RESUMEN
It is well known that the aminoglycoside antibiotic gentamicin is capable of causing damage to kidney cells. Given the known involvement of Ca2+ in the nephrotoxic action of gentamicin, the purpose of this study was to establish a relationship between the concentration of intracellular Ca2+ ([Ca2+]i) and cellular cytotoxicity using MDCK-C11 cells, a clone that has several properties that resemble those of intercalated cells of the distal nephron. Changes in [Ca2+]i was determined using fluorescence microscopy. Cell viability was evaluated by the neutral red method, and cell cytotoxicity by the MTT method. The [Ca2+]i gradually increased when cells were exposed to 0.1 mM gentamicin for 10, 20, and 30 min. The presence of extracellular Ca2+ was found to be necessary to stimulate the increase in [Ca2+]i induced by gentamicin, since this stimulus disappeared by using 1.8 mM EGTA (a Ca2+ chelator). Morphological changes were observed with scanning electron microscopy in epithelial cells exposed to the antibiotic. Furthermore, with the MTT method, a decrease in metabolic activity induced by gentamicin was observed, which indicates a cytotoxic effect. In conclusion, gentamicin was able to alter [Ca2+]i, change the morphology of MDCK-C11 cells, and promote cytotoxicity.
Asunto(s)
Antibacterianos/toxicidad , Calcio/metabolismo , Gentamicinas/toxicidad , Células de Riñón Canino Madin Darby/efectos de los fármacos , Pruebas de Toxicidad/métodos , Animales , Membrana Celular/metabolismo , Supervivencia Celular/efectos de los fármacos , Células Clonales , Perros , Células de Riñón Canino Madin Darby/metabolismo , Células de Riñón Canino Madin Darby/ultraestructura , Microscopía Electrónica de Rastreo , Modelos Animales , Nefronas/citología , Nefronas/efectos de los fármacosRESUMEN
It is well known that the aminoglycoside antibiotic gentamicin is capable of causing damage to kidney cells. Given the known involvement of Ca2+ in the nephrotoxic action of gentamicin, the purpose of this study was to establish a relationship between the concentration of intracellular Ca2+ ([Ca2+]i) and cellular cytotoxicity using MDCK-C11 cells, a clone that has several properties that resemble those of intercalated cells of the distal nephron. Changes in [Ca2+]i was determined using fluorescence microscopy. Cell viability was evaluated by the neutral red method, and cell cytotoxicity by the MTT method. The [Ca2+]i gradually increased when cells were exposed to 0.1 mM gentamicin for 10, 20, and 30 min. The presence of extracellular Ca2+ was found to be necessary to stimulate the increase in [Ca2+]i induced by gentamicin, since this stimulus disappeared by using 1.8 mM EGTA (a Ca2+ chelator). Morphological changes were observed with scanning electron microscopy in epithelial cells exposed to the antibiotic. Furthermore, with the MTT method, a decrease in metabolic activity induced by gentamicin was observed, which indicates a cytotoxic effect. In conclusion, gentamicin was able to alter [Ca2+]i, change the morphology of MDCK-C11 cells, and promote cytotoxicity.
Asunto(s)
Animales , Perros , Gentamicinas/toxicidad , Calcio/metabolismo , Pruebas de Toxicidad/métodos , Células de Riñón Canino Madin Darby/efectos de los fármacos , Antibacterianos/toxicidad , Microscopía Electrónica de Rastreo , Membrana Celular/metabolismo , Supervivencia Celular/efectos de los fármacos , Células Clonales , Modelos Animales , Células de Riñón Canino Madin Darby/metabolismo , Células de Riñón Canino Madin Darby/ultraestructura , Nefronas/citología , Nefronas/efectos de los fármacosRESUMEN
The interaction between H+ extrusion via H+-ATPase and Cl- conductance was studied in the C11 clone of MDCK cells, akin to the intercalated cells of the collecting duct. Cell pH (pHi) was measured by fluorescence microscopy using the fluorescein-derived probe BCECF-AM. Control recovery rate measured after a 20 mM NH4Cl acid pulse was 0.136 ± 0.008 pH units/min (dpHi/dt) in Na+ Ringer and 0.032 ± 0.003 in the absence of Na+ (0 Na+). With 0 Na+ plus the Cl- channel inhibitor NPPB (10 æM), recovery was reduced to 0.014 ± 0.001 dpHi/dt. 8-Br-cAMP, known to activate CFTR Cl- channels, increased dpHi/dt in 0 Na+ to 0.061 ± 0.009 and also in the presence of 46 nM concanamycin and 50 æM Schering 28080. Since it is thought that the Cl- dependence of H+-ATPase might be due to its electrogenic nature and the establishment of a +PD (potential difference) across the cell membrane, the effect of 10 æM valinomycin at high (100 mM) K+ was tested in our cells. In Na+ Ringer, dpHi/dt was increased, but no effect was detected in 0 Na+ Ringer in the presence of NPPB, indicating that in intact C11 cells the effect of blocking Cl- channels on dpHi/dt was not due to an adverse electrical gradient. The effect of 100 æM ATP was studied in 0 Na+ Ringer solution; this treatment caused a significant inhibition of dpHi/dt, reversed by 50 æM Bapta. We have shown that H+-ATPase present in MDCK C11 cells depends on Cl- ions and their channels, being regulated by cAMP and ATP, but not by the electrical gradient established by electrogenic H+ transport.
Asunto(s)
Animales , Canales de Cloruro/metabolismo , ATPasas de Translocación de Protón/metabolismo , Línea Celular , Células Clonales , Fluoresceínas , Colorantes Fluorescentes , Concentración de Iones de Hidrógeno , Microscopía FluorescenteRESUMEN
The interaction between H(+) extrusion via H(+)-ATPase and Cl(-) conductance was studied in the C11 clone of MDCK cells, akin to the intercalated cells of the collecting duct. Cell pH (pHi) was measured by fluorescence microscopy using the fluorescein-derived probe BCECF-AM. Control recovery rate measured after a 20 mM NH(4)Cl acid pulse was 0.136 +/- 0.008 pH units/min (dpHi/dt) in Na(+) Ringer and 0.032 +/- 0.003 in the absence of Na(+) (0 Na(+)). With 0 Na(+) plus the Cl(-) channel inhibitor NPPB (10 microM), recovery was reduced to 0.014 +/- 0.001 dpHi/dt. 8-Br-cAMP, known to activate CFTR Cl(-) channels, increased dpHi/dt in 0 Na(+) to 0.061 +/- 0.009 and also in the presence of 46 nM concanamycin and 50 microM Schering 28080. Since it is thought that the Cl(-) dependence of H(+)-ATPase might be due to its electrogenic nature and the establishment of a +PD (potential difference) across the cell membrane, the effect of 10 microM valinomycin at high (100 mM) K(+) was tested in our cells. In Na(+) Ringer, dpHi/dt was increased, but no effect was detected in 0 Na+ Ringer in the presence of NPPB, indicating that in intact C11 cells the effect of blocking Cl(-) channels on dpHi/dt was not due to an adverse electrical gradient. The effect of 100 microM ATP was studied in 0 Na(+) Ringer solution; this treatment caused a significant inhibition of dpHi/dt, reversed by 50 microM Bapta. We have shown that H(+)-ATPase present in MDCK C11 cells depends on Cl(-) ions and their channels, being regulated by cAMP and ATP, but not by the electrical gradient established by electrogenic H(+) transport.
Asunto(s)
Canales de Cloruro/metabolismo , ATPasas de Translocación de Protón/metabolismo , Animales , Línea Celular , Células Clonales , Fluoresceínas , Colorantes Fluorescentes , Concentración de Iones de Hidrógeno , Microscopía FluorescenteRESUMEN
In several tissues ammonium ions are able to use the transport pathways of other ions, particularly of K+. We investigated this possibility in the C11 clone of MDCK cells, thought to represent intercalated cells, in control and 0 Cl- conditions. Cell pH was measured by ratiometric fluorescence microscopy using the pH indicator BCECF. After preincubating the cells for 10 min in control or 0 Cl- (substituted by gluconate) Ringer, an ammonium pulse was applied to induce cell acidification. The magnitude of the initial alkalinization (DeltapH) was 0.24+/-0.03 ( n=28) pH units in controls, which fell to 0.023+/-0.01 ( n=12) in 0 Cl-, suggesting uptake of NH4+ balancing the alkalinization by NH3. Addition of 10(-3) M bumetanide or furosemide to the 0 Cl- medium, or 10(-4 )M hexamethylene amiloride, did not alter DeltapH. However, with 5 mM Ba+, DeltapH increased to 38% of control. When 2.5x10(-4) M ouabain, an inhibitor of Na+-K+ ATPase, was used, DeltapH increased to 46% of control. Inhibition of H+-K+ ATPase by SCH28080 or by omeprazol caused significant increase in DeltapH. In 0 Cl- solution, these cells underwent a mean volume reduction (-d V) of -10.24+/-1.96% per 10 min as measured by confocal microscopy. To investigate if NH4+ influx was regulated by cell volume or by cell Cl-, volume reduction was avoided by two procedures. When preincubating with NPPB, a Cl- channel blocker, in 0 Cl-, volume reduction was inhibited (d V=-2.12% per 10 min), and DeltapH was 0.24+/-0.04 ( n=5). When the cells were preincubated in hypotonic 0 Cl- (260 mosmol/l), cell volume reduction was abolished (d V=+2.6% per 10 min) and DeltapH was 0.52+/-0.07 ( n=7). Thus, activation of NH4+ influx by several transporters was due to volume reduction rather than to [Cl-] alteration.