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  • 1
    ISSN: 1432-2013
    Keywords: Isolated perfused tubule ; cTAL ; Na+ 2Cl− K+ cotransporter ; piretanide ; macromolecular probe
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract Piretanide blocks the Na+ 2Cl− K+ cotransporter protein in the thick ascending limb (TAL) of the loop of Henle reversibly. When tested from the luminal side in isolated perfused cTAL segments it leads to a half maximal inhibition (IC50) of the equivalent short circuit current (Isc) at a concentration of 10−6 mol/l. From the basolateral side it has no effect on Isc up to 10−4 mol/l. The present study was designed to search for high affinity blockers of the Na+ 2Cl− K+ cotransporter with large molecular weight in an attempt to use these macromolecules for antibody-labelling or affinity separation of this transport-protein. Amino-ethyl-dextran or amino-ethyl-polyethylene glycol (M.W. 5kd) were coupled to isothiocyanato-piretanide (ISO-PIR) at room temperature in DMSO. The resulting compounds dextran-sulfonylurea-piretanide (PIR-DEX) and polyethylene glycol-sulfonylurea-piretanide (PIR-PEG) (M.W. 5.38kd) were purified and tested in isolated perfused cTAL segments. IC50 values for ISO-PIR, PIR-DEX and PIR-PEG were estimated from dose response curves after their addition to the lumen or bath perfusate, respectively. ISO-PIR, PIR-DEX and PIR-PEG acted from the lumen side at 3·10−6, 6·10−6 and 2·10−6 mol/l. The inhibitory effect was easily reversible. From the basolateral side no effect for any compound was seen at up to 10−4 mol/l. In clearance experiments PIR-DEX was given to female Wistar rats as an i.v. bolus (25 μmol/kg) and the diuretic urine was collected. After dialysis (exclusion limit 2.5kd) the dialysed urine and the dialysate were tested in isolated perfused cTAL segments. The dialysates had no effect on Isc, but the dialysed urine inhibited Isc by 35% from the luminal side. The present data show: High molecular derivatives of piretanide with dextran or polyethylene glycol moieties block the Na+ 2Cl− K+ cotransporter in cTAL segments at roughly the same low concentration as piretanide itself. Our data exclude a metabolism of these piretanide compounds in the kidney. Since these macromolecular probes can probably not enter the cell their inhibitory effect indicates that the binding site for piretanide diuretics on the Na+ 2Cl− K+ cotransporter is exposed on the surface of the luminal cell membrane.
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  • 2
    ISSN: 1432-2013
    Keywords: Isolated perfused tubule ; Macula densa ; Intracellular voltage ; Furosemide ; Tubuloglomerular feedback
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract The macula densa cells of the juxtaglomerular apparatus probably serve as the sensor cells for the signal which leads to the appropriate tubuloglomerular feedback response. The present study reports basolateral membrane voltage (PDbl) measurements in macula densa cells. We isolated and perfused in vitro thick ascending limb segments with the glomerulus, and therefore the macula densa cells, and the early distal tubule still attached. Macula densa cells were impaled with microelectrodes under visual control. PDbl was recorded in order to examine how these cells sense changes in luminal NaCl concentrations. The addition of furosemide, a specific inhibitor of the Na+2Cl−K+ cotransporter in the thick ascending limb, to the lumen of the perfused thick ascending limb hyperpolarized PDbl from −55±5 mV to −79±4 mV (n=7). Reduction of NaCl in the lumen perfusate from 150 mmol/l to 30 mmol/l also hyperpolarized PDbl from −48±3 mV to −66±5 mV (n=4). A Cl− concentration step in the bath from 150 mmol/l to 30 mmol/l resulted in a 24±4 mV (n=4) depolarization of PDbl. This depolarization of PDbl was absent when furosemide was present during the Cl− concentration step. These data suggest that the macula densa cells sense changes in luminal NaCl concentration via coupled uptake of Na+ and Cl−. The transport pathways for NaCl transport in macula densa cells are probably identical to those in the thick ascending limb: the (Na++K+)-ATPase in the basolateral membrane drives Na+ and Cl− uptake via the luminal Na+2Cl−K+ cotransport, Cl− leaves the cell via basolateral Cl− channels and K+ recycles across the apical membrane via K+ channels. Changes in intracellular Cl− activity as a result of altered luminal NaCl uptake, and thus voltage changes of the basolateral membrane are probably the first signal in the tubuloglomerular feedback regulation.
    Type of Medium: Electronic Resource
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  • 3
    ISSN: 1432-2013
    Keywords: Glucagon ; Transepithelial ion net fluxes ; Na+, Cl−, K+, Ca2+, Mg2+ transport ; Electron microprobe ; Mouse kidney ; In vitro microperfusion ; Cortical and medullary thick ascending limb of Henle's loop ; In vivo micropuncture study
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract The effects of glucagon on transepithelial Na+, Cl−, K+, Ca2+ and Mg2+ net fluxes were investigated in isolated perfused cortical (cTAL) and medullary (mTAL) thick ascending limbs of Henle's loop of the mouse nephron. Transepithelial ion net fluxes (J Na +,J Cl −,J K +,J Ca 2+,J Mg 2+) were determined by electron probe analysis of the collected tubular fluid. Simultaneously the transepithelial voltage (PDte) and the transepithelial resistance (R te) were recorded. In cTAL-segments (n=8), glucagon (1.2×10−8 mol · l−1) stimulated significantly the reabsorption of Na+, Cl−, Ca2+ and Mg2+∶J Na + increased from 204±20 to 228±23 pmol · min−1 · mm−1,J Cl − from 203±18 to 234±21 pmol · min−1 · mm−1,J Ca 2+ from 0.52±0.13 to 1.34±0.30 pmol · min−1 · mm−1 andJ Mg 2+ from 0.51±0.08 to 0.84±0.08 pmol · min−1 · mm−1.J K+ remained unchanged: 3.2±1.3 versus 4.0±1.9 pmol · min−1 · mm−1. Neither PDte (16.3±1.5 versus 15.9±1.4 mV) norR te (22.5±3.0 versus 20.3±2.6 Ωcm2) were changed significantly by glucagon. However, in the post-experimental periods a significant decrease in PDte and increase inR te were noted. In mTAL-segments (n=9), Mg2+ and Ca2+ transports were close to zero and glucagon elicited no significant effect. The reabsorptions of Na+ and Cl−, however, were strongly stimulated:J Na + increased from 153±17 to 226±30 pmol · min−1 · mm−1 andJ Cl − from 151±23 to 243±30 pmol · min−1 · mm−1. The rise in NaCl transport was accompanied by an increase in PDte from 10.3±1.1 to 12.3±1.2 mV and a decrease inR te from 19.1±2.7 to 17.8±2.0 Ωcm2. No net K+ movement was detectable either in the absence or in the presence of glucagon. A micropuncture study carried out in hormone-deprived rats indicated that glucagon stimulates Na+, Cl−, K+, Mg2+ and Ca2+ reabsorptions in the loop of Henle. In conclusion our data demonstrate that glucagon stimulates NaCl reabsorption in the mTAL segment and to a lesser extent in the cTAL segment whereas it stimulates Ca2+ and Mg2+ reabsorptions only in the cortical part of the thick ascending limb of the mouse nephron. These data are in good agreement with, and extend, those obtained in vivo on the rat with the hormone-deprived model.
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