Nephrol Dial Transplant (2002) 17: 1742-1750
© 2002 European Renal Association-European Dialysis and Transplant Association
Characterization of a Na+–Ca2+ exchanger in podocytes
1 University Hospital Freiburg, Department of Medicine, Division of Nephrology and General Medicine, Freiburg, Germany and 2 Nephrological Center, Medical Policlinic, Ludwig-Maximilians-University Munich, Munich, Germany
Background. Knowledge about Ca2+ extrusion mechanisms in podocytes is limited. The aim of the study was to test whether a Na+–Ca2+ exchanger (NCX) is present in differentiated podocytes and if so to examine its regulatory properties.
Methods. Intracellular Ca2+ concentration ([Ca2+]i) and intracellular pH were measured microspectrofluorometrically in single podocytes. Expression of NCX mRNA was studied by reverse transcription–polymerase chain reaction. NCX protein expression was investigated by immunocytochemistry.
Results. Substitution of extracellular Na+ (from 145 to 0, 5, 10, 20, and 30 mM) with N-methyl-D-glucamine resulted in a Na+ concentration-dependent, reversible increase of [Ca2+]i. Complete extracellular Na+ substitution (0 Na+) increased [Ca2+]i reversibly from 95±5 to 275±16 and back to 66±5 nM (n=205). Raising the intracellular Na+ concentration by application of 50 µM monensin increased [Ca2+]i from 105±22 to 192±45 nM (n=12). The [Ca2+]i response induced by a low Na+ concentration required extracellular Ca2+ and did not correlate with changes of intracellular pH. The effect was blocked by the NCX inhibitor benzamil (IC50
100 nM). Neither flufenamate (100 µM, n=6), a blocker of non-selective cation channels, nor Hoe 694 (1 µM, n=6), an inhibitor of the Na+–H+ exchanger, did significantly influence the [Ca2+]i response induced by extracellular Na+ depletion. Activation of protein kinase C (PKC) by short-term application (5 min) of phorbol 12-myristate-13-acetate (PMA; 10 nM, n=4; 100 nM, n=7) inhibited Na+–Ca2+ exchange, whereas PKC inhibition by long-term incubation (24 h) with PMA (100 nM, n=9) or bisindolylmaleimide I (100 nM, n=11) both increased Na+–Ca2+ exchange, respectively. Expression of NCX mRNA was detected both in cultured differentiated podocytes and in podocytes directly pulled off from glomeruli ex vivo. NCX protein expression was detected by immunocytochemistry. In a different series of experiments, we studied the potential involvement of the exchanger in podocyte injury induced by the aminonucleoside puromycin. Pre-treatment of podocytes with 0.3 mM puromycin for 24 h significantly reduced the [Ca2+]i response induced by extracellular Na+ depletion (n=56). Compared with mRNA expression of the housekeeping gene GAPDH, NCX mRNA expression was significantly reduced by puromycin.
Conclusion. Our results demonstrate the presence of a Na+–Ca2+ exchanger in podocytes and its regulation by PKC. Inhibition of Na+–Ca2+ exchange by puromycin may contribute to podocyte injury in PAN nephrosis.
Keywords: immunocytochemistry; intracellular Ca2+ concentration; intracellular pH; Na+–Ca2+ exchange; podocyte; protein kinase C; puromycin aminonucleoside
Correspondence and offprint requests to: Dr Karl-Georg Fischer, MD, University Hospital Freiburg, Department of Medicine, Division of Nephrology and General Medicine, Hugstetter Str. 55, D-79106 Freiburg, Germany. Email: fischer{at}med1.ukl.uni\|[hyphen]\|freiburg.de
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