Marc Paulais

An inward rectifier K+ channel at the basolateral membrane of the mouse distal convoluted tubule: similarities with Kir4‐Kir5.1 heteromeric channels

In this study, K+ channels present in the basolateral membrane of the distal convoluted tubule (DCT) were investigated using patch‐clamp methods. In addition, Kir4.1, Kir4.2 and Kir5.1 inward rectifier channels were investigated using RT‐PCR and immunohistochemistry (Kir4.1). DCTs were microdissected from collagenase‐treated mouse kidneys. One type of K+ channel was detected in about 50 % of cell‐attached patches from the DCT basolateral membrane; this channel was inwardly rectifying and had an inward conductance (gin) of ∼40 pS at an external [K+] of 145 mm. The current‐voltage relationship was linear when inside‐out patches were exposed to a Mg2+‐free medium. Mg2+ at a concentration of 1.2 mm considerably reduced the outward conductance (gout), yielding a gin/gout ratio of ∼4.7. The polycation spermine (5 × 10−7m) reduced the open probability (Po) by 50 %. Channel activity was dependent upon the intracellular pH, with acid pH decreasing, and basic pH increasing, Po. Internal ATP (2 mm) and Ca2+ (up to 10−3m) had no effect. Channel activity declined irreversibly when the inner side of the patch was exposed to Mg2+. Kir4.1, Kir4.2 and Kir5.1 mRNAs were all detected in the DCT. The Kir4.1 protein co‐localised with the Na+‐Cl− cotransporter, which is specific to the DCT, and was located on basolateral membranes. The DCT K+ channel differs from other functionally identified renal K+ channels with regard to its inhibition by spermine and insensitivity to internal ATP and Ca2+. At the current state of knowledge, the channel is similar to Kir4.1‐Kir5.1 and Kir4.2‐Kir5.1 heteromeric channels, but not to Kir4.1 or Kir4.2 homomeric channels.

Inhibitors of vacuolar H+-ATPase impair the preferential accumulation of daunomycin in lysosomes and reverse the resistance to anthracyclines in drug-resistant renal epithelial cells.

It has been suggested that the inappropriate sequestration of weak-base chemotherapeutic drugs in acidic vesicles by multidrug-resistance (MDR) cells contributes to the mechanisms of drug resistance. The function of the acidic lysosomes can be altered in MDR cells, and so we investigated the effects of lysosomotropic agents on the secretion of lysosomal enzymes and on the intracellular distribution of the weak-base anthracycline daunomycin in drug-resistant renal proximal tubule PKSV-PR(col50) cells and their drug-sensitive PKSV-PR cell counterparts. Imaging studies using pH-dependent lysosomotropic dyes revealed that drug-sensitive and drug-resistant cells exhibited a similar acidic lysosomal pH (around 5.6-5.7), but that PKSV-PR(col50) cells contained more acidic lysosomes and secreted more of the lysosomal enzymes N -acetyl-beta-hexosaminidase and beta-glucuronidase than their parent PKSV-PR cells. Concanamycin A (CCM A), a potent inhibitor of the vacuolar H(+)-ATPase, but not the P-glycoprotein modulator verapamil, stimulated the secretion of N -acetyl-beta-hexosaminidase in both drug-sensitive and drug-resistant cells. Fluorescent studies and Percoll density gradient fractionation studies revealed that daunomycin accumulated predominantly in the lysosomes of PKSV-PR(col50) cells, whereas in PKSV-PR cells the drug was distributed evenly throughout the nucleo-cytoplasmic compartments. CCM A did not impair the cellular efflux of daunomycin, but induced the rapid nucleo-cytoplasmic redistribution of the drug in PKSV-PR(col50) cells. In addition, CCM A and bafilomycin A1 almost completely restored the sensitivity of these drug-resistant cells to daunomycin, doxorubicin and epirubicin. These findings indicate that lysosomotropic agents that impair the acidic-pH-dependent accumulation of weak-base chemotherapeutic drugs may reverse anthracycline resistance in MDR cells with an expanded acidic lysosomal compartment.

Kir4.1/Kir5.1 channel forms the major K+ channel in the basolateral membrane of mouse renal collecting duct principal cells

K(+) channels in the basolateral membrane of mouse cortical collecting duct (CCD) principal cells were identified with patch-clamp technique, real-time PCR, and immunohistochemistry. In cell-attached membrane patches, three K(+) channels with conductances of approximately 75, 40, and 20 pS were observed, but the K(+) channel with the intermediate conductance (40 pS) predominated. In inside-out membrane patches exposed to an Mg(2+)-free medium, the current-voltage relationship of the intermediate-conductance channel was linear with a conductance of 38 pS. Addition of 1.3 mM internal Mg(2+) had no influence on the inward conductance (G(in) = 35 pS) but reduced outward conductance (G(out)) to 13 pS, yielding a G(in)/G(out) of 3.2. The polycation spermine (6 x 10(-7) M) reduced its activity on inside-out membrane patches by 50% at a clamp potential of 60 mV. Channel activity was also dependent on intracellular pH (pH(i)): a sigmoid relationship between pH(i) and channel normalized current (NP(o)) was observed with a pK of 7.24 and a Hill coefficient of 1.7. By real-time PCR on CCD extracts, inwardly rectifying K(+) (Kir)4.1 and Kir5.1, but not Kir4.2, mRNAs were detected. Kir4.1 and Kir5.1 proteins cellularly colocalized with aquaporin 2 (AQP2), a specific marker of CCD principal cells, while AQP2-negative cells (i.e., intercalated cells) showed no staining. Dietary K(+) had no influence on the properties of the intermediate-conductance channel, but a Na(+)-depleted diet increased its open probability by approximately 25%. We conclude that the Kir4.1/Kir5.1 channel is a major component of the K(+) conductance in the basolateral membrane of mouse CCD principal cells.

Renal phenotype in mice lacking the Kir5.1 (Kcnj16) K+ channel subunit contrasts with that observed in SeSAME/EAST syndrome.

The heteromeric inwardly rectifying Kir4.1/Kir5.1 K+ channel underlies the basolateral K+ conductance in the distal nephron and is extremely sensitive to inhibition by intracellular pH. The functional importance of Kir4.1/Kir5.1 in renal ion transport has recently been highlighted by mutations in the human Kir4.1 gene (KCNJ10) that result in seizures, sensorineural deafness, ataxia, mental retardation, and electrolyte imbalance (SeSAME)/epilepsy, ataxia, sensorineural deafness, and renal tubulopathy (EAST) syndrome, a complex disorder that includes salt wasting and hypokalemic alkalosis. Here, we investigated the role of the Kir5.1 subunit in mice with a targeted disruption of the Kir5.1 gene (Kcnj16). The Kir5.1−/− mice displayed hypokalemic, hyperchloremic metabolic acidosis with hypercalciuria. The short-term responses to hydrochlorothiazide, an inhibitor of ion transport in the distal convoluted tubule (DCT), were also exaggerated, indicating excessive renal Na+ absorption in this segment. Furthermore, chronic treatment with hydrochlorothiazide normalized urinary excretion of Na+ and Ca2+, and abolished acidosis in Kir5.1−/− mice. Finally, in contrast to WT mice, electrophysiological recording of K+ channels in the DCT basolateral membrane of Kir5.1−/− mice revealed that, even though Kir5.1 is absent, there is an increased K+ conductance caused by the decreased pH sensitivity of the remaining homomeric Kir4.1 channels. In conclusion, disruption of Kcnj16 induces a severe renal phenotype that, apart from hypokalemia, is the opposite of the phenotype seen in SeSAME/EAST syndrome. These results highlight the important role that Kir5.1 plays as a pH-sensitive regulator of salt transport in the DCT, and the implication of these results for the correct genetic diagnosis of renal tubulopathies is discussed.

A cation channel in the thick ascending limb of Henle's loop of the mouse kidney: inhibition by adenine nucleotides.

1. Patch‐clamp single‐channel current recordings were used to study the inhibition of Ca2+‐activated non‐selective cation channels by internal nucleotides in patches excised from basolateral membranes of the thick ascending limb of Henles loop of the mouse kidney. 2. The application of ATP, ADP or AMP to the cytoplasmic face of excised inside‐out membrane patches reduced the open‐state probability of the channels (Po) in a dose‐dependent way without effect upon the unitary current amplitude. Dose‐response curves gave half‐maximal inhibitory concentrations of 20, 21 and 2.5 microM for ATP, ADP and AMP, respectively, while the Hill coefficient was close to one in all three cases. 3. Cyclic AMP partially inhibited channel activity (Po = 35 +/‐ 17% of control) only at high, unphysiological concentrations (10(‐3) M) while adenosine (10(‐3) M) had very little effect (Po = 83 +/‐ 7% of control). 4. Replacement of adenine with other purines (guanine, hypoxanthine) or pyrimidine (uridine) bases very largely reduced inhibitory activity. Cyclic GMP had no effect. 5. Non‐hydrolysable analogues of ATP, AMP‐PNP (10(‐3) M) and ATP‐gamma‐S (5 x 10(‐4) M), were effective inhibitors of the channel (Po = 24 +/‐ 7 and 9 +/‐ 4% of control, respectively.

cAMP-activated chloride channel in the basolateral membrane of the thick ascending limb of the mouse kidney

SummaryThe properties of an anion-selective channel observed in basolateral membranes of microdissected, collagenase-treated, cortical thick ascending limbs of Henles loop from mouse kidney were investigated using patch-clamp single-channel recording techniques. In basal conditions, single Cl− currents were detected in 8% of cell-attached and excised, inside-out, membrane patches whereas they were observed in 24% of cell-attached and 67% of inside-out membrane patches when tubular fragments were preincubated with Forskolin (10−5m) or 8-bromo-cAMP (10−4m) and isobutylmethylxanthine (10−5m). The channel exhibited a linear current-voltage relationship with conductances of about 40 pS in both cell-attached and cell-free membrane configurations. APNa+PCl−ratio of 0.05 was estimated in the presence of a 142/42mm NaCl concentration gradient applied to inside-out membrane patches. Anionic selectivity of the channel followed the sequence Cl−>Br−>No3−≫F−; gluconate was not a permeant species. The open-state probability of the channel increased with membrane depolarization in cell-attached, i.e.,in situ membrane patches. In excised, inside-out, membrane patches, the channel was predominantly open with the open-state probability close to 0.8 over the whole range of potentials tested (−60 to +60 mV). The channel activity was not a function of internal calcium concentration between 10−9 and 10−3m. We suggest that this Cl− channel, whose properties are distinct from those in other epithelia, could account for the well-documented conductance which mediates Cl− exit in the basolateral step of NaCl absorption in thick ascending limb of Henles loop.

A Chloride Channel at the Basolateral Membrane of the Distal-convoluted Tubule: a Candidate ClC-K Channel

The distal-convoluted tubule (DCT) of the kidney absorbs NaCl mainly via an Na+-Cl− cotransporter located at the apical membrane, and Na+, K+ ATPase at the basolateral side. Cl− transport across the basolateral membrane is thought to be conductive, but the corresponding channels have not yet been characterized. In the present study, we investigated Cl− channels on microdissected mouse DCTs using the patch-clamp technique. A channel of ∼9 pS was found in 50% of cell-attached patches showing anionic selectivity. The NP o in cell-attached patches was not modified when tubules were preincubated in the presence of 10−5 M forskolin, but the channel was inhibited by phorbol ester (10−6 M). In addition, NP o was significantly elevated when the calcium in the pipette was increased from 0 to 5 mM (NP o increased threefold), or pH increased from 6.4 to 8.0 (NP o increased 15-fold). Selectivity experiments conducted on inside-out patches showed that the Na+ to Cl− relative permeability was 0.09, and the anion selectivity sequence Cl− ∼ I−> Br− ∼ NO3 − > F−. Intracellular NPPB (10−4 M) and DPC (10−3 M) blocked the channel by 65% and 80%, respectively. The channel was inhibited at acid intracellular pH, but intracellular ATP and PKA had no effect. ClC-K Cl− channels are characterized by their sensitivity to the external calcium and to pH. Since immunohistochemical data indicates that ClC-K2, and perhaps ClC-K1, are present on the DCT basolateral membrane, we suggest that the channel detected in this study may belong to this subfamily of the ClC channel family.

A Ca2-activated cation-selective channel in the basolateral membrane of the cortical thick ascending limb of Henle's loop of the mouse

The patch-clamp technique was used to investigate the properties of a cation-selective channel in the basolateral membrane of microdissected collagenase-treated fragments of cortical thick ascending limbs of Henles loop from mouse kidney. The channel activity was seldom observed in cell-attached patches (2 out 15 studied cases). In inside-out excised patches immersed in symmetrical NaCl Ringers solutions, the unit channel conductance was ohmic and ranged from 22 to 33 pS (mean, 26.8 +/- 0.6 pS, n = 24). When NaCl was replaced by KCl (n = 8) or sodium gluconate (n = 2) on the cytoplasmic side of the membrane, single-channel currents still reversed at 0 mV and the conductance was unchanged. The reversal potential was +28.8 +/- 0.4 mV (n = 8) when a NaCl concentration (140 vs. 42 mmol/l) gradient was applied, close to the expected value (approx. 30 mV) for a cation selective channel. The channel was found to discriminate poorly between Na+, K+, Cs+, and Li+ ions. The activity of the channel was not clearly voltage-dependent but was dependent upon the free Ca2+ concentration on the cytoplasmic side of the membrane. We conclude that the channel resembles the non-selective cation channel which has been previously described in several tissues.

A Na+- and Cl−-activated K+ Channel in the Thick Ascending Limb of Mouse Kidney

This study investigates the presence and properties of Na+-activated K+ (KNa) channels in epithelial renal cells. Using real-time PCR on mouse microdissected nephron segments, we show that Slo2.2 mRNA, which encodes for the KNa channels of excitable cells, is expressed in the medullary and cortical thick ascending limbs of Henles loop, but not in the other parts of the nephron. Patch-clamp analysis revealed the presence of a high conductance K+ channel in the basolateral membrane of both the medullary and cortical thick ascending limbs. This channel was highly K+ selective (PK/PNa ∼ 20), its conductance ranged from 140 to 180 pS with subconductance levels, and its current/voltage relationship displayed intermediate, Na+-dependent, inward rectification. Internal Na+ and Cl− activated the channel with 50% effective concentrations (EC50) and Hill coefficients (nH) of 30 ± 1 mM and 3.9 ± 0.5 for internal Na+, and 35 ± 10 mM and 1.3 ± 0.25 for internal Cl−. Channel activity was unaltered by internal ATP (2 mM) and by internal pH, but clearly decreased when internal free Ca2+ concentration increased. This is the first demonstration of the presence in the epithelial cell membrane of a functional, Na+-activated, large-conductance K+ channel that closely resembles native KNa channels of excitable cells. This Slo2.2 type, Na+- and Cl−-activated K+ channel is primarily located in the thick ascending limb, a major renal site of transcellular NaCl reabsorption.

The ClC-K2 Chloride Channel Is Critical for Salt Handling in the Distal Nephron.

Chloride transport by the renal tubule is critical for blood pressure (BP), acid-base, and potassium homeostasis. Chloride uptake from the urinary fluid is mediated by various apical transporters, whereas basolateral chloride exit is thought to be mediated by ClC-Ka/K1 and ClC-Kb/K2, two chloride channels from the ClC family, or by KCl cotransporters from the SLC12 gene family. Nevertheless, the localization and role of ClC-K channels is not fully resolved. Because inactivating mutations in ClC-Kb/K2 cause Bartter syndrome, a disease that mimics the effects of the loop diuretic furosemide, ClC-Kb/K2 is assumed to have a critical role in salt handling by the thick ascending limb. To dissect the role of this channel in detail, we generated a mouse model with a targeted disruption of the murine ortholog ClC-K2. Mutant mice developed a Bartter syndrome phenotype, characterized by renal salt loss, marked hypokalemia, and metabolic alkalosis. Patch-clamp analysis of tubules isolated from knockout (KO) mice suggested that ClC-K2 is the main basolateral chloride channel in the thick ascending limb and in the aldosterone-sensitive distal nephron. Accordingly, ClC-K2 KO mice did not exhibit the natriuretic response to furosemide and exhibited a severely blunted response to thiazide. We conclude that ClC-Kb/K2 is critical for salt absorption not only by the thick ascending limb, but also by the distal convoluted tubule.