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Disruption of cell contact sites in renal epithelial cells contributes to organ dysfunction after ischemia. We hypothesized that heat shock protein 27 (Hsp27), a known cytoprotectant protein, preserves cell architecture and cell contact site function during ischemic stress. To test this hypothesis, renal epithelial cells were subjected to transient ATP depletion, an in vitro model of ischemia-reperfusion injury. Compared with control, selective Hsp27 overexpression significantly preserved cell-cell junction function during metabolic stress as evidenced by reduced stress-mediated redistribution of the adherens junction protein E-cadherin, higher transepithelial electrical resistance, and lower unidirectional flux of lucifer yellow. Hsp27 overexpression also preserved paxillin staining within focal adhesion complexes and significantly decreased cell detachment during stress. Surprisingly, Hsp27, an F-actin-capping protein, only minimally reduced stress induced actin cytoskeleton collapse. In contrast to Hsp27 overexpression, siRNA-mediated knockdown had the opposite effect on these parameters. Since ischemia activates c-Src, a tyrosine kinase that disrupts both cell-cell and cell-substrate interactions, the relationship between Hsp27 and c-Src was examined. Although Hsp27 and c-Src did not coimmunoprecipitate and Hsp27 overexpression failed to inhibit whole cell c-Src activation during injury, manipulation of Hsp27 altered active c-Src accumulation at cell contact sites. Specifically, Hsp27 overexpression reduced, whereas Hsp27 knockdown increased active [p-.sup.416]Src detected at contact sites in intact cells as well as in a purified cell membrane fraction. Together, this evidence shows that Hsp27 overexpression prevents sublethal REC injury at cell contact sites possibly by a c-Src-dependent mechanism. Further exploration of the biochemical link between Hsp27 and c-Src could yield therapeutic interventions for ameliorating ischemic renal cell injury and organ dysfunction. ischemia; renal failure; acute kidney injury; ATP depletion; renal epithelial cells

[H.sup.+] transport in the collecting duct is regulated by exocytic insertion of [H.sup.+]-ATPase-laden vesicles into the apical membrane. The soluble N-ethylmaleimide-sensitive fusion protein attachment protein (SNAP) receptor (SNARE) proteins are critical for exocytosis. Syntaxin 1A contains three main domains, SNARE N, H3, and carboxy-terminal transmembrane domain. Several syntaxin isoforms form SNARE fusion complexes through the H3 domain; only syntaxin 1A, through its H3 domain, also binds [H.sup.+]-ATPase. This raised the possibility that there are separate binding sites within the H3 domain of syntaxin 1A for [H.sup.+]-ATPase and for SNARE proteins. A series of truncations in the H3 domain of syntaxin 1A were made and expressed as glutathione S-transferase (GST) fusion proteins. We determined the amount of [H.sup.+]-ATPase and SNARE proteins in rat kidney homogenate that complexed with GST-syntaxin molecules. Full-length syntaxin isoforms and syntaxin-1A[DETA]C [amino acids (aa) 1-264] formed complexes with [H.sup.+]-ATPase and SNAP23 and vesicle-associated membrane polypeptide (VAMP). A cassette within the H3 portion was found that bound [H.sup.+]-ATPase (aa 235-264) and another that bound SNAP23 and VAMP (aa 190-234) to an equivalent degree as full-length syntaxin. However, the aa 235-264 cassette alone without the SNARE N (aa 1-160) does not bind but requires ligation to the SNARE N to bind [H.sup.+]-ATPase. When this chimerical construct was transected into inner medullary collecting duct cells it inhibited intracellular pH recovery, an index of [H.sup.+]-ATPase mediated secretion. We conclude that within the H3 domain of syntaxin 1A is a unique cassette that participates in the binding of the [H.sup.+]-ATPase to the apical membrane and confers specificity of syntaxin 1A in the process of [H.sup.+]-ATPase exocytosis. soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor proteins; exocytosis; [H.sup.++] transport

Mouse proximal tubular cells (BUMPT), when cultured in the absence of growth factors, activate a default apoptotic pathway. Although Wnt signaling antagonizes the effect of proapoptotic triggers, its role in regulating the default pathway of apoptosis is less well defined. The present study examines the hypothesis that lithium ([Li.sup.+]) and (2'Z,3'E)-6-bromoindirubin-3'-oxime (BIO), two glycogen synthase kinase-3[beta] (GSK3[beta]) inhibitors, promote survival of growth factor-deprived renal epithelial cells by activating the Wnt pathway. These studies demonstrate that [Li.sup.+] and BIO activate Wnt signaling as indicated by the following changes: phosphorylation (inhibition) of GSK3[beta]; decreased phosphorylation of [beta]-catenin (a GSK3[beta] substrate); nuclear translocation of [beta]-catenin; specific transcriptional activation of Tcf/catenin-responsive pTopflash constructs; and an increase in the expression of cyclin D1 (indicative of a promitogenic cell response). In addition, [Li.sup.+] or BIO significantly increases the phosphorylation (activation) of Akt, an anti-apoptotic protein, and inhibits apoptosis (decreases both annexin-V staining and caspase-3 activation), during serum deprivation. Inhibition of phosphatidylinositol 3-kinase (responsible for Akt activation) either by wortmanin or LY-294002 prevented [Li.sup.+]-or BIO-induced Akt phosphorylation and reduces cell survival without altering the phosphorylation state of GSK3[beta]. [Li.sup.+] or BIO also increases the expression of insulin-like growth factor-II (IGF-II), a potent proliferative signaling protein. [Li.sup.+] or BIO-free conditioned medium harvested from [Li.sup.+] or BIO-exposed cells also induced Akt phosphorylafion, mimicking the protective effect of the two GSK3[beta] inhibitors on serum-starved cells. Furthermore, the effect of conditioned medium on Akt phosphorylation could be inhibited by either LY-294002 or IGF-binding protein. BIO, a specific GSK3[beta] inhibitor, replicated the protective effect of [Li.sup.+] on cell viability, suggesting that GSK3[beta] activation is important for initiating the apoptotic pathway. Taken together, these data suggest that [Li.sup.+] or BIO promotes renal epithelial cell survival by inhibiting apoptosis through GSK3[beta]-dependent activation of the Wnt pathway and subsequent release of IGF-II. Extracellular IGF-II serves as an autocrine survival factor that is responsible, in part, for activating the anti-apoptotic phosphatidylinositol-3-kinase-Akt pathway during serum deprivation. serum deprivation; renal epithelial cells; insulin-like growth factors; [beta]-catenin; glycogen synthase kinase-3[beta]; apoptosis

Munc-18-2 regulates exocytosis of [H.sup.+]-ATPase in rat inner medullary collecting duct cells. Am J Physiol Cell Physiol 287: C1366-C1374, 2004. First published July 7, 2004; doi:10.1152/ ajpcell.00588.2003.--Exocytic insertion of [H.sup.+]-ATPase into the apical membrane of inner medullary collecting duct (IMCD) cells is dependent on a soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein target receptor (SNARE) complex. In this study we determined the role of Munc-18 in regulation of IMCD cell exocytosis of [H.sup.+]-ATpase. We compared the effect of acute cell acidification (the stimulus for IMCD exocytosis) on the interaction of syntaxin 1A with Munc-18-2 and the 31-kDa subunit of [H.sup.+]-ATPase. Immunoprecipitation revealed that cell acidification decreased green fluorescent protein (GFP)-syntaxin 1A and Munc-18-2 interaction by 49 [+ or -] 7% and increased the interaction between GFP-syntaxin 1A and [H.sup.+]-ATPase by 170 [+ or -] 23%. Apical membrane Munc-18-2 decreased by 27.5 [+ or -] 4.6% and [H.sup.+]-ATPase increased by 246 [+ or -] 22%, whereas GP-135, an apical membrane marker, did not increase. Pretreatment of IMCD cells with a PKC inhibitor (GO-6983) diminished the previously described changes in Munc-18-2-syntaxin 1A interaction and redistribution of [H.sup.+]-ATPase. In a pull-down assay of [H.sup.+]-ATPase by glutathione S-transferase (GST)-syntaxin 1A bound to beads, preincubation of beads with an approximately twofold excess of His-Munc-18-2 decreased [H.sup.+]-ATPase pulled down by 64 [+ or -] 16%. IMCD cells that overexpress Munc-18-2 had a reduced rate of proton transport compared with control cells. We conclude that Munc-18-2 must dissociate from the syntaxin 1A protein for the exocytosis of [H.sup.+]-ATPase to occur. This dissociation leads to a conformational change in syntaxin 1A, allowing it to interact with [H.sup.+]-ATPase, synaptosome-associated protein (SNAP)-23, and vesicle-associated membrane protein (VAMP), forming the SNARE complex that leads to the docking and fusion of [H.sup.+]-ATPase vesicles. soluble N-ethylmaleimide-sensitive factor attachment protein target receptor; cell pH; acid secretion

Inhibition of the mitochondrial release and nuclear translocation of apoptosis-inducing factor (AIF) by heat stress protein (HSP)72 may ameliorate apoptosis in renal epithelial cells exposed to a metabolic inhibitor. To evaluate this hypothesis, cells were transiently exposed to 5 mM sodium cyanide in the absence of medium glucose, a maneuver known to induce apoptosis. ATP depletion for 1-2 h resulted in the progressive accumulation of mitochondrial AIF in the cytosol of samples obtained by selectively permeabilizing the plasma membrane with digitonin. During recovery from ATP depletion, time-dependent nuclear AIF accumulation (but not cytochrome c, an [F.sub.0][F.sub.1] ATP synthase subunit, or talin) was observed in isolated nuclei. Nuclear AIF accumulation was associated with peripheral chromatin condensation and DNA degradation. Prior heat stress (HS) significantly reduced AIF leakage into the cytosol, decreased nuclear accumulation of AIF, and inhibited DNA degradation. HS also increased the interaction between AIF and HSP72 detected by immunoprecipitation. In ATP depleted cells, selective overexpression of human HSP72 reduced the leakage of mitochondrial AIF in a dose-dependent manner (r = 0.997). This study suggests that mitochondrial membrane injury and subsequent AIF release contribute to nuclear injury and apoptosis in ATP-depleted renal cells. HSP72, an antiapoptotic protein, inhibits cell injury in part by preventing mitochondrial AIF release and perhaps by decreasing its nuclear accumulation. heat stress; adenovirus; metabolic inhibitors; heat stress protein 60; DNA degradation

Cyanide (CN)-induced chemical anoxia of cultured mouse proximal tubular (MPT) cells increased the kinase activity of c-Src by approximately threefold. 4-Amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2), a specific inhibitor of c-Src, prevented Src activation. CN also increased the permeability of MPT cell monolayers, an event ameliorated by PP2. During CN treatment, the proteins of the zonula adherens (ZA; E-cadherin and the catenins) disappeared from their normal location at cell-cell borders and appeared within the cytosol. CN also resulted in the appearance of c-Src at cell-cell borders. PP2 prevented these CN-induced alterations in the distribution of ZA proteins and c-Src. CN also increased the association of c-Src with [beta]-catenin and p120 and induced a substantial increase in tyrosine phosphorylation of both catenins. PP2 prevented the CN-induced phosphorylation of these catenins. In summary, we show that CN-induced chemical anoxia activates c-Src and induces its translocation to cell-cell junctions where it binds to and phosphorylates [Beta]-catenin and p120. Our findings suggest that these events contribute to the loss of the epithelial barrier function associated with chemical anoxia. ischemia; tight junction; Src kinase; E-cadherin; catenins; [p120.sup.ctn]; renal tubular cells

The 31-KDa subunit of the H+-ATPase was used to investigate the effect of acute cellular acidification on the distribution of vacuolar H+ATPase in cultured rat inner medullary collecting duct cells. The 31-KDa subunits were found to be redistributed from vesicles to the apical membrane. Results also revealed that the transport of cytosolic vesicles containing the subunit into the apical membrane is regulated by cell acidification.

Research shows that clostridial toxins botulinum and tetanus target proteins found in the inner medullary collecting duct cell line. Evidence suggests that clostridial toxins inhibit realkalinization following acid absorption. Experiments using immunoprecipitation and immunoblot techniques to investigate sodium-independent hydrogen transport mechanisms are reported.

A 15.5-kDa renal protein was isolated, purified and characterized. Amino acid sequence analysis identified amino acids 10-117 of alpha2mu-globulin (A2), a major urinary protein produce in the male rat liver. Injection of A2 into female rats resulted in the accumulation of A2 in the renal cortex of female rats. Biochemical analyses of isolated rat proximal tubule revealed a significantly high amount of glucose oxidation and of heart fatty acid binding protein in rats lacking the 15.5-kDa protein. These results suggest that A2 contributes to proximal tubule metabolism.

Cultured rat inner medullary collecting duct (IMCD) cells exhibit carbonic anhydrase (CA) and possess an mRNA which hybridizes with a mouse CA II cDNA. The CA activity seen in these cultured cells is due to the CA isoform CA II. This is confirmed by the presence of a cytosolic immunoreactive protein in a rabbit antibody to human CA II and the identification of suitable protein band isolated from cytosolic fractions of homogenized IMCD cells by Western blot analysis.

A study was conducted to determine the heat stress protein (HSP) 72 response of inner medullary collecting duct (IMCD) cells in vitro and the influence of a 43 degrees celsius or a 50 degrees celsius thermal insult on mitochondrial working and glycolysis connected with exposure to 50 degrees celsius. Lowered cytoprotection corresponded to HSP 72 disappearance.

The function of alterations in lipid metabolism in primary cultures of mouse proximal tubular (MPT) cells which are susceptible to chemical anoxia are defined, and the functions of resistance to impairment of a continuous renal cell line, the Madin-Darby canine kidney (MDCK) cell, are investigated. The results reveal that the resistance of MDCK cells to anoxia are due to factors which do not rely on glycolytic adaptation. Resistance of MDCK cells to injury is more than that shown by MPT cells because of decreased free fatty acid (FFA) discharge. The theory that variations in phospholipase A2 (PLA2) activity caused by anoxia between the cells are due to differences in FFA accumulation and cell impairments, was formed.

Banerjee, Abhijit, Guangmu Li, Edward A. Alexander, and John H. Schwartz. Role of SNAP-23 in trafficking of [H.sup.+]-ATPase in cultured inner medullary collecting duct cells. Am J Physiol Cell Physiol 280: C775-C781, 2001.--The trafficking of [H.sup.+]-ATPase vesicles to the apical membrane of inner medullary collecting duct (IMCD) cells utilizes a mechanism similar to that described in neurosecretory cells involving soluble N-ethylmaleimide-sensitive factor attachment protein target receptor (SNARE) proteins. Regulated exocytosis of these vesicles is associated with the formation of SNARE complexes. Clostridial neurotoxins that specifically cleave the target (t-) SNARE, syntaxin-1, or the vesicle SNARE, vesicle-associated membrane protein-2, reduce SNARE complex formation, [H.sup.+]-ATPase translocation to the apical membrane, and inhibit [H.sup.+] secretion. The purpose of these experiments was to characterize the physiological role of a second t-SNARE, soluble N-ethylmaleimide-sensitive factor attachment protein (SNAP)-23, a homologue of the neuronal SNAP-25, in regulated exocytosis of [H.sup.+]-ATPase vesicles. Our experiments document that 25-50 nM botulinum toxin (Bot) A or E cleaves rat SNAP-23 and thereby reduces immunodetectable and [sup.35]S-labeled SNAP-23 by [is greater than] 60% within 60 min. Addition of 25 nM BotE to IMCD homogenates reduces the amount of the 20 S-like SNARE complex that can be immunoprecipitated from the homogenate. Treatment of intact IMCD monolayers with BotE reduces the amount of [H.sup.+]-ATPase translocated to the apical membrane by 52 [+ or -] 2% of control and reduces the rate of [H.sup.+] secretion by 77 [+ or -] 3% after acute cell acidification. We conclude that SNAP-23 is a substrate for botulinum toxin proteolysis and has a critical role in the regulation of [H.sup.+]-ATPase exocytosis and [H.sup.+] secretion in these renal epithelial cells. soluble N-ethylmaleimide-sensitive factor attachment protein target receptor; soluble N-ethylmaleimide-sensitive factor attachment protein; botulinum toxins; proton pumps; hydrogen ion secretion

We hypothesized that nucleophosmin (NPM), a nucleolar phosphoprotein, is critical for Bax-mediated cell death. To test this hypothesis, Bax activation was induced by metabolic stress. During stress, nucleolar NPM translocated into the cytosol, NPM-Bax complexes formed, and both NPM and Bax accumulated in mitochondria. Expression of a cytosol-restricted NPM mutant (NPM-ΔNLS), but not a nucleus-restricted NPM mutant, increased NPM-Bax complex formation, mitochondrial NPM and Bax accumulation, mitochondrial membrane injury, caspase 3 activation, and ischemia-induced cell death. Coexpression of NPM-ΔNLS with constitutively active Bax mutants caused nearly universal cell death in the absence of metabolic stress, whereas expression of active Bax or NPM-ΔNLS alone did not. A Bax peptide that disrupts NPM-Bax interaction significantly reduced cell death caused by exposure to metabolic inhibitors in vitro and preserved kidney function after ischemia in vivo. Thus, NPM-Bax interaction enhances mitochondrial Bax accumulation, organelle injury, and cell death. NPM-Bax complex formation is a novel target for preventing ischemic tissue injury. [ABSTRACT FROM AUTHOR]