L. Robson

Role of cell volume and protein kinase C in regulation of a Cl‐ conductance in single proximal tubule cells of Rana temporaria.

1. The whole‐cell patch clamp technique was used to investigate Cl‐ currents in single proximal tubule cells isolated from kidneys of Rana temporaria. 2. Immediately following establishment of the whole‐cell clamp, the Cl‐ conductance (gCl) of the cells was low. However, with 2 mM ATP in the pipette there was a time‐dependent activation of gCl. Such activation was inhibited when the bath contained a hypertonic Ringer solution. 3. The Cl‐ conductance was not directly dependent on cell volume; gCl increased with hypotonic shock and decreased with hypertonic shock, but only in the presence of ATP. 4. Activation of gCl by ATP was dependent on extracellular Ca2+; however, the conductance was not directly Ca2+ sensitive. Activation was inhibited by Gd3+, which also had a direct inhibitory action on gCl. 5. Inhibition of protein kinase C (PKC), by 10 microM PKC pseudo‐substrate (PKC‐ps), completely abolished the ATP‐dependent activation of gCl, while stimulation of PKC, by the PKC activator 4 beta‐phorbol 12‐myristate, 13‐acetate (PMA), increased the degree of activation typically observed with ATP. 6. We propose that gCl is activated by PKC‐mediated phosphorylation and plays a role in volume regulation of the cells.

Volume-activated, gadolinium-sensitive whole-cell currents in single proximal cells of frog kidney.

Stretch-activated channels (SACs) have been implicated in the control of epithelial cell volume. Such channels are generally sensitive to the trivalent lanthanide, gadolinium (Gd3+). In this study, using Gd3+ sensitivity and volume activation as indices, we have looked for ionic currents attributable to SACs using the wholecell-patch clamp technique in freshly isolated proximal tubule cells of the frog. Hypotonic shock caused a reversible increase in whole-cell conductance, which was inhibited by Gd3+. In conjunction with this increase in conductance, cell length (measured using an optical technique) also increased. We observed two types of volume and Gd3+-sensitive currents: voltage-dependentIVD and voltage-independent IVI. IVD was found in all cells, activated by depolarisation and hypotonic shock, and was inhibited reversibly by 10 μM Gd3+. The conductance did not discriminate between Na+ and K+ but was slightly anion-selective and was Ca2+-permeable.IVI was observed in only 50% of cells and was also inhibited by Gd3+. Although the inhibition was irreversible, it was dose-dependent, suggesting a specific effect of Gd3+ onIVI. Cells that showed IVI had a significantly higher conductance than those that did not (38.7±4.4,n=20, and 20.5±0.7,n=15, μS · cm−2 respectively). In contrast toIVD,IVI was mildly cation-selective, Ca2+-permeable, and also selective for Na+ over K+. As withIVD, volume-induced increases inIVI were inhibited by Gd3+. Both of these currents are activated during hypotonic shock and may be involved in volume-regulatory processes in frog proximal cells.

Volume regulatory responses in frog isolated proximal cells

Cells respond to increases in volume by activating solute efflux pathways, resulting in water loss and restoration of the original cell volume. The solute efflux pathways underlying these volume regulatory decrease (VRD) responses have been relatively well studied. However, the transduction pathways whereby the change in cell volume is converted into an intracellular signal resulting in VRD are much less well understood. We have examined VRD in isolated proximal tubule cells from the frog, with particular attention to the roles of stretch-activated channels, Ca2+ and protein kinases. Cell length was taken as an index of cell volume, and was measured continuously using a photodiode array. VRD was observed in approximately 50% of cells, and was inhibited by Ba2+, Gd3+ and 4,4′-diisothiocyanatostilbene 2,2′-disulphonic acid (DIDS), and removal of extracellular Ca2+. VRD was accelerated by the active phorbol ester, phorbol 12-myristate 13-acetate (PMA), and the phosphatase inhibitor F−; on the other hand, VRD was prolonged by 4α-phorbol 12,13-didecanoate (PDC), an inactive phorbol ester), and inhibited by PMA and Gd3+, PMA and 0 Ca2+, and staurosporine. Volume regulation was unaffected by di-butyryl cAMP and 3-isobutyl-1-methyl-xanthene (IBMX). These data suggest that Ca2+ and PKC, via protein phosphorylation, play a stimulatory role in VRD.

Adaptive downregulation of a quinidine-sensitive cation conductance in renal principal cells of TWIK-1 knockout mice

TWIK-1, a member of the two-pore domain K+ channel family, is expressed in brain, kidney, and lung. The aim of this study was to examine the effect of loss of TWIK-1 on the renal cortical collecting duct. Ducts were isolated from wild-type and TWIK-1 knockout mice by enzyme digestion and whole-cell clamp obtained via the basolateral membrane. Current- and voltage-clamp approaches were used to examine K+ conductances. No difference was observed between intercalated cells from wild-type or knockout ducts. In contrast, knockout principal cells were hyperpolarized compared to wild-type cells and had a reduced membrane conductance. This was a consequence of a fall in a barium-insensitive, quinidine-sensitive conductance (GQuin). GQuin demonstrated outward rectification and had a relatively low K+ to Na+ selectivity ratio. Loss of GQuin would be expected to lead to the hyperpolarization observed in knockout ducts by increasing fractional K+ conductance and Na+ uptake by the cell. Consistent with this hypothesis, knockout ducts had an increased diameter in comparison to wild-type ducts. These data suggest that GQuin contributes to the resting membrane potential in the cortical collecting duct and that a fall in GQuin could be an adaptive response in TWIK-1 knockout ducts.

Renal proximal tubule function is preserved in Cftrtm2camΔF508 cystic fibrosis mice

1 Changes in proximal tubule function have been reported in cystic fibrosis patients. The aim of this study was to investigate proximal tubule function in the Cftrtm2camΔF508 cystic fibrosis (CF) mouse model. A range of techniques were used including renal clearance studies, in situ microperfusion, RT‐PCR and whole‐cell patch clamping. 2 Renal Na+ clearance was similar in wild‐type (1.4 ± 0.3 μl min−1, number of animals, N= 12) and CF mice (1.6 ± 0.4 μl min−1, N= 7) under control conditions. Acute extracellular volume expansion resulted in significant natriuresis in wild‐type (7.0 ± 0.8 μl min−1, N= 8) and CF mice (9.3 ± 1.4 μl min−1, N= 9); no difference between genotypes was observed. 3 In situ microperfusion revealed that fluid absorptive rate (Jv) was similar under control conditions between wild‐type (2.2 ± 0.4 nl mm−1 min−1, n= 10) and CF mice (1.9 ± 0.3 nl mm−1 min−1, n= 11). Addition of a forskolin‐dibutyryl cAMP (db‐cAMP) cocktail to the perfusate caused no significant change in Jv in either wild‐type (2.6 ± 0.7 nl mm−1 min−1, n= 10) or Cftrtm2camΔF508 mice (2.0 ± 0.5 nl mm−1 min−1, n= 10). 4 CFTR expression was confirmed in samples of outer cortex using RT‐PCR. However, no evidence for functional CFTR was obtained when outer cortical cells were stimulated with protein kinase A or forskolin‐db‐cAMP using whole‐cell patch clamping. 5 In conclusion, no functional deficit in proximal tubule function was found in Cftrtm2camΔF508 mice. This may be a consequence of a lack of whole‐cell cAMP‐dependent Cl− conductance in mouse proximal tubule cells.

TWO K+-SELECTIVE CONDUCTANCES IN SINGLE PROXIMAL TUBULE CELLS ISOLATED FROM FROG KIDNEY ARE REGULATED BY ATP

1. The whole‐cell and single channel patch clamp techniques were used to identify K(+)‐selective conductances in single proximal tubule cells isolated from frog kidney and to examine their ATP sensitivity. Whole‐cell currents were inhibited by the K+ channel inhibitors Ba2+ and quinidine in a dose‐dependent manner. Addition of Ba2+ alone, quinidine alone, or both inhibitors together revealed two separate conductances, one of which was blocked by both Ba2+ and quinidine (GBa)1, the other being sensitive to quinidine alone (Gquin). 2. With Na(+)‐containing Ringer solution in the bath and K(+)‐containing Ringer solution in the pipette, both currents were selective for K+ over Na+. The K+ : Na+ selectivity ratio of GBa was around 50:1, while that of Gquin was 4:1. In symmetrical KCl solutions GBa showed inward rectification, while Gquin demonstrated outward rectification. 3. In the absence of pipette ATP, both GBa and Gquin ran down over 10 min. However, when 2 mM ATP was included in the pipette GBa increased, while Gquin remained unchanged. 4. Single channel studies demonstrated that a basolateral K+ channel shared several of the characteristics of GBa. It was inhibited by both Ba2+ and quinidine, underwent run‐down in excised patches in the absence of ATP, and was activated by ATP. 5. We conclude that cells of the frog proximal tubule contain at least two distinct K(+)‐selective conductances, both of which are regulated by ATP, and which may be involved in pump‐leak coupling.

A Kir2.3-like K + Conductance in Mouse Cortical Collecting Duct Principal Cells

K+ channels play an important role in renal collecting duct cell function. The current study examined barium (Ba2+)-sensitive whole-cell K+ currents (IKBa) in mouse isolated collecting duct principal cells. IKBa demonstrated strong inward rectification and was inhibited by Ba2+ in a dose- and voltage-dependent fashion, with the Kd decreasing with hyperpolarization. The electrical distance of block by Ba2+ was around 8.5%. As expected for voltage-dependent inhibition, the association constant increased with hyperpolarization, suggesting that the rate of Ba2+ entry was increased at negative potentials. The dissociation constant also increased with hyperpolarization, consistent with the movement of Ba2+ ions into the intracellular compartment at negative potentials. These properties are not consistent with ROMK but are consistent with the properties of Kir2.3. Kir2.3 is thought to be the dominant basolateral K+ channel in principal cells. This study provides functional evidence for the expression of Kir2.3 in mouse cortical collecting ducts and confirms the expression of Kir2.3 in this segment of the renal tubule using reverse-transcriptase polymerase chain reaction. The conductance described here is the first report of a macroscopic K+ conductance in mouse principal cells that shares the biophysical profile of Kir2.3. The properties and dominant nature of the conductance suggest that it plays an important role in K+ handling in the principal cells of the cortical collecting duct.

Volume regulation is defective in renal proximal tubule cells isolated from KCNE1 knockout mice

The membrane protein KCNE1 has been implicated in cell volume regulation. Using a knockout mouse model, this study examined the role of KCNE1 in regulatory volume decrease (RVD) in freshly isolated renal proximal tubule cells. Cell diameter was measured using an optical technique in response to hypotonic shock and stimulation of Na+‐alanine cotransport in cells isolated from wild‐type and KCNE1 knockout mice. In HEPES buffered solutions 64% of wild‐type and 56% of knockout cells demonstrated RVD. In HCO−3 buffered solutions 100% of the wild‐type cells showed RVD, while in the knockout cells the proportion of cells displaying RVD remained unchanged. RVD in the knockout cells was rescued by valinomycin, a K+ ionophore. In wild‐type HCO−3 dependent cells the K+ channel inhibitors barium and clofilium inhibited RVD. These data suggest that mouse renal proximal tubule is comprised of two cell populations. One cell population is capable of RVD in the absence of HCO−3, whereas RVD in the other cell population has an absolute requirement for HCO−3. The HCO−3 dependent RVD requires the normal expression of KCNE1.

Stable, polarised, functional expression of Kir1.1b channel protein in Madin-Darby canine kidney cell line.

1 The family of Kir1.1 (ROMK) channel proteins constitute a secretory pathway for potassium in principal cells of cortical collecting duct and thick ascending limb of Henles loop. Mutations in Kir1.1 account for some types of Bartters syndrome. 2 Here we report that stable transfection of Kir1.1b (ROMK2) in Madin‐Darby canine kidney (MDCK) cell line results in expression of inwardly rectifying K+ currents and transmonolayer electrical and transport properties appropriate to Kir1.1 function. When grown on permeable supports, transfected monolayers secreted K+ into the apical solution. This secretion was inhibited by application of barium to the apical membrane, or by reduction in expression temperature from 37 to 26°C. However, whole‐cell voltage clamp electrophysiology showed that K+ conductance was higher in cells expressing Kir1.1b at 26°C. 3 To investigate this further, Kir1.1b was tagged with (EGFP), a modification that did not affect channel activity. Protein synthesis was inhibited with cycloheximide. Spectrofluorimetry was used to compare protein degradation at 37 and 26°C. The increased level of Kir1.1b at the plasma membrane at 26°C was due to an increase in protein stability. 4 Confocal microscopic investigation of EGFP‐Kir1.1b fluorescence in transfected cells showed that the channel protein was targeted to the apical domain of the cell. 5 These results demonstrate that Kir1.1b is capable of appropriate trafficking and function in MDCK cell lines at physiological temperatures. In addition, expression of Kir1.1b in MDCK cell lines provides a useful and convenient tool for the study of functional activity and targeting of secretory K+ channels.

The role of Ca2+ in volume regulation induced by Na+‐coupled alanine uptake in single proximal tubule cells isolated from frog kidney

1 It has been suggested that epithelial cells maintain cell volume and function, in the face of changes in the rate of transepithelial transport, by activation of volume‐regulatory pathways. 2 The aim of the following study was to examine directly the effect of an alteration in Na+‐coupled alanine transport on cell length in single proximal tubule cells isolated from frog kidney. 3 An optical technique was used to examine the change in cell length induced by 5 mM L‐alanine. 4 On addition of L‐alanine to the bath there was an initial increase in cell length to a peak value. This was followed by two types of response. In eighteen out of thirty‐one cells a typical volume‐regulatory response was observed. The remaining cells showed no volume regulation. 5 Volume regulation was not affected by the removal of extracellular Ca2+. The mean degrees of recovery were 159 ± 21 % (n= 18) and 144 ± 18 % (n= 8) in the presence and absence of Ca2+, respectively. 6 Volume regulation was inhibited by depletion of intracellular Ca2+ stores, or in the presence of either Gd3+ or DIDS. The mean degrees of regulation were 55.4 ± 9.2 % (n= 7), 68.2 ± 18.8 % (n= 7) and 69.1 ± 14.3 % (n= 7), respectively. 7 The alanine‐induced increases in cell length were both stereospecific and Na+ dependent. 8 The evidence suggests that volume regulation induced by Na+‐coupled alanine uptake may be dependent on the release of Ca2+ from intracellular stores. This is in contrast to volume regulation induced by hypotonic shock, which appears to require extracellular Ca2+. Results obtained using a hypotonic shock should, therefore, be viewed with caution.