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Induced alterations in calcium uptake rate in normoxic rate proximal tubules
T J Burke1, J K Joseph, D Bunnachak
1Department of Medicine, University of Colorado School of Medicine, Denver 80262, USA.
Abstract:
This study is well-oxygenated, freshly isolated rat proximal tubules (RPT), examined the effects of several drugs that alter the transmembrane K+ and Na+ gradients across cell membranes, including valinomycin (VAL), amphotericin B (AMPHO), and ouabain (OUAB). The effects of high extracellular potassium chloride (KCl) concentrations (45 mM) and low extracellular sodium concentration (100mM) were also studied. After 10 min of drug exposure Ca2+ uptake rate (nmol/mg/min) increased from 2.7 to 3.8 with VAL (p < .02), from 2.9 to 3.7 with AMPHO (p < .05), from 3.6 to 4.1 with OUAB (p < .05), and from 3.2 to 4.8 with 45 mM KCl (p < .001). Ca2+ uptake rate was sustained at these high levels at 20 min in all treated RPT except those exposed to OUAB. LDH release averaged less than 15% in control tubules and did not increase significantly except in RPT treated with VAL, where LDH release at 10 min was 48% and at 20 min was 57% (both p < .001). Of importance, only in VAL-treated RPT did ATP decrease to low levels (6.7 nmol/mg in control to 2.0 +/- 0.3 nmol/mg in VAL, p < .001). Treatment with verapamil reduced Ca2+ uptake rates at 10 min in VAL-treated RPT (from 3.8 to 3.1, p < .02, in AMPHO-treated RPT (from 3.8 to 3.1 p < .001), in OUAB-treated tubules (from 4.0 to 3.4, p < .01), and in KCl-treated RPT (from 3.7 to 3.2, p < .01). These results indicate that acute changes in the transmembrane ion gradient in RPT are accompanied by increased Ca2+ uptake rates. Ca2+ uptake rates are also increased during O2 deprivation in RPT, a situation in which the transmembrane ion gradient is likewise altered. The increased Ca2+ uptake rate observed in the present study and during hypoxia may have a common basis, that is, altered transmembrane ion gradients or some function thereof.
Insights
Altering transmembrane ion gradients in rat proximal tubules (RPT) with drugs like valinomycin (VAL) or high potassium chloride (KCl) significantly increases calcium (Ca2+) uptake. This suggests a common mechanism linking ion gradient changes to elevated Ca2+ influx, potentially relevant during hypoxia.
Area of Science:
- Biochemistry
- Cell Physiology
- Renal Physiology
Background:
- Transmembrane ion gradients are critical for cellular function in renal proximal tubules (RPT).
- Disruptions in these gradients can impact cellular processes, including ion transport and energy metabolism.
- Calcium (Ca2+) homeostasis is vital for kidney tubule function and is influenced by ion gradients.
Purpose of the Study:
- To investigate the effects of altered transmembrane potassium (K+) and sodium (Na+) gradients on Ca2+ uptake in isolated rat proximal tubules (RPT).
- To examine the impact of specific ionophores (valinomycin, amphotericin B) and ion concentration changes (high KCl, low Na+) on Ca2+ influx.
- To explore the relationship between altered ion gradients, Ca2+ uptake, and cellular integrity markers like lactate dehydrogenase (LDH) release and ATP levels.
Main Methods:
- Isolated, well-oxygenated rat proximal tubules (RPT) were exposed to valinomycin (VAL), amphotericin B (AMPHO), ouabain (OUAB), high extracellular KCl (45 mM), or low extracellular Na+ (100 mM).
- Calcium (Ca2+) uptake rates were measured after 10 and 20 minutes of exposure.
- Lactate dehydrogenase (LDH) release and adenosine triphosphate (ATP) levels were assessed to evaluate cell viability and metabolic status. Verapamil was used to assess Ca2+ channel involvement.
Main Results:
- Exposure to VAL, AMPHO, OUAB, and high KCl significantly increased Ca2+ uptake rates in RPT within 10 minutes.
- This elevated Ca2+ uptake was generally sustained at 20 minutes, except for OUAB treatment.
- Valinomycin (VAL) treatment led to significant LDH release and a marked decrease in ATP levels, indicating cellular damage and metabolic compromise. Verapamil attenuated the increased Ca2+ uptake across all treatment groups.
Conclusions:
- Acute alterations in transmembrane ion gradients in RPT are directly associated with increased Ca2+ uptake rates.
- The observed increase in Ca2+ uptake during ion gradient disruption may share a common mechanism with that seen during oxygen deprivation (hypoxia).
- These findings highlight the sensitivity of RPT Ca2+ handling to changes in ion gradients and cellular energy status.