title: Characterization of a Na(+)-Ca(2+) exchanger in podocytes.
abstract: BACKGROUND
  Knowledge about Ca(2+) extrusion mechanisms in podocytes is limited. The aim of the study was to test whether a Na(+)-Ca(2+) exchanger (NCX) is present in differentiated podocytes and if so to examine its regulatory properties.
  
  
  METHODS
  Intracellular Ca(2+) concentration ([Ca(2+)](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 [Ca(2+)](i). Complete extracellular Na(+) substitution (0 Na(+)) increased [Ca(2+)](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 micro M monensin increased [Ca(2+)](i) from 105+/-22 to 192+/-45 nM (n=12). The [Ca(2+)](i) response induced by a low Na(+) concentration required extracellular Ca(2+) and did not correlate with changes of intracellular pH. The effect was blocked by the NCX inhibitor benzamil (IC(50) approximately 100 nM). Neither flufenamate (100 micro M, n=6), a blocker of non-selective cation channels, nor Hoe 694 (1 micro M, n=6), an inhibitor of the Na(+)-H(+) exchanger, did significantly influence the [Ca(2+)](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(+)-Ca(2+) 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(+)-Ca(2+) 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 [Ca(2+)](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.
  
  
  CONCLUSIONS
  Our results demonstrate the presence of a Na(+)-Ca(2+) exchanger in podocytes and its regulation by PKC. Inhibition of Na(+)-Ca(2+) exchange by puromycin may contribute to podocyte injury in PAN nephrosis.
authors.affiliation: University Hospital Freiburg, Department of Medicine, Division of Nephrology and General Medicine, Freiburg, Germany. fischer@med1.ukl.uni-freiburg.de
authors.forename: Karl-Georg
authors.initials: KG
authors.lastname: Fischer
authors.forename: Nils
authors.initials: N
authors.lastname: Jonas
authors.forename: Florian
authors.initials: F
authors.lastname: Poschenrieder
authors.forename: Clemens
authors.initials: C
authors.lastname: Cohen
authors.forename: Matthias
authors.initials: M
authors.lastname: Kretzler
authors.forename: Stefan
authors.initials: S
authors.lastname: Greiber
authors.forename: Hermann
authors.initials: H
authors.lastname: Pavenstädt
mesh_terms: D000584:Amiloride / Q000031:analogs & derivatives* / Q000494:pharmacology; D000818:Animals; D002118:Calcium / Q000378:metabolism; D002478:Cells, Cultured; D003600:Cytosol / Q000378:metabolism; D004789:Enzyme Activation / Q000502:physiology; D005110:Extracellular Space / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007668:Kidney / Q000166:cytology* / Q000378:metabolism*; D051379:Mice; D009994:Osmolar Concentration; D011493:Protein Kinase C / Q000378:metabolism; D011692:Puromycin Aminonucleoside / Q000494:pharmacology; D012333:RNA, Messenger / Q000378:metabolism; D012964:Sodium / Q000378:metabolism; D019831:Sodium-Calcium Exchanger / Q000037:antagonists & inhibitors / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*
chemical_list: D012333:RNA, Messenger; D019831:Sodium-Calcium Exchanger; C013407:benzamil; D011692:Puromycin Aminonucleoside; D000584:Amiloride; D012964:Sodium; D011493:Protein Kinase C; D002118:Calcium