Peritubular Cell Membrane Research Articles

MO082THE EFFECTS OF DIFFERENT MICROMOLAR CONCENTRATIONS OF CADMIUM IONS ON THE PERITUBULAR MEMBRANE POTENTIAL OF PROXIMAL TUBULAR CELLS IN PERFUSED FROG KIDNEYS

Abstract Background and Aims Cadmium (Cd2+) is toxic metal and environmental pollutant. Accumulation of cadmium in the kidney results initially in proximal tubule dysfunction. Although Cd2+ toxicity is well documented, all mechanisms that are involved in the early stages of nephrotoxicity, especially considering low micromolar concentrations of Cd2+ ions are still unknown. The Aim of this study was to investigate the effects acute exposure to different peritubular micromolar concentrations of cadmium (0.25, 0.50, 1.0, 2.0, 3.0, 5.0 μmol/L) on the peritubular cell membrane potential in proximal tubular cells of frog kidney. Method The experiments were performed on isolated, doubly perfused kidneys of Rana esculenta of both sexes. Aortic and portal vein were cannulated in order to perfusate luminal and peritubular cell membraine. In controled conditions, Ringer solution was simultaneously used to perfusate both cell membraines. Cadmium chloride (different concentrations: 0.25, 0.50, 1.0, 2.0, 3.0, 5.0 μmol/L) were added to the peritubular perfusate separately, by switching the peritubular perfusate from the control Ringer solution to Ringer solution with addition of cadmium ions. Peritubular cell membrane potentials (PD) were measured with conventional 3 mol/L KCl microelectrodes. Results The peritubular application of different micromolar Cd2+ concentrations led to a rapid, sustained, reversible hyperpolarization of the peritubular cell membrane: 0.25 µmol/L, by −3.3±0.4 mV (n=8, p<0.001); 0.50 µmol/L, by −3.0±0.5 mV (n=11, p<0.001); 1.0 µmol/L, by −2.9±0.6 mV (n=8, p<0.01); 2.0 µmol/L, by −4.2±0.4 mV (n=13, p<0.01); 3.0 µmol/L, by −3.4±0.3 mV (n=14, p<0.001); 5.0 µmol/L, by −3.0±0.4 mV (n=10, p<0.001). After switching the perfusion from Ringer solution with addition of cadmium ions to control Ringer, the peritubular membraine potential returned to the average values that were maintained before the peritubular Cd2+ application (p>0.05). Comparing the effect of different Cd2+ concentrations, there was no difference in the hyperpolarization of the peritubular cell membrane (p>0.05).Each cell served as its own control. Conclusion Different low micromolar concentrations of Cd2+ provoked rapid and sustained hyperpolarization of peritubular membrane potential that did not show concentration-dependent response.

Peritubular membrane potential in kidney proximal tubular cells of spontaneously hypertensive rats

Peritubular membrane potential in kidney proximal tubular cells of spontaneously hypertensive rats (SHR-Okamoto strain adult rats) was measured with conventional 3 mol KCl microelectrodes, in vivo. Peritubular cell membrane potential was not different in SHR (-66.5 ± 0.7 mV) as compared with normotensive control Wistar rats (-67.5 ± 1.2 mV). To test the effects of possible altered sodium membrane transport in SHR on proximal tubule peritubular membrane potential, we allowed SHR and control rats to drink 1% NaCl for two weeks. Again, proximal tubule peritubular membrane potential was not different in SHR on 1% NaCl (-67.0 ± 1.0 mV) as compared with control rats on 1% NaCl (-64.7 ± 1.3 mV). From these results we concluded that peritubular membrane potential in kidney proximal tubular cells of SHR was not different from normotensive Wistar control rats, and if some alteration of sodium transport in kidney proximal tubular cells of SHR could exist, that was not possible to evaluate from the measurements of peritubular membrane potential in kidney proximal tubular cells.

Modeling Proximal Tubule Cell Homeostasis: Tracking Changes in Luminal Flow

During normal kidney function, there are routinely wide swings in proximal tubule fluid flow and proportional changes in Na(+) reabsorption across tubule epithelial cells. This "glomerulotubular balance" occurs in the absence of any substantial change in cell volume, and is thus a challenge to coordinate luminal membrane solute entry with peritubular membrane solute exit. In this work, linear optimal control theory is applied to generate a configuration of regulated transporters that could achieve this result. A previously developed model of rat proximal tubule epithelium is linearized about a physiologic reference condition; the approximate linear system is recast as a dynamical system; and a Riccati equation is solved to yield the optimal linear feedback that stabilizes Na(+) flux, cell volume, and cell pH. The first observation is that optimal feedback control is largely consigned to three physiologic variables, cell volume, cell electrical potential, and lateral intercellular hydrostatic pressure. Parameter modulation by cell volume stabilizes cell volume; parameter modulation by electrical potential or interspace pressure act to stabilize Na(+) flux and cell pH. This feedback control is utilized in a tracking problem, in which reabsorptive Na(+) flux varies over a factor of two, in order to represent a substantial excursion of glomerulotubular balance. The resulting control parameters consist of two terms, an autonomous term and a feedback term, and both terms include transporters on both luminal and peritubular cell membranes. Overall, the increase in Na(+) flux is achieved with upregulation of luminal Na(+)/H(+) exchange and Na(+)-glucose cotransport, with increased peritubular Na(+)-3HCO(3)(-) and K(+)-Cl(-) cotransport, and with increased Na(+), K(+)-ATPase activity. The configuration of activated transporters emerges as a testable hypothesis of the molecular basis for glomerulotubular balance. It is suggested that the autonomous control component at each cell membrane could represent the cytoskeletal effects of luminal flow.

Modeling Proximal Tubule Cell Homeostasis: Tracking Changes in Luminal Flow

Normally, there are wide swings in proximal tubule fluid flow and proportional changes in Na reabsorption across tubule epithelial cells. This “glomerulotubular balance” occurs in the absence of substantial change in cell volume, and is a challenge to coordinate luminal solute entry with peritubular solute exit. In this work, linear optimal control theory is applied to generate a configuration of regulated transporters that could achieve this result. A model of rat proximal tubule epithelium is linearized about a reference condition; the approximate linear system is recast as a dynamical system; and a Riccati equation is solved to yield the optimal linear feedback that stabilizes Na flux, cell volume, and cell pH. The first observation is that optimal feedback control is largely consigned to three variables: cell volume, cell electrical potential, and lateral intercellular hydrostatic pressure. Parameter modulation by cell volume stabilizes cell volume; parameter modulation by electrical potential or interspace pressure stabilizes Na flux and cell pH. This feedback control is utilized in a tracking problem, in which reabsorptive Na flux varies over a factor of two. The resulting control parameters consist of two terms, an autonomous term and a feedback term, and both terms include transporters on both luminal and peritubular cell membranes. The increase in Na flux is achieved with upregulation of luminal Na/H exchange and Na‐glucose cotransport, with increased peritubular Na‐3HCO3 and K‐Cl cotransport, and with increased Na,K‐ATPase activity. The configuration of activated transporters emerges as testable hypothesis of the molecular basis for glomerulotubular balance. It is suggested that the autonomous control component at each cell membrane could represent cytoskeletal effects of luminal flow. Supported by NIH RO1‐DK‐29857.

Low micromolar concentrations of cadmium and mercury ions activate peritubular membrane K + conductance in proximal tubular cells of frog kidney

The present study was designed to investigate the acute effects of extracellular low micromolar concentrations of cadmium and mercury ions on the peritubular cell membrane potential and its potassium selectivity in proximal tubular cells of the frog kidney. Peritubular exposure to 3 µmol/L Cd 2+ or 1 µmol/L Hg 2+ led to a rapid, sustained and reversible hyperpolarization of the peritubular cell membrane, paralleled by an increase in fractional K + conductance. Peritubular barium abolished hyperpolarization of the peritubular cell membrane to peritubular 3 µmol/L Cd 2+ or 1 µmol/L Hg 2+. Perfusing the lumen with 10 mmol/L l-alanine plus/minus 3 µmol/L Cd 2+ or Hg 2+ did not modify rapid depolarization and rate of slow repolarization of the peritubular cell membrane potential. In conclusion, low micromolar concentrations of Cd 2+ and Hg 2+ increase K + conductive pathway in the peritubular cell membrane and in this way can enhance ability of proximal renal tubular cells to maintain the driving force for electrogenic Na + and substrate reabsorption.

Absence of KCNQ1-dependent K + fluxes in proximal tubular cells of frog kidney

The present study was designed to investigate the functional significance of KCNQ1-mediated K + secretory fluxes in proximal tubular cells of the frog kidney. To this end, we investigated the effects on rapid depolarization and slow repolarization of the peritubular membrane potential after luminal addition of l-phenylalanine or l-alanine plus/minus KCNQ1 channel blockers. Perfusing the lumen with 10 mmol/L l-phenylalanine plus/minus luminal 293B, a specific blocker of KCNQ1, did not modify the rapid depolarization and the rate of slow repolarization. Perfusing the lumen with 10 mmol/L l-alanine plus/minus luminal HMR-1556, a more potent KCNQ1 channel blocker, did not also alter the rapid depolarization and the rate of slow repolarization. Pretreatment (1 h) of the lumen with HMR-1556 also failed to modify rapid depolarization and rate of slow repolarization upon luminal 10 mmol/L l-alanine. Perfusing the lumen with 1 mmol/L l-alanine plus/minus luminal HMR-1556 did not change the rapid depolarization and the rate of slow repolarization. The pretreatment (1 h) with luminal HMR-1556 did not modify the rapid depolarization and the rate of slow repolarization upon luminal 1 mmol/L l-alanine. The pretreatment (1 h) of the lumen with HMR-1556 did not change transference number for K + of peritubular cell membrane. Finally, luminal barium blunted the rapid depolarization upon application of luminal 1 mmol/L l-alanine. RT-PCR showed that KCNQ1 mRNA was not expressed in frog kidney. In conclusion, the KCNQ1-dependent K + secretory fluxes are absent in proximal tubule of frog kidney.

Characterization of Luminal and Peritubular Membrane K+ Selectivity in Proximal Tubular Cells of Frog Kidney

The functional significance of KCNQ1/KCNE1 K(+) secretory fluxes and the effects of 3 micromol/L Cd(2+) on the peritubular cell membrane potential and its potassium selectivity were investigated in proximal tubular cells of the frog kidney. Perfusing the lumen with l-phenylalanine plus/minus chromanol 293B, a specific blocker of KCNQ1, did not modify rapid depolarization and rate of slow repolarization of the peritubular cell membrane. Peritubular exposure to Cd(2+) led to a sustained, reversible hyperpolarization of the peritubular membrane, paralleled by an increase in fractional K(+) conductance. Peritubular barium blunted hyperpolarization of the peritubular cell membrane to Cd(2+). Thus, the KCNQ1/KCNE1 K(+) secretory fluxes are absent in proximal tubule of frog kidney, while Cd(2+) increases K(+) selectivity of the peritubular cell membrane.

Biochemical heterogeneity of arginine metabolism along kidney proximal tubules

By using an in vitro single tubule micromethod of high specificity, in four different species of mammals it has been observed that (a) arginine synthesis from citrulline (arginine synthase activity, E.C. 6.3.4.5 and E.C. 4.3.2.1) is restricted to the early portions of proximal convoluted tubules, whereas (b) urea production from arginine (arginase activity, E.C. 3.5.3.1.) is present mainly in the cortical (CPST) and even more in the outer medullary (OSPST) portions of straight proximal tubules. The data suggest that (a) in early PCT cells, the citrulline reabsorbed from glomerular filtrate is converted into arginine, which in turn crosses peritubular cell membranes together with reabsorbed arginine, and (b) the urea formed in CPST and OSPST cells might passively diffuse into the luminal fluid entering Henle's loops. Such urea secretion might contribute to sustain the process of urea recycling in kidney medulla and thereby participate in the mechanism of urine concentration.

Mathematical models of tubular transport.

Mathematical models of the renal tubule are classified as epithelial, tubular, or multitubular. The simplest epithelial models are single equations that represent the overall transepitheiiai solute and water fluxes as functions of the luminal and peritubular bath conditions. More detailed models represent both luminal and peritubular cell membranes, in parallel with a tight junction, all in series with an epithelial basement membrane. In these models, the presence of transport components in series renders the cell and interspace concentrations unknown variables that must be determined via solution of a set of conservation equations. The tubular models allow prediction of the effect of epithelial transport to modify the luminal solution and, conversely, the effect of altered luminal composition on transepithelial fluxes. Here the conservation equations constitute a set of ordinary differential equations, with initial data that must be integrated along the tubule length. The lining epithelium may be represented by single flux equations, or by a compart­ )

Ammonia transport in a mathematical model of rat proximal tubule

Pathways for ammonia transport have been incorporated within a model of rat proximal tubule [A. M. Weinstein. Am. J. Physiol. 263 (Renal Fluid Electrolyte Physiol. 32): F784-F798, 1992]. The luminal membrane includes a Na+/NH4+ exchanger, while at the peritubular membrane there is uptake of NH4+ on the Na(+)-K(+)-adenosinetriphosphatase (Na(+)-K(+)-ATPase); both luminal and peritubular cell membranes contain conductive pathways for NH4+. The model equations have been expanded to include cellular ammoniagenesis. The principal focus of this study is the interplay of forces that can raise proximal tubule fluid total ammonia concentration 10-fold higher than in arterial plasma. Analysis of a cellular model reveals that luminal membrane Na+/NH4+ exchange, cellular production of ammonia, and peritubular membrane NH4+ uptake (via Na(+)-K(+)-ATPase or via K+ channel) all act in parallel to drive ammonia secretion. This derives from the cellular interconversion of NH4+ and NH3 and the free permeation of NH3 across cell membranes. It implies that inhibition of the luminal membrane transporter does not block the contribution of peritubular uptake to the overall active transport of ammonia. Conversely, when inhibition of the luminal membrane Na+/NH4+ entry (i.e., Na+/H+ inhibition) depresses transcellular Na+ flux, then the decrease of NH4+ flux through the peritubular Na+ pump enhances the apparent importance of the luminal membrane pathway. This analysis is confirmed in the numerical calculations and is a departure from the Ussing paradigm of series membrane Na+ transport. Although active secretion of ammonia by this tubule is substantial, the relative contribution of luminal Na+/NH4+ exchange and of peritubular uptake via the Na+ pump remains uncertain. The determination of peritubular capillary NH4+ concentration will be crucial to resolving this uncertainty, with lower concentration (i.e., closer to systemic arterial ammonia) obligating greater luminal membrane Na+/NH4+ exchange.

Anion Channels in the Apical Membrane of Collecting Duct Principal Cells in Culture

Ion channels in the apical cell membrane of monolayers of rabbit renal collecting duct principal cells in primary culture were investigated with the patch-clamp technique. Besides three or more types of cation channels published previously, three types of anion channels were observed. Most frequent was an outwardly rectifying Cl– selective channel with the slope conductance of 30.4 ± 4.8 pS (n = 6) in symmetrical isotonic NaCl solutions at 0 mV holding potential. This channel was only observed in excised patches. Its open probability was independent of the calcium concentration at the cytoplasmic surface of the membrane patch. The channel had complex open/close kinetics and was blocked by 5-nitro-2-(3-phenylpropylamino) benzoic acid from the extracellular surface. Inhibition increased at more cytoplasm-positive membrane potentials. Secondly, a high conductance, poorly selective anion channel was observed. It had a linear current voltage (I/V) relation with a conductance of 267 ± 46 pS (n = 4) and exhibited at least four substates. The open probability of this channel decreased sharply with increasing membrane potentials of either polarity. Thirdly, and least frequently, a bursting Cl– selective channel was observed which displayed a peculiar I/V relation in isotonic NaCl solutions. Near 0 mV it had a slope conductance of ≈ 50 pS which increased to 113 ± 11 pS (n = 4) at potentials exceeding ± 50 mV. Since the peritubular cell membrane of principal cells is Cl– permeable, it is conceivable that one or more of these channels contribute to collecting duct Cl– absorption particularly in situations in which the luminal Cl– concentration and the transepithelial potential difference are high.

Influence of mepacrine, indomethacin, and nordihydroguaiaretic acid on the electrical properties of frog renal proximal tubules

In proximal renal tubules of the frog kidney, stimulation of sodium-coupled transport leads to a depolarization of the peritubular cell membrane, followed by partial repolarization. These alterations of the potential difference across the peritubular cell membrane (PDpt) are in part the result of altered peritubular potassium conductance. The repolarization has been blunted by the phospholipase A2 inhibitor mepacrine, but not by the cyclooxygenase inhibitor indomethacin. In the present study the effect of mepacrine, indomethacin and the lipoxygenase inhibitor nordihydroguaiaretic acid on the electrical properties of proximal renal tubules has been tested in the presence and absence of stimulated sodium-coupled transport. In the absence of inhibitors, addition of 10 mmol/l phenylalanine to the luminal perfusate leads to a rapid depolarization and partial repolarization of the peritubular cell membrane, a decrease of the luminal cell membrane resistance (Ra) and a small increase of the cellular core resistance (Rc). Removal of phenylalanine leads to rapid hyperpolarization, increase of Ra and decline Rc. Mepacrine (100 mumols/l) depolarizes the cell membrane and increases the peritubular cell membrane resistance (Rb), Rc and the intracellular pH. In the presence of mepacrine, phenylalanine leads to a sustained depolarization and a transient decrease of Ra. Indomethacin (10 mumol/l) does not significantly modify PDpt, the lumped resistance of both cell membranes (Rm) or Rc in the presence or absence of phenylalanine. Nordihydroguaiaretic acid (50 mumols/l) does not alter significantly PDpt, Ra, Rb or Rc prior to phenylalanine.(ABSTRACT TRUNCATED AT 250 WORDS)

Chloride Transport in the Proximal Renal Tubule

Our knowledge of chloride transport along the nephron has greatly expanded. Whereas for a long time it was assumed that chloride ions were reabsorbed entirely passively with sodium--the mendicant role of chloride, more recent studies suggest that several distinct reabsorptive transport mechanisms operate in parallel. Accordingly, a new model of proximal chloride transport has evolved that includes both active, transcellular as well as passive, intercellular transport pathways. Transcellular chloride reabsorption involves anion exchange mechanisms in both the luminal and peritubular cell membranes, processes that also depend on sodium, hydrogen, and bicarbonate ions. Chloride transport is thus intimately related to sodium and fluid transport as well as to cell acid-base metabolism. Unresolved problems concern the relative magnitude of transcellular and paracellular chloride transport and the details of luminal and basolateral chloride translocation steps.

Fused cells of frog proximal tubule: II. Voltage-dependent intracellular pH.

Experiments were performed in intact proximal tubules of the doubly perfused kidney and in fused proximal tubule cells of Rana esculenta to evaluate the dependence of intracellular pH (pHi) on cell membrane potential applying pH-sensitive and conventional microelectrodes. In proximal tubules an increase of the K+ concentration in the peritubular perfusate from 3 to 15 mmol/liter decreased the peritubular cell membrane potential from -55 +/- 2 to -38 +/- 1 mV paralleled by an increase of pHi from 7.54 +/- 0.02 to 7.66 +/- 0.02. The stilbene derivative DIDS hyperpolarized the cell membrane potential from -57 +/- 2 to -71 +/- 4 mV and led to a significant increase of the K+-induced cell membrane depolarization, but prevented the K+-induced intracellular alkalinization. Fused proximal tubule cells were impaled by three microelectrodes simultaneously and cell voltage was clamped stepwise while pHi changes were monitored. Cell membrane hyperpolarization acidified the cell cytoplasm in a linear relationship. This voltage-induced intracellular acidification was reduced to about one-third when HCO-3 ions were omitted from the extracellular medium. We conclude that in proximal tubule cells pHi depends on cell voltage due to the rheogenicity of the HCO-3 transport system.

Chloride Transport in the Proximal Renal Tubule

Our knowledge of chloride transport along the nephron has greatly expanded. Whereas for a long time it was assumed that chloride ions were reabsorbed entirely passively with sodium--the "mendicant" role of chloride, more recent studies suggest that several distinct reabsorptive transport mechanisms operate in parallel. Accordingly, a new model of proximal chloride transport has evolved that includes both active, transcellular as well as passive, intercellular transport pathways. Transcellular chloride reabsorption involves anion exchange mechanisms in both the luminal and peritubular cell membranes, processes that also depend on sodium, hydrogen, and bicarbonate ions. Chloride transport is thus intimately related to sodium and fluid transport as well as to cell acid-base metabolism. Unresolved problems concern the relative magnitude of transcellular and paracellular chloride transport and the details of luminal and basolateral chloride translocation steps.

Regulation of cytosolic free calcium in isolated perfused proximal tubules of Necturus.

To study the role of intracellular Ca2+ in regulating renal tubular transport of ions and water, cytosolic calcium ion activity (aiCa), cytosolic sodium ion activity (aiNa), and intracellular pH (pHi) in cells of isolated perfused proximal tubules of Necturus kidney were measured with Ca2+-, Na+-, and H+-selective microelectrodes, respectively. In control conditions, i.e., HCO3-Ringer solution on both sides of the epithelium, aiCa was 82 +/- 7 (SE) nM (n = 54), aiNa averaged 12.8 +/- 0.4 mM (n = 53), and pHi was 7.33 +/- 0.03 (n = 27). When the Na-K pump was inhibited by nominally K-free Ringer circumfusion, aiCa increased from a control level of 75 +/- 13 to 237 +/- 40 nM (paired t test; n = 16; P less than 0.001); in a different set of tubules, aiNa rose from 11.3 +/- 0.6 to 51.5 +/- 5.8 mM (n = 11; P less than 0.001). When organic solutes were deleted in the luminal perfusate, aiCa decreased from 73 +/- 11 to 61 +/- 11 nM (n = 9; P less than 0.001) and aiNa decreased from 14.6 +/- 0.6 to 8.3 +/- 0.7 mM (n = 9; P less than 0.001). Depolarization of the peritubular cell membrane with high-K, low-Na Ringer decreased aiCa from 90 +/- 12 to 55 +/- 9 nM (n = 13; P less than 0.001) and reduced aiNa from 13.1 +/- 1.0 to 7.5 +/- 0.6 mM (n = 16; P less than 0.001). Ionomycin (2 X 10(-6) M) increased aiCa from 67 +/- 10 to 158 +/- 26 nM (n = 10; P less than 0.001) and pHi from 7.33 +/- 0.03 to 7.39 +/- 0.03 (n = 27; P less than 0.001) but reduced aiNa from 11.8 +/- 0.9 to 10.3 +/- 0.7 mM (n = 11; P less than 0.001). The data are consistent with the view that aiCa is determined, in part, by the magnitude of the electrochemical potential gradient for Na ions across the basolateral cell membrane.

PH-dependent electrical properties and buffer permeability of the Necturus renal proximal tubule cell.

Necturus kidneys were perfused with Tris-buffered solutions at three different pH values, i.e. 7.5, 6.0 and 9.0. A significant drop in fluid absorption occurred at pH 6.0, whereas pH 9.0 did not increase volume flow significantly. When acute unilateral, i.e. either in the lumen or the peritubular capillaries, and bilateral pH changes were elicited in both directions from 7.5 to 9.0 at a constant Tris-butyrate buffer concentration, both peritubular membrane potential difference V1 and transepithelial potential difference V3 hyperpolarized, independently of the side where the change in pH was brought about. Acid perfusions at pH 6.0 caused a similar response but of opposite sign. Analysis of the potential changes shows that pH influences not only the electromotive force and resistance of the homolateral membrane, but also the electrical properties of the paracellular path. Interference of pH with Na, Cl or K conductance was assessed. Any appreciable role for sodium or chloride was excluded, whereas the potassium transference number (tK) of the peritubular membrane increased 16% in alkaline pH. However, this increase accounts only for 19 to 36% of the observed hyperpolarization. Since changes in Tris-butyrate buffer concentration at constant pH do not affect V1 or V3 considerably, the hyperpolarization in pH 9 cannot be explained by an elevation in internal pH only, or by a Tris-H+ ion diffusion potential only. The role of the permeability of the buffers: bicarbonate, butyrate and phosphate, in determining electrical membrane parameters was evaluated. Transport numbers of the buffer anions ranked as follows: tHCO3 greater than tbutyrate greater than tphosphate. It is concluded that modulation of membrane potential by extracellular pH is mediated primarily by a change in peritubular cell membrane tK and additionally by membrane currents carried by buffer anions.

On the nature of delayed repolarization during sustained sodium coupled transport in frog proximal tubules.

In proximal tubules of the frog kidney, stimulation of coupled transport of sodium with phenylalanine leads to depolarization of the cell membrane, followed by repolarization within a few minutes. The repolarization is due to a delayed increase of potassium conductance at the peritubular cell membrane. The present study was designed to test for the role of depolarization, of calmodulin and of arachidonic acid metabolites for the delayed increase of potassium conductance. To this end, the potential difference across the peritubular cell membrane of proximal convoluted tubules (PDpt) has been recorded continuously during exposure of the lumen to phenylalanine or during galvanic current injection into a neighbouring cell. During control conditions, PDpt averages -68.6 +/- 1.0 mV (n = 45). Phenylalanine leads to a depolarization of the peritubular cell membrane by +31.5 +/- 1.3 mV (n = 20), followed by a repolarization by -12.9 +/- 1.1 mV (n = 20) within 3 min. Injection of currents from 10 to 80 nAmps leads to a depolarization by +0.83 +/- 0.01 mV/nAmps which is again followed by repolarization. A linear correlation is observed between the magnitude of depolarization (dep) and repolarization (rep) within 3 min: rep (mV) = -(0.24 +/- 0.01) dep (mV) +(2.45 +/- 0.12) mV (r = 0.90). Thus, depolarization is capable to trigger delayed repolarization. The extent of repolarization is a function of the magnitude of depolarization. The possible involvement of calmodulin or arachidonic acid metabolites has been tested for by inducing sodium coupled transport in the presence of 100 mumol/l mepacrine, 10 mumol/l indomethacin or 10 mumol/l trifluoperazine.

Axial heterogeneity of sodium-bicarbonate cotransport in proximal straight tubule of rabbit kidney.

Intracellular microelectrodes were used to investigate rheogenic Na+ (HCO3-)n cotransport in different segments of isolated proximal straight tubule (PST) of rabbit kidney. In the first portion (S2 segment) the peritubular cell membrane potential Vb averaged -46.0, SE +/- 1.3 mV (n = 20), while in terminal portion (S3 segment) it averaged -68.3, SE +/- 2.5 mV (n = 10). This difference may reflect different modes of anion permeation across the peritubular cell membrane. In S2 segments, sudden 10:1 reduction of bath HCO3- concentration caused a fast transient cell depolarization, delta Vb = -45.8, SE +/- 1.2 mV (n = 33) as expected from the presence of Na+ (HCO3-)n cotransport. As the puncture site moved further distally, delta Vb declined and gradually changed its time course by superposition of a slower secondary depolarization. In this region the transient cell depolarization could be recuperated by inhibiting the peritubular K+ conductance with Ba2+ (1 mmol/l). In S3 segments, however, the HCO3(-)-dependent transient cell depolarization was completely lost both in the absence and presence of Ba2+. In addition, sudden reduction of bath Na+ concentration did not acidify the cell, as it did in the S2 segment. The data indicate that the expression of Na+ (HCO3-)n cotransport in the peritubular cell membrane gradually diminishes towards the end of the S2 segment and is lost in the S3 segment.

Effect of temperature on peritubular cell membrane potential and potassium conductance in isolated perfused proximal tubules of the mouse

In order to test for the effect of temperature on the electrical properties of renal tubule cells, electrophysiological measurements were made in segments of proximal straight tubules of the mouse, which were dissected free and perfused in vitro. The transepithelial potential difference was −1.6±0.1 mV ( n = 29). The potential difference across the peritubular cell membrane ( U m,bl ) recorded with microelectrodes, amounts to −62 ± 1 mV ( n = 118). Rapid increases of peritubular potassium concentration from 5 to 20 mmol/dm 3 lead to a depolarization of the basolateral cell membrane (Δ U m,bl by +24 ± 1 mV ( n = 103) which is indicative for a high relative potassium conductance of the basolateral cell membrane. Rapid cooling leads to an initial hyperpolarization from −61 ± 1 mV to −65 ± 1 mV ( n = 33) followed by a gradual decline of U m,bl . A parallel decline of Δ U m,bl indicates that the depolarization is at least partially due to decreasing peritubular potassium conductance. Rewarming causes a transient depolarization from −61±1 to −34±2 mV ( n = 20) followed by a complete repolarization within 1 minutes. Phlorizin hyperpolarizes U m,bl from −59±2 to −66±1 mV ( n = 13) and blunts the hyperpolarizing effect of hypothermia and the depolarizing effect of rewarming. Ouabain, like hypothermia, leads to a parallel decline of U m,bl and Δ U m,bl . In conclusion, inhibition of the sodium/potassium-ATPase by hypothermia or ouabain leads to a significant reduction of the peritubular potassium conductance which leads to the depolarization of U m,bl In addition, hypothermia inhibits the sodium-coupled glucose transport across the luminal cell membrane, an effect which tends to hyperpolarize the cell membrane.