Expression of calcineurin activity and [alpha] subunit isoforms in
specific segments of the rat nephron.
Tumlin, J. A., J. T. Someren, C. E. Swanson, and J. P. Lea.
Renal Division, Department of Medicine, Emory University School of
Medicine, Atlanta, GA 30322
APStracts 2:0074F, 1995.
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 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 the 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
ug/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. We conclude that 1) the resistance of proximal tubule Na/K
-ATPase to CsA and FK-506 is related to higher calcineurin activity;
and 2) expression of calcineurin [alpha] subunit isoforms varies
between individual segments of the nephron.
Received 28 September 1994; accepted in final form 2 May 1995.
APS Manuscript Number F345-4.
Article publication pending Am. J. Physiol. (Renal Fluid Electrolyte
Physiology).
ISSN 1080-4757 Copyright 1995 The American Physiological Society.
Published in APStracts on 9 May 1995.