Expression of calcineurin activity and alpha-subunit isoforms in specific segments of the rat nephron

Calcineurin activity and alpha-subunit expression were studied in microdissected proximal tubules (S2), medullary thick ascending limbs (MTAL), cortical collecting ducts (CCD), connecting tubules (CNT), and outer medullary collecting ducts (OMCD). We have shown that cyclosporin A (CsA) and FK-506 inhibit sodium-potassium-adenosinetriphosphatase (Na-K-ATPase) activity in CCD, OMCD, and MTAL but did not uncover the mechanism for resistance of proximal tubule segments to these drugs. Because cells expressing high calcineurin activity are relatively resistant to the biological effects of CsA and FK-506, we hypothesized that the resistance of proximal tubules may be linked to increased calcineurin expression. Consequently, we measured calcineurin activity in microdissected tubules using a calcineurin-specific substrate. Calcineurin activity in S2 proximal tubule segments was 10-fold higher than in CCD, CNT, OMCD, or MTAL. FK-506 (6.0 ng/ml) inhibited calcineurin activity in CCD, CNT, and MTAL but not S2; 250 ng/ml FK-506 inhibited S2 calcineurin activity by 50%. Likewise, high concentrations of CsA (25 micrograms/ml) and FK-506 (250 ng/ml) inhibited S2 Na-K-ATPase activity by 77 and 73%, respectively. To investigate whether the resistance of S2 segments might be due to differential expression of calcineurin alpha-subunit isoforms, we determined the isoform expression by Western blot analysis using isoform-specific antibodies against the alpha 1-, alpha 2-, and alpha 3-isoforms. We found that alpha 1 expression in S2 was significantly greater than in the CCD and MTAL, whereas alpha 2 expression in the S2 was significantly less than in CCD and MTAL. No alpha 3 was detected in any nephron segment tested.(ABSTRACT TRUNCATED AT 250 WORDS)