Characterization of a Na+-Ca2+ exchanger in podocytes

Abstract

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 microspectro-fluorometrically 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 ∼ 100nM). 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 (100nM, n = 9) or bisindolylmaleimide I (100nM, 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 immunocyto-chemistry. 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.