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Aldosterone-sensitive distal nephron (ASDN) including the distal convoluted tubule (DCT), connecting tubule (CNT) and collecting duct (CD) plays an important role in the regulation of hormone-dependent Na + reabsorption and dietary K + -intake dependent K + excretion. The major Na + transporters in the ASDN are thiazide-sensitive Na-Cl cotransporter (NCC), epithelial Na + channel (ENaC), pendrin/Na + -dependent Cl - -bicarbonate exchanger (NDCBE). Whereas major K + channels in the ASDN are Kir4.1 and Kir5.1 in the basolateral membrane; and Kir1.1 (ROMK) and Ca 2+ activated big conductance K + channel (BK) in the apical membrane. Although a variety of in vitro cell lines of the ASDN is available and these cell models have been employed for studying Na + and K + channels, the biophysical properties and the regulation of Na + and K + channels in vitro cell models may not be able to recapitulate those in vivo conditions. Thus, the studies performed in the native ASDN are essential for providing highly physiological relevant information and for understanding the Na + and K + transport in the ASDN. Here we provide a detailed methodology describing how to perform the electrophysiological measurement in the native DCT, CNT and cortical collecting duct (CCD)., (© 2019 Elsevier Inc. All rights reserved.)

With-no-lysine kinase 4 (WNK4) and kidney-specific (KS)-WNK1 regulate ROMK (Kir1.1) channels in a variety of cell models. We now explore the role of WNK4 and KS-WNK1 in regulating ROMK in the native distal convoluted tubule (DCT)/connecting tubule (CNT) by measuring tertiapin-Q (TPNQ; ROMK inhibitor)-sensitive K + currents with whole cell recording. TPNQ-sensitive K + currents in DCT2/CNT of KS- WNK1 -/- and WNK4 -/- mice were significantly smaller than that of WT mice. In contrast, the basolateral K + channels (a Kir4.1/5.1 heterotetramer) in the DCT were not inhibited. Moreover, WNK4 -/- mice were hypokalemic, while KS- WNK1 -/- mice had normal plasma K + levels. High K + (HK) intake significantly increased TPNQ-sensitive K + currents in DCT2/CNT of WT and WNK4 -/- mice but not in KS- WNK1 -/- mice. However, TPNQ-sensitive K + currents in the cortical collecting duct (CCD) were normal not only under control conditions but also significantly increased in response to HK in KS- WNK1 -/- mice. This suggests that the deletion of KS-WNK1-induced inhibition of ROMK occurs only in the DCT2/CNT. Renal clearance study further demonstrated that the deletion of KS-WNK1 did not affect the renal ability of K + excretion under control conditions and during increasing K + intake. Also, HK intake did not cause hyperkalemia in KS- WNK1 -/- mice. We conclude that KS-WNK1 but not WNK4 is required for HK intake-induced stimulation of ROMK activity in DCT2/CNT. However, KS-WNK1 is not essential for HK-induced stimulation of ROMK in the CCD, and the lack of KS-WNK1 does not affect net renal K + excretion.

Chloride channels located on the basolateral membrane are known to be involved in chloride absorption in several parts of the renal tubule, and particularly in the thick ascending limb and distal convoluted tubule. The data available suggest that the ClC-K channels play the major role in this process. We provide here a description of the electrophysiological properties of these channels, still very incomplete at this stage, and we attempt to compare ClC-Ks to three chloride channels that we have identified in the basolateral membrane of microdissected fragments of the mouse renal tubule using the patch-clamp technique. Based on anion selectivity and dependence on external pH and calcium shown by the ClC-Ks, we propose candidate ClC-K1 and ClC-K2 in native tissue. We also discuss the possibility that chloride channels that do not belong to the ClC family may also be involved in the absorption of chloride across the cortical thick ascending limb., (Copyright (c) 2005 S. Karger AG, Basel.)

The distal convoluted tubule (DCT) is a heterogeneous segment subdivided into early (DCT1) and late (DCT2) parts, depending on the distribution of various transport systems. We do not have an exhaustive picture of the Cl(-) channels on the basolateral side: the presence of ClC-K2 channels is generally accepted, whereas that of ClC-K1 remains controversial. We used here single-cell RT-PCR and patch clamp to probe Cl(-) channel heterogeneity in microdissected mouse DCT at the molecular and functional levels. Our findings show that 63% of the DCT cells express ClC-K2 mRNA, either alone (type 1 cells: 47 and 23% in DCT1 and DCT2, respectively), or combined with ClC-K1, mostly in DCT2 (type 2 cells: 33%), but 37% of DCT1 and DCT2 cells do not express any ClC-K. Patch-clamp experiments revealed that a Cl(-) channel, with 9-pS conductance and Cl(-) > NO(3)(-) = Br(-) anion selectivity sequence, is present in the DCT1 and DCT2 basolateral membranes (87 and 71% of the patches, respectively). This dominant channel is likely to be ClC-K2 in type 1 cells. In type 2 cells, it could be ClC-K2 and/or ClC-K1 homodimers, but also ClC-K1/ClC-K2 heterodimers, or a mixture of all combinations. A second, distinct Cl(-) channel (13% of DCT1 patches, 29% of DCT2 patches) also displayed 9-pS conductance but had a completely different anion selectivity (I(-) > NO(3)(-) > Br(-) > Cl(-)), which was not compatible with that of the ClC-Ks. This indicates that a Cl(-) channel that is unlikely to belong to the ClC family may also be involved in Cl(-) absorption in the DCT2.

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 (g(in)) of approximately 40 pS at an external [K(+)] of 145 mM. The current-voltage relationship was linear when inside-out patches were exposed to a Mg(2+)-free medium. Mg(2+) at a concentration of 1.2 mM considerably reduced the outward conductance (g(out)), yielding a g(in)/g(out) ratio of approximately 4.7. The polycation spermine (5 x 10(-7) M) reduced the open probability (P(o)) by 50 %. Channel activity was dependent upon the intracellular pH, with acid pH decreasing, and basic pH increasing, P(o). Internal ATP (2 mM) and Ca(2+) (up to 10(-3) M) had no effect. Channel activity declined irreversibly when the inner side of the patch was exposed to Mg(2+). 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 Ca(2+). 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.

Background: The fine control of NaCl absorption takes place in the distal parts of the renal tubule, but the regulation of Cl(-) transport in this region has not been fully elucidated. We have analysed the effects of dD-arginine vasopressin (dDAVP) on Cl(-) fluxes in cultured mouse distal convoluted tubule (mpkDCT), cortical collecting duct (mpkCCD) and inner medullary collecting duct (mpkIMCD) cell lines., Methods: RT-PCR and Western blotting were used to detect the amiloride-sensitive sodium channel (ENaC) and cystic fibrosis transmembrane conductance regulator (CFTR) mRNAs and protein in cultured mpkDCT, mpkCCD and mpkIMCD cells. Cl(-) fluxes were analysed by measuring the short-circuit current (I(sc)) and bidirectional (36)Cl(-) fluxes on confluent cells grown on filters., Results: All three cell lines expressed ENaC and CFTR and had I(sc) stimulated by dDAVP. The rise in I(sc) caused by dDAVP (10(-8) M) was inhibited by amiloride, and to a lesser extent by 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB) in all three cell lines. The dDAVP-dependent I(sc) measured under apical Na(+)-free condition was reduced by Cl(-) channel blockers with a profile (NPPB>glibenclamide>DIDS), similar to that for rat CFTR. dDAVP stimulated the apical-to-basal (36)Cl(-) flux and to a lesser extent the basal-to-apical (36)Cl(-) flux under open-circuit condition in all three cultured cell lines. Adding NPPB to the apical side reduced the basal-to-apical (36)Cl(-) flux but not the opposite (36)Cl(-) flux from dDAVP-treated cells., Conclusion: These results indicate that dDAVP stimulates the bi-directional flux of Cl(-), resulting in net Cl(-)absorption, in these cultured mouse distal and collecting duct cells. I(sc) experiments also suggest the presence of a minor component of electrogenic Cl(-) secretion, possibly mediated by CFTR.

The effects of extracellular nucleotides on the cytosolic free Ca2+ concentration ([Ca2+]i) of mouse cortical thick ascending limb (CTAL) segments were investigated using the Ca(2+)-sensitive fluorescent probe fura 2. ATP (50% effective dose, ED50, 40 microM) transiently increased [Ca2+]i, while adenosine (a P1 purinoceptor agonist), N6-cyclohexyladenosine (an A1 agonist), AMP, ADP (a P2t agonist), beta, gamma-methyleneadenosine 5'-triphosphate (a P2x agonist), or 2-methylthioadenosine 5'-triphosphate (a P2y agonist) all had little or no effect. CTAL tubules were also sensitive to UTP. The responses to 100 microM ATP and UTP were similar but not additive. Both [Ca2+]i responses were strongly inhibited by 300 microM suramin (a P2 purinoceptor antagonist). Adenosine 5'-O-(3- thiotriphosphate) and ITP were slightly less potent than ATP, while GTP and CTP had no effect. The absence of external Ca2+ or the presence of 50 microM nifedipine similarly and markedly reduced the ATP- and UTP-evoked [Ca2+]i transients. We conclude that mouse CTAL tubules possess nucleotide receptors that are equally sensitive to ATP and UTP and that transiently elevate [Ca2+]i by triggering Ca2+ entry via a nifedipine-sensitive pathway.

The transepithelial potential difference (VTE) and transepithelial resistance (RTE) were determined along the length of the distal tubule of the amphibian Triturus alpestris. The site of impalements was determined at the end of each experiment by latex injection and microdissection. Two segments, differing by their distribution at the surface of the kidney, by their respective diameters and by their electrical properties could be identified: the early distal tubule (EDT) and the late distal tubule (LDT). VTE was invariably positive in the EDT; it increased from approximately + 5 mV to approximately + 22 mV within the first 30% of this segment and remained roughly constant distally to this site. RTE was estimated at 57 omega X cm2 in the EDT. The LDT exhibited essentially negative VTE figures with the exception of its very initial portion; the peak negativity at the end of the LDT was -35 mV. RTE was assessed from 8 measurements performed in the first 30% of the LDT; the tentative transepithelial resistance was 530 omega X cm2. Provisional evidence suggests that the transepithelial resistance of the terminal portion of the LDT may be substantially larger than the resistance measured in other parts of this segment.

Double-barreled microelectrodes selective to either potassium or sodium were used to determine the transepithelial potential difference (VTE) and the intraluminal activity of potassium (alpha LuK) or sodium (alpha LuNa) in the early distal tubule (EDT) of Triturus waltlii in vivo; luminal activities were compared with the corresponding plasma ion activities, alpha PtK and alpha PtNa. The transepithelial equilibrium potentials for potassium (EK(TE] and sodium (ENa(TE] were computed from the respective transmural chemical distributions: they were used to assess the transepithelial electrochemical potential differences [(V-EK)TE and (V-ENa)TE]. By dividing the raw data into three groups of 30% total tubular length (0-30, 31-60, 61-90%), the following results were obtained. 1) VTE increases from +15 to +20 mV (lumen positive) between the first and second portion of the EDT but remains constant thereafter. 2) The alpha LuK/alpha PtK ratio decreases steadily along the EDT from 1.92 to 1.66 and then to 1.32. 3) The values of alpha LuNa/alpha PtNa in the same three subdivisions are 0.79, 0.44, and 0.45. 4) The (V-EK)TE difference is largely positive along the whole EDT: +32, +33, and +27 mV. 5) The (V-ENa)TE difference declines from +9 mV (first portion) to values statistically not different from zero in the last two thirds of the EDT.(ABSTRACT TRUNCATED AT 250 WORDS)