Immunohistochemical localization of CA-VI in the pig kidney was performed as reported previously (Nishita et al., 2011). To estimatetheCA-VIconcentrationsinurine,Westernblottingwasperformed following sodium dodecyl sulfate polyacrylamide gel electrophoresis and used anti-pig CA-VI serum that had been produced in our laboratory. To quantify the amount of CA-VI in the kidneys, renal tissues from the healthy pigs (6 month-old females, n=7) that contained both the cortex and medulla were homogenized with 0.01M Tris-HCl (pH 8.0), and the homogenates were centrifuged at 4°C for 30min at 30,000g. Urine samples from 56 clinically normal domestic female pigs (2–5 years old) and 65 pigs with kidney disease (1–5 years old) were collected using a urethral catheter and were stored at −20°C until use.