To determine if
ischemia induces alterations in renal proximal tubule surface membranes, brush border (BBM) and basolateral membranes (BLM) were isolated simultaneously from the same cortical homogenate after 50 min of renal pedicle clamping.
Ischemia caused a selective decrease in the specific activity of BBM marker
enzymes leucine aminopeptidase and
alkaline phosphatase, but did not effect enrichment (15 times). Neither specific activity nor enrichment (10 times) of BLM NaK-
ATPase was altered by
ischemia. Contamination of BBM by intracellular organelles was also unchanged, but there was an increase in the specific activity (41.1 vs. 60.0, P less than 0.01) and enrichment (2.3 vs. 4.3, P less than 0.01) of NaK-
ATPase in the ischemic BBM fraction.
Ischemia increased BLM
lysophosphatidylcholine (1.3 vs. 2.5%, P less than 0.05) and
phosphatidic acid (0.4 vs. 1.3%, P less than 0.05).
Ischemia also decreased BBM
sphingomyelin (38.5 vs. 29.6%, P less than 0.01) and
phosphatidylserine (16.1 vs. 11.4%, P less than 0.01), and increased
phosphatidylcholine (17.2 vs. 29.7%, P less than 0.01),
phosphatidylinositol (1.8 vs. 4.6%, P less than 0.01), and
lysophosphatidylcholine (1.0 vs. 1.8%, P less than 0.05). The large changes in BBM
phospholipids did not result from new
phospholipid synthesis, since the specific activity (32P dpm/nmol Pi) of prelabeled individual and total
phospholipids was unaltered by
ischemia. We next evaluated if these changes were due to inability of ischemic cells to maintain surface membrane polarity. Cytochemical evaluation showed that while NaK-
ATPase could be detected only in control BLM, specific deposits of reaction product were present in the BBM of ischemic kidneys. Furthermore, using continuous
sucrose gradients, the enzymatic profile of ischemic BBM NaK-
ATPase shifted away from ischemic BLM NaK-
ATPase and toward the BBM enzymatic marker
leucine aminopeptidase. Taken together, these data suggest that NaK-
ATPase activity determined enzymatically and cytochemically was located within ischemic BBM. We propose that
ischemia impairs the ability of cells to maintain surface membrane polarity, and also results in the accumulation of putative
calcium ionophores.