title: Effect of chronic metabolic acidosis on calbindin expression along the rat distal tubule.
abstract: Calbindin D28k has been reported to be involved in the transcellular calcium transport along the rat distal tubule. It has also been shown that chronic metabolic acidosis (CMA) induces significant hypercalciuria. The present study investigated whether CMA affects the mRNA and the protein expression of calbindin D28k along isolated distal tubule (DT) of rats. The animals were made acidotic by adding 0.28 mol/L NH4Cl to the drinking water for 7 d. This maneuver was associated with an increase in plasma ionized calcium. Inulin clearance experiments demonstrated that metabolic acidosis did not affect GFR, but it significantly increased both total and fractional urinary calcium excretion. To define the role of calbindin D28k, total RNA was extracted from DT, identified, and microdissected from collagenase-treated kidneys. cDNA was synthesized from RNA using reverse transcriptase and oligo(dT)(12-18) primers. Calbindin D28k mRNA abundance was semiquantified by a competitive reverse transcription-PCR, using an internal standard of cDNA that differed from the wild-type calbindin D28k by a deletion of 86 bp. The reverse transcription-PCR was performed starting from the same amount of total RNA. For each set of experiments, control and acidotic rats were studied in parallel. The identity of the DT was further verified by the presence of the thiazide-sensitive NaCl cotransporter (rTSC1) mRNA. Calbindin D28k mRNA abundance was 0.89 +/- 0.21 amol/ng total RNA in DT of CMA rats (n = 5) compared with 0.30 +/- 0.12 amol/ng total RNA of control rats (n = 5) (P < 0.05). Using specific rabbit polyclonal anti-calbindin D28k antibody, Western blotting was performed starting from thin slices of outer cortex. Densitometric analysis revealed that in acidotic rats (n = 7) there was a 17 +/- 5% (P < 0.05) increase in calbindin D28k protein abundance compared with controls (n = 7). These results indicate that in the rat, ammonium chloride loading induces an increase in filtered ionized calcium load that is associated with a significant upregulation of calbindin D28k both at the mRNA and protein level. These last effects will help to reduce the concomitant hypercalciuria, thus mitigating the consequence of CMA on calcium metabolism.
authors.affiliation: Chair of Nephrology, Second University of Napoli, Italy.
authors.forename: Maria
authors.initials: M
authors.lastname: Rizzo
authors.affiliation: Chair of Nephrology, Second University of Napoli, Italy.
authors.forename: Giovambattista
authors.initials: G
authors.lastname: Capasso
authors.affiliation: Institute of Cellular Signalling, University of Nijmegen, The Netherlands.
authors.forename: Markus
authors.initials: M
authors.lastname: Bleich
authors.affiliation: Chair of Nephrology, Second University of Napoli, Italy.
authors.forename: Angelo
authors.initials: A
authors.lastname: Pica
authors.affiliation: Chair of Nephrology, Second University of Napoli, Italy.
authors.forename: Domenico
authors.initials: D
authors.lastname: Grimaldi
authors.affiliation: Institute of Physiology, Albert Ludwigs University, Freiburg, Germany.
authors.forename: René J M
authors.initials: RJM
authors.lastname: Bindels
authors.affiliation: Institute of Cellular Signalling, University of Nijmegen, The Netherlands.
authors.forename: Rainer
authors.initials: R
authors.lastname: Greger
mesh_terms: D000138:Acidosis / Q000378:metabolism* / Q000503:physiopathology; D000818:Animals; D015153:Blotting, Western; D064092:Calbindin 1; D064026:Calbindins; D002908:Chronic Disease; D066298:In Vitro Techniques; D007686:Kidney Tubules, Distal / Q000378:metabolism*; D008297:Male; D009399:Nephrons / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D064030:S100 Calcium Binding Protein G / Q000235:genetics / Q000378:metabolism*
chemical_list: C576500:Calb1 protein, rat; D064092:Calbindin 1; D064026:Calbindins; D012333:RNA, Messenger; D064030:S100 Calcium Binding Protein G