Evidence that mouse renal proximal tubule cells produce nephrocalcin

Nephrocalcin (NC) is a glycoprotein inhibitor of calcium oxalate monohydrate crystal growth present in urine and kidney tissue. To determine if kidney cells can produce NC, we have isolated proximal tubules from mouse kidney and cultured them in a serum-free medium containing supplements. The tubules accumulate p-aminohippurate and respond with increase in adenosine 3',5'-cyclic monophosphate to parathyroid hormone but not to arginine vasopressin. They produce 1,25 dihydroxyvitamin D3 when 25 hydroxyvitamin D3 is added as substrate. Medium conditioned for 3 days reacted by direct enzyme-linked immunosorbent assay (ELISA) with a rabbit antiserum to human urinary NC; the same antiserum was localized to the cells using immunoperoxidase staining. Using ion exchange and molecular sieve chromatography, we purified a glycoprotein calcium oxalate crystal-growth inhibitor the chromatographic behavior and amino acid and carbohydrate compositions of which closely resembled that of purified human urinary NC. Its dissociation constant toward the calcium oxalate crystal was 0.4-10 X 10(-7) M. Purified fractions of the protein cross-reacted with the antiserum tested by ELISA. Cultured proximal tubule cells produce a molecule that resembles NC from human urine, rat urine, and rat kidneys; proximal tubule is a probable site of NC production in vivo.