Abstract. Ankyrins are a family of large, membraneassociated proteins that mediate the linkage of the cytoskeleton to a variety of membrane transport and receptor proteins. A repetitive 33-residue motif characteristic of domain

Abstract. In nonerythroid cells the distribution of the cortical membrane skeleton composed of fodrin (spectrin), actin, and other proteins varies both temporally with cell development and spatially within the cell and on the membrane. In monolayers of Madin-Darby canine kidney (MDCK) cells, it has previously been shown that fodrin and Na,K-ATPase are codistributed asymmetrically at the basolateral margins of the cell, and that the distribution of fodrin appears to be regulated posttranslationally when confluence is achieved (Nelson, W. J., and P. I. Veshnock. 1987. J. Cell Biol. 104:1527-1537. The molecular mechanisms underlying these changes are poorly understood. We find that (a) in confluent MDCK cells and intact kidney proximal tubule cells, Na,K-ATPase, fodrin, and analogues of human erythrocyte ankyrin are precisely colocalized in the basolateral domain at the ultrastructural level. (b) This colocalization is only achieved in MDCK cells after confluence is attained. (c) Erythrocyte ankyrin binds saturably to Na,K-ATPase in a molar ratio of ",d ankyrin to 4 Na,K-ATPase's, with a kD of 2.6/~M. (d) The binding of ankyrin to Na,K-ATPase is inhibited by the 43-kD cytoplasmic domain of erythrocyte band 3. (e) ~25I-labeled ankyrin binds to the alpha subunit of Na,K-ATPase in vitro. There also appears to be a second minor membrane protein of • 240 kD that is associated with both erythrocyte and kidney membranes that binds ~25I-labeled ankyrin avidly. The precise identity of this component is unknown. These results identify a molecular mechanism in the renal epithelial cell that may account for the polarized distribution of the fodrin-based cortical cytoskeleton.

Active K absorption in the rat distal colon is energized by an apical membrane H-K-ATPase, whereas K absorption in the distal collecting duct is generally believed to be modulated by a related renal H-K-ATPase. Experiments were performed to establish the mechanism(s) by which dietary Na depletion (with resulting elevated aldosterone levels) and K depletion stimulate K absorption. A colonic H-K-ATPase-specific cDNA probe and a polyclonal antibody were utilized to measure mRNA (Northern blot analyses) and protein (Western blot and immunofluorescence studies) abundance in the distal and proximal colon and renal collecting ducts and cortex of dietary Na- and K-depleted rats. Dietary Na depletion, but not K depletion, upregulated H-K-ATPase-specific mRNA and protein expression in the distal and proximal colon; Na depletion also stimulated H-K-ATPase activity in the distal colon. In contrast to the distal colon, H-K-ATPase-specific protein level in the outer medulla was enhanced by dietary K depletion, but not by Na depletion. This study establishes that 1) dietary Na depletion stimulates colonic H-K-ATPase activity most likely by a transcriptional process and 2) the regulation of colonic H-K-ATPase expression by dietary Na depletion and dietary K depletion is not identical in the large intestine and differs in the kidney from the colon, suggesting the presence of two (or more) H-K-ATPase isoforms in the rat colon.

A putative cDNA for the colonic K-ATPase has recently been cloned (Crowson, M. S., and G. E. Shull. 1992. J. Biol. Chem. 267:13740-13748). Considerable evidence exists that there are two K-ATPases and active K absorptive processes in the rat distal colon: one that is ouabain sensitive and the other ouabain insensitive. The present study used the baculovirus expression system to express K-ATPase activity in insect Spodoptera frugiperda (Sf 9) cells and a polyclonal antibody (M-1), developed against a fusion protein produced from the 327 nucleotide fragment from 5' coding region of the putative K-ATPase cDNA, to identify the specific localization of the K-ATPase protein. K-ATPase activity (28.7+1.2 nmol inorganic phosphate/mg protein min) was expressed in plasma membranes isolated from Sf 9 cells infected with baculovirus containing recombinant DNA with the putative K-ATPase cDNA. Km for K for the K-ATPase was 1.2 mM. The expressed K-ATPase activity was not inhibited by ouabain (1 mM); while the K; for vanadate inhibition was 8.3 ,uM. Western blot analysis with the M-1 antibody identified a 100-kD protein in apical membranes prepared from distal, but not proximal, rat colon. Immunohistochemical studies with M-1 antibody localized K-ATPase only in the apical membrane of surface cells, while an mAb (c464.6) against Na,K-ATPase localized basolateral membranes of both surface and crypt cells of rat distal colon. In conclusion, the putative K-ATPase cDNA encodes an ouabain-insensitive K-ATPase that is present only in the apical membrane of surface cells of rat distal colon. (J. Clin.

To determine whether heat shock proteins (HSPs) might be active in cellular recovery following transient ischemia, we examined rat kidneys for 70-kDa HSP (HSP-70) mRNA expression, protein elaboration, and intracellular localization after 45 min of renal ischemia and reflow of 15 min, 2, 6, and 24 h. Inducible HSP-70 mRNA is present at 15 min of reperfusion, peaks between 2 and 6 h, and falls by 24 h. Inducible 72-kDa HSP (HSP-72) protein accumulates progressively through 24 h and is found in both soluble and microsomal fractions following ischemia. Within proximal tubules, immunofluorescent localization of HSP-72 is restricted to the apical domain at 15 min, is dispersed through the cytoplasm in a vesicular pattern at 2 and 6 h, and has migrated away from the apical domain at 24 h. A portion of the vesicular HSP-72 is associated with lysosomes; no intranuclear HSP-72 is detected. The course of mRNA induction, protein elaboration, and HSP-72 localization coincides with previously described changes in proximal tubule morphology and polarity following sublethal ischemic injury. HSP-72 may be instrumental in cellular remodeling and restitution of epithelial polarity during recovery from ischemic renal injury.

Abstract. The purpose of the study was to determine whether Hsp27 interacts with actin and could protect against selected manifestations of injury from energy depletion in renal epithelia.

Renal ischemia results in both a profound fall in cellular ATP and a rapid induction of the 70 kD heat-shock protein family, HSP-70. The present studies examined the relationship between cellular ATP and induction of the stress response in renal cortex. Cellular ATP, continuously monitored by in vivo 31P-NMR spectroscopy, was reduced and maintained at specific, stable levels in renal cortex by partial aortic occlusion for 45 min. Activation of heat-shock transcription factor (HSF) was detected by gel retardation assay and transcription was confirmed by Northern analysis. Activation of HSF was not present, and HSP-70 mRNA induction did not occur when ATP levels were maintained above 60% preocclusion (control) levels. Reduction in cortical ATP levels to 35-50% preocclusion values resulted in HSF activation and low-level expression of inducible HSP-70 mRNA. Cellular ATP of 20-25% control values resulted in a greater level of HSF activation and subsequent HSP-70 mRNA elaboration. HSF was activated at the end of 15 min of total occlusion. The studies indicate that a 50% reduction in cellular ATP in the renal cortex must occur before the stress response is detectable, that reduction of ATP below 25% control levels produces a more vigorous response, and that reperfusion is not required for initiation of a heatshock response in the kidney. Cellular ATP, or the metabolic consequences associated with ATP depletion, may be a threshold factor for initiation of a stress response in the kidney. (J. Clin. Invest. 1994. 94:1518-1523

Renal ischemia causes redistribution of Na(+)-K(+)-adenosinetriphosphatase (Na(+)-K(+)-ATPase) to the apical membrane of proximal tubules. We determined the time course of regeneration of Na(+)-K(+)-ATPase polarity and sought evidence of increased enzyme production during recovery as a means to restore polarity. Anesthetized rats underwent 45 min renal ischemia and reflow of 15 min, 2 h, 6 h, and 24 h. Immunofluorescent and electron microscopy showed loss of strict basolateral localization of Na(+)-K(+)-ATPase at 15 min reflow with repolarization by 24 h in sublethally injured cells. Both alpha 1- and beta-subunits were only in microsomal fractions at all reflow intervals. Immunodetectable levels of both subunits declined to 60-70% of control by 24 h reflow. Levels of mRNA for each subunit declined in parallel through 24 h to 55% of control. Overall transcription was profoundly depressed through 6 h but had recovered to near control by 24 h. Specific transcription of alpha 1- and beta-subunit mRNA was markedly decreased after ischemia and only partially recovered by 24 h. These results suggest that recycling of misplaced units rather than new Na(+)-K(+)-ATPase production is the means by which renal epithelia initially repolarize after ischemic injury.