; protein phosphatases are important in the resensitization process. We conclude that a defect in the post-translational modification of the D1A receptor is important in the pathogenesis of spontaneous hypertension.

The aquaporin family of water channels in kidney

The longstanding puzzle of membrane water permeability was advanced by the discovery of channel-forming integral protein (CHIP). This protein was shown to function as a water channel when expressed in Xenopus oocytes or when reconstituted into synthetic membranes. Site-directed mutagenesis and electron crystallography reveal tetrameric organization of CHIP, and the two halves of CHIP are tandem repeats folded into an obversely symmetric structure which resembles an hourglass. Each tetramer is comprised of functionally independent subunits. CHIP is the archetypal member of a newly-recognized family of membrane water transporters known as the "Aquaporins" (AQPs). AQP1 (CHIP) is abundant in the apical and basolateral membranes of renal proximal tubules and descending thin limbs, and is also present in a number of extra renal tissues. In the collecting duct, AQP2 is the predominant vasopressin-sensitive water channel. AQP2 is localized in the apical membrane and in intracellular vesicles which are targeted to the apical plasma membranes when stimulated by antidiuretic hormone. Humans are identified with mutations in AQP1 and AQP2 and exhibit contrasting clinical phenotypes. AQP3 resides in the basolateral membranes of collecting duct principal cells providing an exit pathway for water, and AQP4 is abundant in brain, where it apparently functions as the hypothalamic osmoreceptor responsible for secretion of antidiuretic hormone. Continued analysis of the aquaporins is providing detailed molecular insight into the fundamental physiological problems of water balance and water balance disorders.

Drug excretion mediated by a new prototype of polyspecific transporter.

Cationic drugs of different types and structures (antihistaminics, antiarrhythmics, sedatives, opiates, cytostatics and antibiotics, for example) are excreted in mammals by epithelial cells of the renal proximal tubules and by hepatocytes in the liver. In the proximal tubules, two functionally disparate transport systems are involved which are localized in the basolateral and luminal plasma membrane and are different from the previously identified neuronal monoamine transporters and ATP-dependent multidrug exporting proteins. Here we report the isolation of a complementary DNA from rat kidney that encodes a 556-amino-acid membrane protein, OCT1, which has the functional characteristics of organic cation uptake over the basolateral membrane of renal proximal tubules and of organic cation uptake into hepatocytes. OCT1 is not homologous to any other known protein and is found in kidney, liver and intestine. As OCT1 translocates hydrophobic and hydrophilic organic cations of different structures, it is considered to be a new prototype of polyspecific transporters that are important for drug elimination.

A putative cytoprotective receptor in the kidney: Relation to the neuronal strychinine-sensitive glycine receptor

The neutral amino acid glycine has been demonstrated to prevent cell death in numerous cell types exposed to a variety of toxic insults. Recently, the central nervous system (CNS) glycine antagonist strychine was demonstrated to bind specifically to the plasma membrane of renal proximal tubules (RPT) and mimic glycine cytoprotection. Further, it has been demonstrated in RPT that glycine and strychnine block chloride influx in the late stages of cell injury. The aim of this study was to determine if the RPT cytoprotective site is related to neuronal glycine receptors. Only antagonists tot he CNS strychnine-sensitive glycine receptor (strychnine, brucine), and not antagonists to the glycine modulatory site of the NMDA receptor (DCQX, 7-CKA, HA-966) or the GABA A receptor (bicuculline methiodide, picrotoxin), prevented mitochondrial inhibitor (antimycin A)-induced RPT cell death. Using immunoblot analysis, proteins corresponding to the 58kDa β-subunit of the strychnine-sensitive glycine receptor and the associated protein gephyrin were identified in rabbit kidney cortical membrane fractions and RPT. No protein corresponding to the 48 kDa α-subunit was identified. Thus, glycine and strychnine may exert their cytoprotective effects via a putative plasma membrane receptor that is related to the strychnine-sensitive glycine receptor found in the CNS.

NHE3: a Na+/H+ exchanger isoform of renal brush border.

Na+/H+ exchangers in the brush-border (luminal, apical) membrane of renal proximal tubules are responsible for active, transcellular reabsorption of NaHCO3 and NaCl. Although well characterized kinetically, the protein that mediates Na+/H+ exchange in the renal brush border has not been identified. Several Na+/H+ exchanger genes, including NHE1, NHE2, NHE3, and NHE4, are expressed in the kidney. To identify the NHE3 gene product and to determine its cellular and subcellular localization in the rabbit kidney, an NHE3-isoform-specific antibody was prepared. Guinea pigs were immunized with purified fusion protein containing the carboxy-terminal 40 amino acids of NHE3 (fpNHE3-C40). After affinity purification, immune sera demonstrated specific reactivity to the NHE3 sequence within the fusion protein as well as to an 80-kDa polypeptide expressed in NHE3-transfected LAP1 cells. Western blot analysis showed that anti-fpNHE3-C40 specifically reacted with an 80-kDa protein that is relatively enriched in renal brush-border membrane compared with basolateral membrane. Immunocytochemical studies confirmed that the Na+/H+ exchanger isoform NHE3 is expressed along the microvillar membrane of the brush border of proximal tubule cells in the rabbit kidney.

Monoclonal antibodies against luminal membranes of renal proximal tubules which are kidney-specific

After immunization with porcine brush-border membrane proteins, 11 monoclonal antibodies were generated which react with proximal tubules. Their antigenic polypeptides were characterized with respect to apparent molecular weight, histochemical localization in porcine and human kidney, and tissue distribution in pig. In porcine kidney, six antibodies bind selectively to the proximal tubule whereas the others also react with other nephron segments. With the exception of one antibody which reacts with the luminal and the basolateral membrane of the porcine proximal tubule, the other antibodies specific for the proximal tubule only stain the brush-border membrane. Four of them react along the entire length of the porcine proximal tubule, whereas one (R1A2) binds to the S3-segment in pig and to the entire length of the proximal tubule in man. This indicates that segment-specific expression may be species-dependent. Testing the antibodies in 21 different extrarenal tissues it was found that three of the antibodies, specific for the brush-border membrane in renal proximal tubules, only react in kidney. Two of these are specific for pig kidney whereas one also reacts with human kidney. This antibody (N4A4) is directed against a polypeptide with an apparent molecular weight of 400 000. Electron microscopic immunohistochemistry showed that N4A4 binds to the intervillus region of the brush-border membrane and to subapical vesicles.

Stretch- and volume-activated channels in isolated proximal tubule cells.

Apical and basolateral channels were studied in isolated proximal tubule cells of Necturus kidney. Many of these isolated cells maintained their polarity, with clearly delineated apical and basolateral regions. A 20-pS stretch-activated (SA) cation-selective channel was identified at the apical side of these cells. This channel was permeable to Ca, K, and Na but was not significantly gated by either membrane potential or cytosolic Ca. Negative pipette pressure (15 cmH2O) increased the open probability (Po) of this channel from 0.04 +/- 0.02 to 0.26 +/- 0.08 (n = 6). Two types of Ca-independent, mechanosensitive, K-selective (SAK) channels were identified at the basolateral surface of polarized proximal tubule cells, i.e., a 30-pS long-open time (50 +/- 7 ms) channel (n = 9), and a 46-pS short-open time (1.3 +/- 0.7 ms) channel (n = 10). Pipette suction (-12 cmH2O) increased the Po of the short-open time channels from 0.008 to 0.015 and increased the Po of the long-open time channel from 0.03 to 0.19. The effect of swelling was studied with isolated cells suspended at the tip of patch pipettes. A 50% dilution of the bath doubled cell volume, hyperpolarized the membrane potential by 11 +/- 0.7 mV, and increased the Po of the basolateral SAK channels. This was followed by a spontaneous regulatory volume decrease (RVD), repolarization of the membrane potential, and a decrease in Po. In contrast, isosmotic (bath side) replacement of an impermeant anion (methanesulfonate) with a permeant anion (Cl) doubled cell volume in 5 min but without a subsequent RVD. This sustained swelling hyperpolarized the cell potential by 5.5 +/- 0.7 mV (n = 16) and increased the Po of short-open time channel by a factor of 2.3 from 0.03 +/- 0.01 to 0.07 +/- 0.02 (n = 6). The increase in Po was primarily produced by a reduction in the interburst closed time, which decreased from 142 +/- 43 ms in K methanesulfonate to 36 +/- 11 ms in KCl solutions. These results are consistent with the hypothesis that cell swelling activates Ca-independent K channels at the basolateral membrane of renal proximal tubule. Efflux of K through these channels may partially mediate renal cell volume regulation.

Radiation target sizes of the Na,K-ATPase and p-aminohippurate transport system in the basolateral membrane of renal proximal tubule

Basolateral membrane vesicles made from rabbit kidney proximal tubules were frozen and irradiated with a high energy electron beam and the effects of irradiation on Na,K-ATPase activity, p-aminohippurate (PAH) transport, the membrane diffusion barrier and vesicle volume were measured. The vesicle volume and diffusion barrier were not significantly changed by radiation exposure. Na,K-ATPase activity was inactivated as a simple exponential function of radiation dose. Target size analysis of the data yielded a molecular size of 267 ± 17 kDa, consistent with its existence as a (αβ) 2 dimer. The carrier-mediated PAH uptake by basolateral membrane vesicles was also inactivated as a function of radiation dose. A target molecular size of 74 ± 16 kDa was calculated for the PAH transport system. This study is the first measurement of the functional size of the organic acid transport system based directly on flux measurements.

Potassium permeability of luminal and peritubular membranes in the proximal tubule of bullfrog kidneys.

Using double-barreled K(+)-selective or conventional voltage microelectrode, K+ permeability of the luminal membrane of renal proximal tubule was investigated in comparison with that of the peritubular membrane in doubly-perfused bullfrog kidneys. A decade change in K+ concentration of the luminal perfusate from 3.5 (control) to 35 mM (high K+) induced depolarization in the luminal membrane potential by 11.2 mV with a slight elevation of intracellular K+ activity, (K)i, while those of the peritubular perfusate depolarized peritubular membrane potential by 31.6 mV with a moderate rise of (K)i by 8.0 mEq. The transport number for K+ (tK) in the luminal membrane was about one-third of that in the peritubular membrane. Both luminal and peritubular administrations of Ba2+ (4 mM) additively depolarized membrane potential. The magnitude of Ba2(+)-induced depolarization in the luminal membrane was about one-fourth of that in the peritubular membrane. The high K(+)-induced depolarization elicited from one-side perfusion was hardly affected by Ba2+ which was given from the other side. From these results we conclude that the transport number of K+ in the luminal and peritubular membranes of the proximal tubular epithelium is in the range of 0.17-0.32 and 0.52-0.66, respectively.