Direct fluorescence measurement of diffusional water permeability in the vasopressin-sensitive kidney collecting tubule.

A fluorescence method has been developed for accurate and instantaneous measurement of transepithelial diffusional water permeability (Pd) in perfused kidney tubules based on the sensitivity of the fluorophore aminonapthelane trisulfonic acid (ANTS) to solution H2O/D2O content. The fluorescence of ANTS was 3.2-fold lower in an H2O buffer than in a D2O buffer. The response of ANTS fluorescence to a change in solution H2O/D2O content occurred in less than 1 ms and was due to a collisional quenching mechanism. Isolated cortical (CCT) and outer medullary (OMCT) collecting tubules from rabbit were perfused with an isosmotic D2O buffer at specified lumen flow rates (2-100 nl/min); tubules were bathed in isosmotic H2O or D2O buffers in which vasopressin (VP) could be added rapidly. Lumen fluorescence was monitored by quantitative epifluorescence microscopy at 380 +/- 5 nm excitation and greater than 530 emission wavelengths. Pd was determined from tubule geometry, lumen flow, ANTS fluorescence, and ANTS fluorescence vs. H2O/D2O calibration relation. The instrument response time for a change in bath H2O/D2O content was less than 4 s. At 37 degrees C, Pd values (mean +/- SE in cm/s x 10(4] were 6.4 +/- 1.0 (-VP, n = 9) and 14.3 +/- 1.1 (+250 microU/ml bath VP, n = 9) in the CCT, and 5.8 +/- 1.0 (-VP, n = 6) and 15.3 +/- 2.0 (+VP, n = 6) in the OMCT; at 23 degrees C, Pd was 5.1 +/- 0.6 (-VP, n = 4) and 7.8 +/- 0.6 (+VP, n = 4) in the CCT.(ABSTRACT TRUNCATED AT 250 WORDS)     

Solvent drag measurement of transcellular and basolateral membrane NaCl reflection coefficient in kidney proximal tubule

The NaCl reflection coefficient in proximal tubule has important implications for the mechanisms of near isosmotic volume reabsorption. A new fluorescence method was developed and applied to measure the transepithelial (sigma NaClTE) and basolateral membrane (sigma NaClcl) NaCl reflection coefficients in the isolated proximal straight tubule from rabbit kidney. For sigma NaClTE measurement, tubules were perfused with buffers containing 0 Cl, the Cl-sensitive fluorescent indicator 6-methoxy-N-[3-sulfopropyl] quinolinium and a Cl-insensitive indicator fluorescein sulfonate, and bathed in buffers of differing cryoscopic osmolalities containing NaCl. The transepithelial Cl gradient along the length of the tubule was measured in the steady state by a quantitative ratio imaging technique. A mathematical model based on the Kedem-Katchalsky equations was developed to calculate the axial profile of [Cl] from tubule geometry, lumen flow, water (Pf) and NaCl (PNaCl) permeabilities, and sigma NaClTE. A fit of experimental results to the model gave PNaCl = (2.25 +/- 0.2) x 10(-5) cm/s and sigma NaClTE = 0.98 +/- 0.03 at 23 degrees C. For measurement of sigma NaClbl, tubule cells were loaded with SPQ in the absence of Cl. NaCl solvent drag was measured from the time course of NaCl influx in response to rapid (less than 1 s) Cl addition to the bath solution. With bath-to-cell cryoscopic osmotic gradients of 0, -60, and +30 mosmol, initial Cl influx was 1.23, 1.10, and 1.25 mM/s; a fit to a mathematical model gave sigma NaClbl = 0.97 +/- 0.04. These results indicate absence of NaCl solvent drag in rabbit proximal tubule. The implications of these findings for water and NaCl movement in proximal tubule are evaluated.     

ULTRASTRUCTURAL STUDIES OF VASOPRESSIN EFFECT ON ISOLATED PERFUSED RENAL COLLECTING TUBULES OF THE RABBIT

Isolated cortical collecting tubules from rabbit kidney were studied during perfusion with solutions made either isotonic or hypotonic to the external bathing medium. Examination of living tubules revealed a reversible increase in thickness of the cellular layer, prominence of lateral cell membranes, and formation of intracellular vacuoles during periods of vasopressin-induced osmotic water transport. Examination in the electron microscope revealed that vasopressin induced no changes in cell structure in collecting tubules in the absence of an osmotic difference and significant bulk water flow across the tubule wall. In contrast, tubules fixed during vasopressin-induced periods of high osmotic water transport showed prominent dilatation of lateral intercellular spaces, bulging of apical cell membranes into the tubular lumen, and formation of intracellular vacuoles. It is concluded that the ultrastructural changes are secondary to transepithelial bulk water flow and not to a direct effect of vasopressin on the cells, and that vasopressin induces osmotic flow by increasing water permeability of the luminal cell membrane. The lateral intercellular spaces may be part of the pathway for osmotically induced transepithelial bulk water flow.     

Calciumdependent mechanisms in vasopressin regulation of osmotic water permeability in the mouse kidney collecting duct cells

In the study, the role of PKC and Ca++ in vasopressin regulation of the plasma membrane water permeability was studied in the cells of the mouse kidney collecting duct. Coefficient of osmotic water permeability of total cell surface (Pf) was calculated from the initial rate of cell swelling following the osmotic shock caused by changing the medium osmolarity from isotonic to hypotonic (300 mOsm to 200 mOsm). Desmopressin (dDAVP 1 nM) increased the Pf in hydrated mice from 168.4 +/- 11.8 microm/s up to 231.3 +/- 14.7 microm/s. The Ca++ chelator BAPTA prevented the desmopressin-induced increase in water permeability. Inhibition of PKC (Ro-31-8220 0.1 microM) also abolished the desmopressin-stimulated increase of plasma membrane water permeability, whereas inhibitor of PKC alone did not suppress the stimulation of the water permeability by db-cAMP. The PKC activity and calciumdependent second messengers seem to be important for regulation of water permeability by vasopressin.     

Water permeabilities and salt reflection coefficients of luminal, basolateral and intracellular membrane vesicles isolated from rabbit kidney proximal tubule

The mechanisms of water transport across the rabbit renal proximal convoluted tubule were approached by measuring osmotic permeabilities and solute reflection coefficients of the brush-border and the basolateral membranes. Plasma and intracellular membrane vesicles were isolated from rabbit renal cortex by centrifugation on a Percoll gradient. Three major turbidity bands were obtained: a fraction of purified basolateral membranes (BLMV), the two others being brush-border (BBMV) and endoplasmic reticulum (ERMV) membrane vesicles. The osmotic permeability (Pf) of the three types of vesicle was measured using stop-flow techniques and their geometry was determined by quasi-elastic light scattering. Pf was equal to 123 +/- 8 microns/s (n = 10) for BBMV, 166 +/- 10 microns/s (n = 10) for BLMV and 156 +/- 9 microns/s (n = 4) for ERMV (T = 26 degrees C). A transcellular water permeability, per unit of apical surface area, of 71 microns/s was calculated considering that the luminal and the basolateral membranes act as two conductances in series. This value is in close agreement, after appropriate normalizations, with previously reported transepithelial water permeabilities obtained using in vitro microperfusion techniques thus supporting the hypothesis of a predominantly transcellular route for water flow across rabbit proximal convoluted tubule. The addition of 0.4 mM HgCl2, a sulfhydryl reagent, decreased Pf about 60% in three types of membrane providing evidence for the existence of proteic pathways. NaCl and KCl reflection coefficients were measured and found to be close to one for plasma and intracellular membranes suggesting that the water channels are not shared by salts.     

Very high water permeability in vasopressin-induced endocytic vesicles from toad urinary bladder

The regulation of transepithelial water permeability in toad urinary bladder is believed to involve a cycling of endocytic vesicles containing water transporters between an intracellular compartment and the cell luminal membrane. Endocytic vesicles arising from luminal membrane were labeled selectively in the intact toad bladder with the impermeant fluid-phase markers 6-carboxyfluorescein (6CF) or fluorescein-dextran. A microsomal preparation containing labeled endocytic vesicles was prepared by cell scraping, homogenization, and differential centrifugation. Osmotic water permeability was measured by a stopped-flow fluorescence technique in which microsomes containing 50 mM mannitol, 5 mM K phosphate, pH 8.5 were subject to a 60-mM inwardly directed gradient of sucrose; the time course of endosome volume, representing osmotic water transport, was inferred from the time course of fluorescence self-quenching. Endocytic vesicles were prepared from toad bladders with hypoosmotic lumen solution treated with (group A) or without (group B) serosal vasopressin at 23 degrees C, and bladders in which endocytosis was inhibited by treatment with vasopressin at 0-2 degrees C (group C), or with vasopressin plus sodium azide at 23 degrees C (group D). Stopped-flow results in all four groups showed a slow rate of 6CF fluorescence decrease (time constants 1.0-1.7 s for exponential fit) indicating a component of nonendocytic 6CF entrapment into sealed vesicles. However, in vesicles from group A only, there was a very rapid 6CF fluorescence decrease (time constant 9.6 +/- 0.2 ms, SEM, 18 separate preparations) with an osmotic water permeability coefficient (Pf) of greater than 0.1 cm/s (18 degrees C) and activation energy of 3.9 +/- 0.8 kcal/mol (16 kJ/mol). Pf was inhibited reversibly by greater than 60% by 1 mM HgCl2. The rapid fluorescence decrease was absent in vesicles in groups B, C, and D. These results demonstrate the presence of functional water transporters in vasopressin-induced endocytic vesicles from toad bladder, supporting the hypothesis that water channels are cycled to and from the luminal membrane and providing a functional marker for the vasopressin-sensitive water channel. The calculated Pf in the vasopressin-induced endocytic vesicles is the highest Pf reported for any biological or artificial membrane.     

Membrane water and solute permeability determined quantitatively by self-quenching of an entrapped fluorophore

Quantitative determination of rapid water and solute transport and solute reflection coefficients by light-scattering methods is complicated by dependence of vesicle or cell light scattering on nonvolume factors including solution refractive index, cell motion, and membrane aggregation. To overcome these difficulties, a fluorescence technique has been developed to measure accurately (1) osmotic water permeability (Pf), (2) solute permeability (Ps), and (3) solute reflection coefficient (sigma). The time course of vesicle volume is determined by the self-quenching of entrapped fluorescein sulfonate (FS), the best of a series of dyes screened for self-quenching, brightness, and vesicle loading/trapping. To validate the method, rabbit renal brush border vesicles (BBV) were loaded with 1-10 mM FS for 12 h at 4 degrees C and washed to remove extravesicular FS. FS leakage occurred over greater than 6 h at 4 degrees C and greater than 30 min at 23 degrees C. FS fluorescence vs vesicle volume was calibrated from the time course of fluorescence decrease (excitation 470 nm, emission greater than 515 nm) in response to a series of inward osmotic gradients in a stopped-flow apparatus. At 23 degrees C Pf was 0.005 +/- 0.001 cm/s, independent of osmotic gradient size, and inhibited 67% by 0.5 mM HgCl2. Urea Ps was 2 x 10(-6) cm/s with sigma 0.95-1.00 on the basis of the fluorescence time course analysis and the extravesicular [urea] required to obtain zero initial volume flow (null method) when vesicles were loaded with sucrose.(ABSTRACT TRUNCATED AT 250 WORDS)     

Functional colocalization of water channels and proton pumps in endosomes from kidney proximal tubule

The apical membrane of mammalian proximal tubule undergoes rapid membrane cycling by exocytosis and endocytosis. Osmotic water and ATP- driven proton transport were measured in endocytic vesicles from rabbit and rat proximal tubule apical membrane labeled in vivo with the fluid phase marker fluorescein-dextran. Osmotic water permeability (Pf) was determined from the time course of fluorescein-dextran fluorescence after exposure of endosomes to an inward osmotic gradient in a stopped- flow apparatus. Pf was 0.009 (rabbit) and 0.029 cm/s (rat) (23 degrees C) and independent of osmotic gradient size. Pf in rabbit endosomes was inhibited reversibly by HgCl2 (KI = 0.2 mM) and had an activation energy of 6.4 +/- 0.5 kcal/mol (15-35 degrees C). Endosomal proton ATPase activity was measured from the time course of internal pH, measured by fluorescein-dextran fluorescence, after the addition of external ATP. Endosomes contained an ATP-driven proton pump that was sensitive to N-ethylmaleimide and insensitive to vanadate and oligomycin. In response to saturating [ATP] the pump acidified the endosomal compartment at a rate of 0.17 (rat) and 0.029 pH unit/s (rabbit); at an external pH of 7.4, the steady-state pH was 6.4 (rat) and 6.5 (rabbit). To examine whether water channels and the proton ATPase were present in the same endosome, the time course of fluorescein-dextran fluorescence was measured in response to an osmotic gradient in the presence and absence of ATP. ATP did not alter endosome Pf, but decreased the amplitude of the fluorescence signal by 43 +/- 3% (rabbit) and 47 +/- 4% (rat).(ABSTRACT TRUNCATED AT 250 WORDS)     

Tetrameric assembly of CHIP28 water channels in liposomes and cell membranes: a freeze-fracture study

Channel forming integral protein of 28 kD (CHIP28) functions as a water channel in erythrocytes, kidney proximal tubule and thin descending limb of Henle. CHIP28 morphology was examined by freeze-fracture EM in proteoliposomes reconstituted with purified CHIP28, CHO cells stably transfected with CHIP28k cDNA, and rat kidney tubules. Liposomes reconstituted with HPLC-purified CHIP28 from human erythrocytes had a high osmotic water permeability (Pf0.04 cm/s) that was inhibited by HgCl2. Freeze-fracture replicas showed a fairly uniform set of intramembrane particles (IMPs); no IMPs were observed in liposomes without incorporated protein. By rotary shadowing, the IMPs had a diameter of 8.5 +/- 1.3 nm (mean +/- SD); many IMPs consisted of a distinct arrangement of four smaller subunits surrounding a central depression. IMPs of similar size and appearance were seen on the P-face of plasma membranes from CHIP28k-transfected (but not mock-transfected) CHO cells, rat thin descending limb (TDL) of Henle, and S3 segment of proximal straight tubules. A distinctive network of complementary IMP imprints was observed on the E-face of CHIP28-containing plasma membranes. The densities of IMPs in the size range of CHIP28 IMPs, determined by non-linear regression, were (in IMPs/microns 2): 2,494 in CHO cells, 5,785 in TDL, and 1,928 in proximal straight tubules; predicted Pf, based on the CHIP28 single channel water permeability of 3.6 x 10(-14) cm3/S (10 degrees C), was in good agreement with measured Pf of 0.027 cm/S, 0.075 cm/S, and 0.031 cm/S, respectively, in these cell types. Assuming that each CHIP28 monomer is a right cylindrical pore of length 5 nm and density 1.3 g/cm3, the monomer diameter would be 3.2 nm; a symmetrical arrangement of four cylinders would have a greatest diameter of 7.2 nm, which after correction for the thickness of platinum deposit, is similar to the measured IMP diameter of approximately 8.5 nm. These results provide a morphological signature for CHIP28 water channels and evidence for a tetrameric assembly of CHIP28 monomers in reconstituted proteoliposomes and cell membranes.     

Expression of mRNA coding for kidney and red cell water channels in Xenopus oocytes

The existence and identity of protein water transporters in biological membranes has been uncertain. Osmotic water permeability (Pf) was measured in defolliculated Xenopus oocytes microinjected with water or mRNA from kidney cortex, kidney papilla, reticulocyte, brain, and muscle. Pf was measured by quantitative image analysis from the time course of oocyte swelling in response to an osmotic gradient. When assayed at 10 degrees C, Pf in water-injected oocytes increased from (3.6 +/- 0.9) x 10(-4) cm/s (S.D., n = 16) to 74 x 10(-4) cm/s with addition of amphotericin B, showing absence of unstirred layers. At 48-72 h after injection of 50 ng of unfractionated mRNA, Pf (in cm/s x 10(-4] was: 4.0 +/- 1.5 (rabbit brain, n = 15), 4.2 +/- 1.8 (rabbit muscle, n = 10), 18.4 +/- 6.3 (rabbit reticulocyte, n = 20), 16.1 +/- 5.6 (rat renal papilla, n = 24), 12.9 +/- 6.3 (rat renal cortex, n = 20), 14.4 +/- 6.1 (rabbit renal papilla, n = 15), and 11.8 +/- 3.4 (rabbit renal cortex, n = 8). In oocytes injected with mRNA from rat renal papilla, Pf was inhibited reversibly by 0.3 mM HgCl2 (4.1 +/- 1.6, n = 10); expressed water channels from kidney and red cell had activation energies of less than 4 kcal/mol. These results show functional oocyte expression of water channels from red cell, kidney proximal tubule (cortex), and the vasopressin-sensitive kidney collecting tubule (papilla), indicating that water channels are proteins, and providing an approach for the expression cloning of water channels.     

High channel-mediated water permeability in rabbit erythrocytes: characterization in native cells and expression in Xenopus oocytes.

Erythrocytes from several mammalian species contain mercurial-sensitive water transporters. By a stopped-flow light scattering technique, osmotic water permeability (Pf) was exceptionally high in rabbit erythrocytes (0.053 +/- 0.002 cm/s) and reversibly inhibited by 98% by p-(chloromercuri)benzenesulfonate (pCMBS). The activation energy (Ea) was 4.6 kcal/mol (15-37 degrees C). pCMBS inhibition was half-maximal at 0.1 mM (60-min incubation); at 1 mM pCMBS, half-maximal inhibition occurred in 8 min. Pf was also inhibited by HgCl2 and pCMB with greater than 90% inhibition in 5 min. There was no inhibition by high concentrations of phloretin, DNDS, cytochalasin B, amiloride, ouabain, furosemide, and several proteases. In defolliculated Xenopus oocytes microinjected with 50 nL of water or unfractionated mRNA (1 mg/mL) from rabbit reticulocytes, oocyte Pf assayed at 10 degrees C after 72-h incubation increased from (4 +/- 1) X 10(-4) cm/s (water injected) to (18 +/- 2) X 10(-4) cm/s (mRNA injected). Pf increased linearly with [mRNA] (0-75 ng/oocyte) and was inhibited slowly and reversibly by pCMBS and immediately by HgCl2 but not by cytochalasin B, phloretin, or DNDS. Ea was 9.6 kcal/mol (water injected) and 2.6 kcal/mol (mRNA injected). These results demonstrate that rabbit erythrocytes have the highest Pf and the greatest percentage inhibition of Pf by mercurials of any mammalian erythrocyte studied. The characteristics of the expressed and native water channels were similar, suggesting that the erythrocyte water channel is a membrane protein suitable for expression cloning.     

Effect of dehydration and dDAVP on water permeability of basolateral membranes of epithelial cells in the kidney collecting tubules

Water permeability of the outer medullary collecting duct's (OMCD) basolateral membrane was determined in vitro in the tubules isolated from hyperhydrated or dehydrated Wistar rats. Oil was injected into the lumen to block apical membrane water permeability. OMCD fragments underwent a hypoosmic shock (600/300 mOsm) and epithelial cells volume increased ad recorded with a digital camera. The latter's rate was used to calculate apparent water permeability of the membrane (Pf). Treatment of the tubules with Hg2Cl2 suppressed the water permeability. Water deprivation and dDAVP induced an increase in the basolateral water permeability. The data obtained suggest that the water permeability of the OMCD basolateral membrane may be stimulated by vasopressin and water deprivation.     

Maturation of rat proximal tubule chloride permeability

We have previously shown that neonate rabbit tubules have a lower chloride permeability but comparable mannitol permeability compared with adult proximal tubules. The surprising finding of lower chloride permeability in neonate proximals compared with adults impacts net chloride transport in this segment, which reabsorbs 60% of the filtered chloride in adults. However, this maturational difference in chloride permeability may not be applicable to other species. The present in vitro microperfusion study directly examined the chloride and mannitol permeability using in vitro perfused rat proximal tubules during postnatal maturation. Whereas there was no maturational change in mannitol permeability, chloride permeability was 6.3 +/- 1.3 x 10(-5) cm/s in neonate rat proximal convoluted tubule and 16.1 +/- 2.3 x 10(-5) cm/s in adult rat proximal convoluted tubule (P < 0.01). There was also a maturational increase in chloride permeability in the rat proximal straight tubule (5.1 +/- 0.6 x 10(-5) cm/s vs. 9.3 +/- 0.6 x 10(-5) cm/s, P < 0.01). There was no maturational change in bicarbonate-to-chloride permeabilities (P(HCO3)/P(Cl)) in the rat proximal straight tubules (PST) and proximal convoluted tubules (PCT) or in the sodium-to-chloride permeability (P(Na)/P(Cl)) in the proximal straight tubule; however, there was a significant maturational decrease in proximal convoluted tubule P(Na)/P(Cl) with postnatal development (1.31 +/- 0.12 in neonates vs. 0.75 +/- 0.06 in adults, P < 0.001). There was no difference in the transepithelial resistance measured by current injection and cable analysis in the PCT, but there was a maturational decrease in the PST (7.2 +/- 0.8 vs. 4.6 +/- 0.1 ohms x cm2, P < 0.05). These studies demonstrate there are maturational changes in the rat paracellular pathway that impact net NaCl transport during development.     

Na-K pump current in the Amphiuma collecting tubule

There is strong evidence supporting the hypothesis of an electrogenic Na-K pump in the basolateral membrane of several epithelia. Thermodynamic considerations and results in nonepithelial cells indicate that the current carried by the pump could be voltage dependent. In order to measure the pump current and to determine its voltage dependence in a tight epithelium, we have used the isolated perfused collecting tubule of Amphiuma and developed a technique for clamping the basolateral membrane potential (Vbl) through transepithelial current injection. The transcellular current was calculated by subtracting the paracellular current (calculated from the transepithelial conductance measured in the presence of luminal amiloride) from the total transepithelial current. Basolateral membrane current-voltage (I-V) curves were obtained in conditions where the ratio of the pump current to the total basolateral membrane current had been maximized by loading the cells with Na+ (exposure to low-K+ bath), and by blocking the basolateral K+ conductance with barium. The pump current was defined as the difference of the current across the basolateral membrane measured before and 10-15 s after the addition of strophanthidin (20 microM) to the bath solution. With a bath solution containing 3 mM K+, the pump current was nearly constant in the Vbl range of -20 to -80 mV (52 +/- 5 microA.cm-2 at -60 mV) but showed a marked voltage dependence at higher negative Vbl (pump current decreased to 5 +/- 9 microA.cm-2 at -180 mV). In a 1.0 mM K bath, the shape of the pump I-V curve was similar but the amplitude of the current was decreased (24 +/- 4 microA.cm-2 at -60 mV). In a 0.1 mM K bath, the pump current was not significantly different from 0. Our results indicate that the basolateral Na-K pump generates a current which depends on the extracellular potassium concentration. With physiological peritubular concentration of K+ and in the physiological range of potential, the pump activity, measured as the pump-generated current, was independent of the membrane potential.     

Transepithelial potential in Malpighian tubules of Rhodnius prolixus: lumen-negative voltages and the triphasic response to serotonin

Previous studies of the Malpighian tubules of Rhodnius reported lumen-negative values of transepithelial potential (TEP), and a characteristic triphasic change in TEP in response to stimulation of tubule fluid secretion by serotonin. TEP was measured using the Ramsay technique, in which electrodes are positioned in bathing and secreted fluid droplets for tubules isolated under paraffin oil. The validity of this method of TEP measurement has been questioned on the grounds that, in tubules of some species, it may permit shunting of current from lumen to bath through the cells or through the thin layer of fluid adherent to the surface of that portion of the tubule in the oil. The triphasic response of TEP to serotonin has been confirmed in this study of tubules of fifth instar Rhodnius prolixus using two different techniques that eliminate the possibility of shunting artefacts. From an initially negative value in unstimulated tubules ( approximately -25 mV, lumen-negative), TEP shifted to approximately -33 mV in phase 1, approximately +30 mV in phase 2 and approximately -32 mV in phase 3. TEP during each phase was similar irrespective of the measurement technique. Ion substitution experiments and the effects of specific pharmacological reagents support the proposal that the three phases of the response of TEP to serotonin correspond to sequential activation of an apical Cl(-) channel, an apical V-type H(+) ATPase and a basolateral Na(+):K(+):2Cl(-) cotransporter.     

Effect of stevioside on PAH transport by isolated perfused rabbit renal proximal tubule

Stevioside, a non-caloric sweetening agent, is used as a sugar substitute. An influence of stevioside on renal function has been suggested, but little is known about its effect on tubular function. Therefore, the present study was designed to explore the direct effect of stevioside on transepithelial transport of p-aminohippurate (PAH) in isolated S2 segments of rabbit proximal renal tubules using in vitro microperfusion. Addition of stevioside at a concentration of 0.45 mM to either the tubular lumen, bathing medium, or both at the same time had no effect on transepithelial transport of PAH. Similarly, a concentration of 0.70 mM (maximum solubility in the buffer) when present in the lumen, had no effect on PAH transport. However, this concentration in the bathing medium inhibited PAH transport significantly by about 25-35%. The inhibitory effect of stevioside was gradually abolished after it was removed from the bath. Addition of 0.70 mM stevioside to both lumen and bathing medium at the same time produced no added inhibitory effect. Stevioside at this concentration has no effect on Na+/K+-ATPase activity as well as cell ATP content. These findings suggest that stevioside, at a pharmacological concentration of 0.70 mM, inhibits transepithelial transport of PAH by interfering with the basolateral entry step, the rate-limiting step for transepithelial transport. The lack of effect of stevioside on transepithelial transport of PAH on the luminal side and its reversible inhibitory effect on the basolateral side indicate that stevioside does not permanently change PAH transport and should not harm renal tubular function at normal human intake levels.     

Cloning, functional analysis and cell localization of a kidney proximal tubule water transporter homologous to CHIP28

The localization and transporting properties of a kidney protein homologous to human erythrocyte protein CHIP28 was evaluated. The cDNA encoding rat kidney protein CHIP28k was isolated from a rat renal cortex cDNA library. A 2.8-kb cDNA was identified which contained an 807 bp open reading frame encoding a 28.8 kD protein with 94% amino acid identity to CHIP28. in vitro translation of CHIP28k cDNA in rabbit reticulocyte lysate generated a 28-kD protein; addition of ER-derived microsomes gave a 32-kD transmembrane glycoprotein. Translation of truncated RNA demonstrated glycosylation of residue Asn42 which is predicted to lie between the first and second transmembrane domains. Expression of in vitro transcribed mRNA encoding CHIP28k in Xenopus oocytes increased oocyte osmotic water permeability (Pf) from (4 +/- 1) x 10(-4) to (33 +/- 4) x 10(-4) cm/s at 10 degrees C; the increase in oocyte Pf was weakly temperature dependent and inhibited by HgCl2. Two- electrode voltage clamp measurements indicated that CHIP28k was not permeable to ions. Oocyte Pf also increased with expression of total mRNA from kidney cortex and papilla; the increase in Pf with mRNA from cortex, but not kidney papilla, was blocked by coinjection with excess antisense CHIP28k cRNA. In situ hybridization of a 150 base cRNA antisense probe to tissue sections from rat kidney showed selective CHIP28k localization to epithelial cells in proximal tubule and thin descending limb of Henle. Pf in purified apical membrane vesicles from rat and human proximal tubule, and in proteoliposomes reconstituted with purified protein, was very high and inhibited by HgCl2; stripping of apical vesicles with N-lauroylsarcosine enriched a 28-kD protein by 25-fold and yielded a vesicle population with high water, but low urea and proton permeabilities. CHIP28k identity was confirmed by NH2- terminus sequence analysis. These results indicate that CHIP28k is a major and highly selective water transporting protein in the kidney proximal tubule and thin descending limb of Henle, but not collecting duct.     

Proton nuclear magnetic resonance measurement of diffusional water permeability in suspended renal proximal tubules.

Diffusional water permeability was measured in renal proximal tubule cell membranes by pulsed nuclear magnetic resonance using proton spin-lattice relaxation times (T1). A suspension of viable proximal tubules was prepared from rabbit renal cortex by Dounce homogenization and differential sieving. T1 measured in a tubule suspension (22% of exchangeable water in the intracellular compartment) containing 20 mM extracellular MnCl2 was biexponential with time constants 1.8 +/- 0.1 ms and 8.3 +/- 0.2 ms (mean +/- SD, n = 8, 37 degrees C, 10 MHz). The slower time constant, representing diffusional exchange of water between intracellular and extracellular compartments, increased to 11.6 +/- 0.6 ms (n = 6) after incubation of tubules with 5 mM parachloromercuribenzene sulfonate (pCMBS) for 60 min at 4 degrees C and was temperature dependent with activation energy Ea = 2.9 +/- 0.4 kcal/mol. To relate T1 data to cell membrane diffusional water permeabilities (Pd), a three-compartment exchange model was developed that included intrinsic decay of proton magnetization in each compartment and apical and basolateral membrane water transport. The model predicted that the slow T1 was relatively insensitive to apical membrane Pd because of low luminal/cell volume ratio. Based on this analysis, basolateral Pd (corrected for basolateral membrane surface convolutions) is 2.0 X 10(-3) cm/s, much lower than corresponding values for basolateral Pf (10-30 X 10(-3) cm/s) measured in the intact tubule and in isolated basolateral membrane vesicles.(ABSTRACT TRUNCATED AT 250 WORDS)     

Estimation of intracellular chloride activity in isolated perfused rabbit proximal convoluted tubules using a fluorescent indicator.

The methodology has been developed to measure cell chloride activity by fluorescence microscopy using the chloride-sensitive dye, 6-methoxy-1-(3-sulfonatopropyl)quinolinium (SPQ). SPQ was loaded into cells of the in vitro microperfused rabbit proximal convoluted tubule by a 10 min luminal perfusion with 20 mM SPQ at 38 degrees C. Fluorescence was excited with a broad band excitation filter (340 and 380 nm) and detected with a 435 nm cut-on filter. The signal to background (autofluorescence) ratio was 4.6 +/- 0.6. The halftime for SPQ leakage from cells at 38 degrees C was 8.6 +/- 1.1 min. In suspended tubules, SPQ did not affect O2 consumption significantly. Intracellular SPQ calibration was performed using the ionophores nigericin and tributyltin, high external potassium concentrations, and varying extracellular chloride concentrations. Cell fluorescence was related to intracellular chloride by a Stern-Volmer relation with a quenching constant of 12 M-1. Apparent chloride concentration in tubules perfused with solutions characteristic for the late proximal convoluted tubule was 27.5 +/- 5 mM (activity 20.6 mM). The halftime of the transient in cell chloride activity upon bath chloride addition was approximately 3 s (38 degrees C). Applications and limitations of this new fluorescence method to study cell chloride transport are discussed.     

Nerve growth factor inhibits HCO3- absorption in renal thick ascending limb through inhibition of basolateral membrane Na+/H+ exchange

Nerve growth factor (NGF) inhibits transepithelial HCO3- absorption in the rat medullary thick ascending limb (MTAL). To investigate the mechanism of this inhibition, MTALs were perfused in vitro in Na+-free solutions, and apical and basolateral membrane Na+/H+ exchange activities were determined from rates of pHi recovery after lumen or bath Na+ addition. NGF (0.7 nM in the bath) had no effect on apical Na+/H+ exchange activity, but inhibited basolateral Na+/H+ exchange activity by 50%. Inhibition of basolateral Na+/H+ exchange activity with ethylisopropyl amiloride (EIPA) secondarily reduces apical Na+/H+ exchange activity and HCO3- absorption in the MTAL (Good, D. W., George, T., and Watts, B. A., III (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 12525-12529). To determine whether a similar mechanism could explain inhibition of HCO3- absorption by NGF, apical Na+/H+ exchange activity was assessed in physiological solutions (146 mM Na+) by measurement of the initial rate of cell acidification after lumen EIPA addition. Under these conditions, in which basolateral Na+/H+ exchange activity is present, NGF inhibited apical Na+/H+ exchange activity. Inhibition of HCO3- absorption by NGF was eliminated in the presence of bath EIPA or in the absence of bath Na+. Also, NGF blocked inhibition of HCO3- absorption by bath EIPA. We conclude that NGF inhibits basolateral Na+/H+ exchange activity in the MTAL, an effect opposite from the stimulation of Na+/H+ exchange by growth factors in other systems. NGF inhibits transepithelial HCO3- absorption through inhibition of basolateral Na+/H+ exchange, most likely as the result of functional coupling in which primary inhibition of basolateral Na+/H+ exchange activity results secondarily in inhibition of apical Na+/H+ exchange activity. These findings establish a role for basolateral Na+/H+ exchange in the regulation of renal tubule HCO3- absorption.