TMIGD1 is a novel adhesion molecule that protects epithelial cells from oxidative cell injury.

Oxidative damage to renal tubular epithelial cells is a fundamental pathogenic mechanism implicated in both acute kidney injury and chronic kidney diseases. Because epithelial cell survival influences the outcome of acute kidney injury and chronic kidney diseases, identifying its molecular regulators could provide new insight into pathobiology and possible new therapeutic strategies for these diseases. We have identified transmembrane and immunoglobulin domain-containing 1 (TMIGD1) as a novel adhesion molecule, which is highly conserved in humans and other species. TMIGD1 is expressed in renal tubular epithelial cells and promotes cell survival. The extracellular domain of TMIGD1 contains two putative immunoglobulin domains and mediates self-dimerization. Our data suggest that TMIGD1 regulates transepithelial electric resistance and permeability of renal epithelial cells. TMIGD1 controls cell migration, cell morphology, and protects renal epithelial cells from oxidative- and nutrient-deprivation-induced cell injury. Hydrogen peroxide-induced oxidative cell injury downregulates TMIGD1 expression and targets it for ubiquitination. Moreover, TMIGD1 expression is significantly affected in both acute kidney injury and in deoxy-corticosterone acetate and sodium chloride (deoxy-corticosterone acetate salt)-induced chronic hypertensive kidney disease mouse models. Taken together, we have identified TMIGD1 as a novel cell adhesion molecule expressed in kidney epithelial cells that protects kidney epithelial cells from oxidative cell injury to promote cell survival.

Glucose-dependent insulinotropic polypeptide-mediated signaling pathways enhance apical PepT1 expression in intestinal epithelial cells.

We have shown recently that glucose-dependent insulinotropic polypeptide (GIP), but not glucagon-like peptide 1 (GLP-1) augments H(+) peptide cotransporter (PepT1)-mediated peptide absorption in murine jejunum. While we observed that inhibiting cAMP production decreased this augmentation of PepT1 activity by GIP, it was unclear whether PKA and/or other regulators of cAMP signaling pathway(s) were involved. This study utilized tritiated glycyl-sarcosine [(3)H-glycyl-sarcosine (Gly-Sar), a relatively nonhydrolyzable dipeptide] uptake to measure PepT1 activity in CDX2-transfected IEC-6 (IEC-6/CDX2) cells, an absorptive intestinal epithelial cell model. Similar to our earlier observations with mouse jejunum, GIP but not GLP-1 augmented Gly-Sar uptake (control vs. +GIP: 154 ± 22 vs. 454 ± 39 pmol/mg protein; P < 0.001) in IEC-6/CDX2 cells. Rp-cAMP (a PKA inhibitor) and wortmannin [phosophoinositide-3-kinase (PI3K) inhibitor] pretreatment completely blocked, whereas neither calphostin C (a potent PKC inhibitor) nor BAPTA (an intracellular Ca(2+) chelator) pretreatment affected the GIP-augmented Gly-Sar uptake in IEC-6/CDX2 cells. The downstream metabolites Epac (control vs. Epac agonist: 287 ± 22 vs. 711 ± 80 pmol/mg protein) and AKT (control vs. AKT inhibitor: 720 ± 50 vs. 75 ± 19 pmol/mg protein) were shown to be involved in GIP-augmented PepT1 activity as well. Western blot analyses revealed that both GIP and Epac agonist pretreatment enhance the PepT1 expression on the apical membranes, which is completely blocked by wortmannin in IEC-6/CDX2 cells. These observations demonstrate that both cAMP and PI3K signaling pathways augment GIP-induced peptide uptake through Epac and AKT-mediated pathways in intestinal epithelial cells, respectively. In addition, these observations also indicate that both Epac and AKT-mediated signaling pathways increase apical membrane expression of PepT1 in intestinal absorptive epithelial cells.

Recognition-dependent signaling events in response to apoptotic targets inhibit epithelial cell viability by multiple mechanisms: implications for non-immune tissue homeostasis.

Apoptosis allows for the removal of damaged, aged, and/or excess cells without harm to surrounding tissue. To accomplish this, cells undergoing apoptosis acquire new activities that enable them to modulate the fate and function of nearby cells. We have shown that receptor-mediated recognition of apoptotic versus necrotic target cells by viable kidney proximal tubular epithelial cells (PTEC) modulates the activity of several signaling pathways critically involved in regulation of proliferation and survival. Generally, apoptotic and necrotic targets have opposite effects with apoptotic targets inhibiting and necrotic targets stimulating the activity of these pathways. Here we explore the consequences of these signaling differences. We show that recognition of apoptotic targets induces a profound decrease in PTEC viability through increased responder cell death and decreased proliferation. In contrast, necrotic targets promote viability through decreased death and increased proliferation. Both target types mediate their effects through a network of Akt-dependent and -independent events. Apoptotic targets modulate Akt-dependent viability in part through a reduction in cellular ß-catenin and decreased inactivation of Bad. In contrast, Akt-independent modulation of viability occurs through activation of caspase-8, suggesting that death receptor-dependent pathways are involved. Apoptotic targets also activate p38, which partially protects responders from target-induced death. The response of epithelial cells varies depending on their tissue origin. Some cell lines, like PTEC, demonstrate decreased viability, whereas others (e.g. breast-derived) show increased viability. By acting as sentinels of environmental change, apoptotic targets allow neighboring cells, especially non-migratory epithelial cells, to monitor and potentially adapt to local stresses.

Glucose-dependent insulinotropic polypeptide regulates dipeptide absorption in mouse jejunum.

Glucose-dependent insulinotropic polypeptide (GIP) secreted from jejunal mucosal K cells augments insulin secretion and plays a critical role in the pathogenesis of obesity and Type 2 diabetes mellitus. In recent studies, we have shown GIP directly activates Na-glucose cotransporter-1 (SGLT1) and enhances glucose absorption in mouse jejunum. It is not known whether GIP would also regulate other intestinal nutrient absorptive processes. The present study investigated the effect of GIP on proton-peptide cotransporter-1 (PepT1) that mediates di- and tripeptide absorption as well as peptidomimetic drugs. Immunohistochemistry studies localized both GIP receptor (GIPR) and PepT1 proteins on the basolateral and apical membranes of normal mouse jejunum, respectively. Anti-GIPR antibody detected 50-, 55-, 65-, and 70-kDa proteins, whereas anti-PepT1 detected a 70-kDa proteins in mucosal homogenates of mouse jejunum. RT-PCR analyses established the expression of GIPR- and PepT1-specific mRNA in mucosal cells of mouse jejunum. Absorption of Gly-Sar (a nondigestible dipeptide) measured under voltage-clamp conditions revealed that the imposed mucosal H(+) gradient-enhanced Gly-Sar absorption as an evidence for the presence of PepT1-mediated H(+):Gly-Sar cotransport on the apical membranes of mouse jejunum. H(+):Gly-Sar absorption was completely inhibited by cephalexin (a competitive inhibitor of PepT1) and was activated by GIP. The GIP-activated Gly-Sar absorption was completely inhibited by RP-cAMP (a cAMP antagonist). In contrast to GIP, the ileal L cell secreting glucagon-like peptide-1 (GLP-1) did not affect the H(+):Gly-Sar absorption in mouse jejunum. We conclude from these observations that GIP, but not GLP-1, directly activates PepT1 activity by a cAMP-dependent signaling pathway in jejunum.

Recognition of apoptotic cells by epithelial cells: conserved versus tissue-specific signaling responses.

During apoptosis, cells acquire new activities that enable them to modulate the fate and function of interacting phagocytes, particularly macrophages (m). Although the best known of these activities is anti-inflammatory, apoptotic targets also influence m survival and proliferation by modulating proximal signaling events, such as MAPK modules and Akt. We asked whether modulation of these same signaling events extends to epithelial cells, a minimally phagocytic cell type. We used BU.MPT cells, a mouse kidney epithelial cell line, as our primary model, but we also evaluated several epithelial cell lines of distinct tissue origins. Like m, mouse kidney epithelial cells recognized apoptotic and necrotic targets through distinct non-competing receptors, albeit with lower binding capacity and markedly reduced phagocytosis. Also, modulation of inflammatory activity and MAPK-dependent signaling by apoptotic and necrotic targets was indistinguishable in kidney epithelial cells and m. In contrast, modulation of Akt-dependent signaling differed dramatically between kidney epithelial cells and m. In kidney epithelial cells, modulation of Akt was linked to target cell recognition, independently of phagocytosis, whereas in m, modulation was linked to phagocytosis. Moreover, recognition of apoptotic and necrotic targets by kidney epithelial cells elicited opposite responses; apoptotic targets inhibited whereas necrotic targets stimulated Akt activity. These data confirm that nonprofessional phagocytes recognize and respond to dying cells, albeit in a manner partially distinct from m. By acting as sentinels of environmental change, apoptotic and necrotic targets may permit neighboring viable cells, especially non-migratory epithelial cells, to monitor and adapt to local stresses.

Induction of heat shock protein 70 inhibits ischemic renal injury.

Heat shock protein 70 (Hsp70) is a potent antiapoptotic agent. Here, we tested whether it directly regulates renal cell survival and organ function in a model of transient renal ischemia using Hsp70 knockout, heterozygous, and wild-type mice. The kidney cortical Hsp70 content inversely correlated with tubular injury, apoptosis, and organ dysfunction after injury. In knockout mice, ischemia caused changes in the activity of Akt and glycogen synthase kinase 3-ß (kinases that regulate the proapoptotic protein Bax), increased active Bax, and activated the proapoptotic protease caspase 3. As these changes were significantly reduced in the wild-type mice, we tested whether Hsp70 influences ischemia-induced apoptosis. An Hsp70 inducer, geranylgeranylacetone, increased Hsp70 expression in heterozygous and wild-type mice, and reduced both ischemic tubular injury and organ dysfunction. When administered after ischemia, this inducer also decreased tubular injury and organ failure in wild-type mice but did not protect the knockout mice. ATP depletion in vitro caused greater mitochondrial Bax accumulation and death in primary proximal tubule cells harvested from knockout compared with wild-type mice and altered serine phosphorylation of a Bax peptide at the Akt-specific target site. In contrast, lentiviral-mediated Hsp70 repletion decreased mitochondrial Bax accumulation and rescued Hsp70 knockout cells from death. Thus, increasing Hsp70 either before or after ischemic injury preserves renal function by attenuating acute kidney injury.

Hexokinase regulates Bax-mediated mitochondrial membrane injury following ischemic stress.

Hexokinase (HK), the rate-limiting enzyme in glycolysis, controls cell survival by promoting metabolism and/or inhibiting apoptosis. Since HK isoforms I and II have mitochondrial targeting sequences, we attempted to separate the protective effects of HK on cell metabolism from those on apoptosis. We exposed renal epithelial cells to metabolic stress causing ATP depletion in the absence of glucose and found that this activated glycogen synthase kinase 3ß (GSK3ß) and Bax caused mitochondrial membrane injury and apoptosis. ATP depletion led to a progressive HK II dissociation from mitochondria, released mitochondrial apoptosis inducing factor and cytochrome c into the cytosol, activated caspase-3, and reduced cell survival. Compared with control, adenoviral-mediated HK I or II overexpression improved cell survival following stress, but did not prevent GSK3ß or Bax activation, improve ATP content, or reduce mitochondrial fragmentation. HK I or HK II overexpression increased mitochondria-associated isoform-specific HK content, and decreased mitochondrial membrane injury and apoptosis after stress. In vivo, HK II localized exclusively to the proximal tubule. Ischemia reduced total renal HK II content and dissociated HK II from proximal tubule mitochondria. In cells overexpressing HK II, Bax and HK II did not interact before or after stress. While the mechanism by which HK antagonizes Bax-mediated apoptosis is unresolved by these studies, one possible scenario is that the two proteins compete for a common binding site on the outer mitochondrial membrane.

GSK3beta promotes apoptosis after renal ischemic injury.

The mechanism by which the serine-threonine kinase glycogen synthase kinase-3beta (GSK3beta) affects survival of renal epithelial cells after acute stress is unknown. Using in vitro and in vivo models, we tested the hypothesis that GSK3beta promotes Bax-mediated apoptosis, contributing to tubular injury and organ dysfunction after acute renal ischemia. Exposure of renal epithelial cells to metabolic stress activated GSK3beta, Bax, and caspase 3 and induced apoptosis. Expression of a constitutively active GSK3beta mutant activated Bax and decreased cell survival after metabolic stress. In contrast, pharmacologic inhibition (4-benzyl-2-methyl-1,2,4-thiadiazolidine-3,5-dione [TDZD-8]) or RNA interference-mediated knockdown of GSK3beta promoted cell survival. Furthermore, RNA interference-mediated knockdown of Bax abrogated the cell death induced by constitutively active GSK3beta. In a cell-free assay, TDZD-8 inhibited the phosphorylation of a peptide containing the Bax serine(163) site targeted by stress-activated GSK3beta. In rats, TDZD-8 inhibited ischemia-induced activation of GSK3beta, Bax, and caspase 3; ameliorated tubular and epithelial cell damage; and significantly protected renal function. Taken together, GSK3beta-mediated Bax activation induces apoptosis and tubular damage that contribute to acute ischemic kidney injury.

Adaptation of intercalated cells along the collecting duct to systemic acid/base changes.

Collecting duct intercalated cells respond to short-term acid/base perturbations by rapidly shuttling H(+)-ATPase to and from the plasma membrane. Purkerson et al. provide information on the regulation of the anion transporters during chronic acidosis and acute recovery (alkalosis). They found that the major mechanism for both acute and chronic states is regulation of both the H(+)-ATPase and the anion exchangers plus changes in the overall expression level of these anion transporters in chronic adaptation.

Beta-catenin promotes survival of renal epithelial cells by inhibiting Bax.

Ischemia activates Bax, a proapoptotic BCL2 protein, as well as the prosurvival beta-catenin/Wnt signaling pathway. To test the hypothesis that beta-catenin/Wnt signaling regulates Bax-mediated apoptosis after induction of metabolic stress, which occurs during renal ischemia, we infected immortalized and primary proximal tubular epithelial cells with adenovirus to express either constitutively active or dominant negative beta-catenin constructs. Constitutively active beta-catenin significantly decreased apoptosis and improved cell survival after metabolic stress. Furthermore, active beta-catenin decreased Bax activation, oligomerization, and translocation to mitochondria, and reduced both organelle membrane injury and apoptosis. Dominant negative beta-catenin had the opposite effects. Because Akt regulates Bax, we examined the effects of the beta-catenin mutants on Akt expression and activation. Constitutively active beta-catenin increased Akt-1 expression and activation before and after stress, and treatment with a phosphatidylinositol-3 kinase inhibitor antagonized the protective effects of beta-catenin on Akt activation, Bax inhibition, and cell survival. In addition, beta-catenin significantly increased the rate of phosphorylation at Bax serine(184), an Akt-specific target. Taken together, these results suggest that beta-catenin/Wnt signaling promotes survival of renal epithelial cells after metabolic stress, in part by inhibiting Bax in a phosphatidylinositol-3 kinase/Akt-dependent manner.

Hsp27 inhibits sublethal, Src-mediated renal epithelial cell injury.

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-(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.

Nucleophosmin, a critical Bax cofactor in ischemia-induced cell death.

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.

Role of mitofusin 2 in the renal stress response.

The role of mitofusin 2 (MFN2), a key regulator of mitochondrial morphology and function in the renal stress response is unknown. To assess its role, the MFN2 floxed gene was conditionally deleted in the kidney of mice (MFN2 cKO) by Pax2 promoter driven Cre expression (Pax2Cre). MFN2 cKO caused severe mitochondrial fragmentation in renal epithelial cells that are critical for normal kidney tubular function. However, despite a small (20%) decrease in nephron number, newborn cKO pups had organ or tubular function that did not differ from littermate Cre-negative pups. MFN2 deficiency in proximal tubule epithelial cells in primary culture induced mitochondrial fragmentation but did not significantly alter ATP turnover, maximal mitochondrial oxidative reserve capacity, or the low level of oxygen consumption during cyanide exposure. MFN2 deficiency also did not increase apoptosis of tubule epithelial cells under non-stress conditions. In contrast, metabolic stress caused by ATP depletion exacerbated mitochondrial outer membrane injury and increased apoptosis by 80% in MFN2 deficient vs. control cells. Despite similar stress-induced Bax 6A7 epitope exposure in MFN2 deficient and control cells, MFN2 deficiency significantly increased mitochondrial Bax accumulation and was associated with greater release of both apoptosis inducing factor and cytochrome c. In conclusion, MFN2 deficiency in the kidney causes mitochondrial fragmentation but does not affect kidney or tubular function during development or under non-stress conditions. However, MFN2 deficiency exacerbates renal epithelial cell injury by promoting Bax-mediated mitochondrial outer membrane injury and apoptosis.

Microscale sample preparation for PCR of C. difficile infected stool.

In this paper, we describe the design of a microfluidic sample preparation chip for human stool samples infected with Clostridium difficile. We established a polymerase chain reaction able to distinguish C. difficile in the presence of several other organisms found in the normal intestinal flora. A protocol for on-chip extraction of nucleic acids from clinical samples is described that can detect target DNA down to 5.0x10(-3) ng of template. The assay and sample preparation chip were then validated using known positive and known negative clinical samples. The work presented has potential applications in both the developed and developing world.

Glucose-dependent insulinotropic polypeptide (GIP) stimulates transepithelial glucose transport.

The purpose of this study was to characterize the effects of glucose-dependent insulinotropic peptide (GIP) on small intestinal glucose transport in vitro. Stripped proximal jejunum from fasted mice was mounted in Ussing chambers. The serosal side was bathed in Regular Ringer solution containing 5 mmol/l glucose, and the mucosal side, with solution containing 10 mmol/l 3-O-methyl glucose (3OMG). Intercellular cyclic adenosine monophosphate (cAMP), mucosa-to-serosa fluxes of 3OMG (J(ms)(3OMG)), and short-circuit current (I(SC)) were measured in the presence and absence of GIP. GIP increased cAMP by 2.5-fold in isolated enterocytes, consistent with a direct effect of GIP on these epithelial cells. GIP also increased I(SC) and J(ms)(3OMG) by 68 and 53%, respectively, indicating that the increase in J(ms)(3OMG) was primarily electrogenic, with a small electroneutral component. The stimulatory effect of GIP on J(ms)(3OMG) was concentration dependent. In addition, 1,000 nmol/l and 10 nmol/l GIP increased J(ms)(3OMG) by 70 and 30% over control, respectively, consistent with receptor activation. Phlorizin (20 mumol/l), an inhibitor of Na(+)-glucose cotransporter (SGLT-1), abolished the increase in I(SC) and decreased J(ms)(3OMG) by approximately 65%. These results indicate that stimulation of SGLT-1 activity by GIP partially accounts for the increase in J(ms)(30MG). These studies are the first to demonstrate direct stimulation of intestinal glucose transport by GIP independent of its insulinotropic properties. GIP stimulates cellular accumulation of cAMP and thereby upregulates glucose transport. The GIP-induced increase in glucose transport appears to be mediated, at least in part, by SGLT-1.

Hsp27 inhibits Bax activation and apoptosis via a phosphatidylinositol 3-kinase-dependent mechanism.

Hsp27 inhibits mitochondrial injury and apoptosis in both normal and cancer cells by an unknown mechanism. To test the hypothesis that Hsp27 decreases apoptosis by inhibiting Bax, Hsp27 expression was manipulated in renal epithelial cells before transient metabolic stress, an insult that activates Bax, induces mitochondrial injury, and causes apoptosis. Compared with control, enhanced Hsp27 expression inhibited conformational Bax activation, oligomerization, and translocation to mitochondria, reduced the leakage of both cytochrome c and apoptosis-inducing factor, and significantly improved cell survival by >50% after stress. In contrast, Hsp27 down-regulation using RNA-mediated interference promoted Bax activation, increased Bax translocation, and reduced cell survival after stress. Immunoprecipitation did not detect Hsp27-Bax interaction before, during, or after stress, suggesting that Hsp27 indirectly inhibits Bax. During stress, Hsp27 expression prevented the inactivation of Akt, a pro-survival kinase, and increased the interaction between Akt and Bax, an Akt substrate. In contrast, Hsp27 RNA-mediated interference promoted Akt inactivation during stress. Hsp27 up- or down-regulation markedly altered the activity of phosphatidylinositol 3-kinase (PI3-kinase), a major regulator of Akt. Furthermore, distinct PI3-kinase inhibitors completely abrogated the protective effect of Hsp27 expression on Akt activation, Bax inactivation, and cell survival. These data show that Hsp27 antagonizes Bax-mediated mitochondrial injury and apoptosis by promoting Akt activation via a PI3-kinase-dependent mechanism.

Vacuolar H+ -ATPase B1 subunit mutations that cause inherited distal renal tubular acidosis affect proton pump assembly and trafficking in inner medullary collecting duct cells.

Point mutations in the B1 subunit of vacuolar H+ -ATPase are associated with impaired ability of the distal nephron to secrete acid (distal renal tubular acidosis). For testing of the hypothesis that these mutations interfere with assembly and trafficking of the H+ -ATPase, constructs that mimic seven known point mutations in inherited distal renal tubular acidosis (M) or wild-type (WT) B1 were transfected into a rat inner medullary collecting duct cell line to express green fluorescence protein (GFP)-B1WT or GFP-B1M fusion proteins. In co-immunoprecipitation studies, GFP-B1WT formed complexes with other H+ -ATPase subunits (c, H, and E), whereas GFP-B1M did not. Proteins that were immunoprecipitated with anti-GFP antibody from GFP-B1WT cells had ATPase activity, whereas proteins from GFP-B1M cells did not. Proton pump-mediated intracellular pH transport was inhibited in GFP-B1M-transfected cells but not in GFP-B1WT cells. GFP-B1WT and GFP-B1M are present in the apical membrane and increased with cellular acidification. In GFP-B1WT cells, the apical membrane fraction of GFP-B1, endogenous B1, and the 31-kD subunits of the H+ -ATPase increased with cell acidification. In GFP-B1M cells, the endogenous B1 and 31-kD subunits did not increase with acidification. B1 point mutations prevent normal assembly of the H+ -ATPase and also may act as an inhibitor of H+ -ATPase function by competing with endogenous intact H+ -ATPase for trafficking in inner medullary collecting duct cells.

Distinct hsp70 domains mediate apoptosis-inducing factor release and nuclear accumulation.

Although hsp70 antagonizes apoptosis-inducing factor (AIF)-mediated cell death, the relative importance of preventing its release from mitochondria versus sequestering leaked AIF in the cytosol remains controversial. To dissect these two protective mechanisms, hsp70 deletion mutants lacking either the chaperone function (hsp70-deltaEEVD) or ATPase function (hsp70-deltaATPase) were selectively overexpressed before exposing cells to a metabolic inhibitor, an insult sufficient to cause mitochondrial AIF release, nuclear AIF accumulation, and apoptosis. Compared with empty vector, overexpression of wild type human hsp70 inhibited bax activation and reduced mitochondrial AIF release after injury. In contrast, mutants lacking either the chaperone function (hsp70-deltaEEVD) or the ATP hydrolytic domain (hsp70-deltaATPase) failed to prevent mitochondrial AIF release. Although hsp70-deltaEEVD did not inhibit bax activation or mitochondrial membrane injury after cell stress, this hsp70 mutant co-immunoprecipitated with leaked AIF in injured cells and decreased nuclear AIF accumulation. In contrast, hsp70-deltaATPase did not interact with AIF either in intact cells or in a cell-free system and furthermore, failed to prevent nuclear AIF accumulation. These results demonstrate that mitochondrial protection against bax-mediated injury requires both intact chaperone and ATPase functions, whereas the ATPase domain is critical for sequestering AIF in the cytosol.

Syntaxin 1A has a specific binding site in the H3 domain that is critical for targeting of H+-ATPase to apical membrane of renal epithelial cells.

H(+) transport in the collecting duct is regulated by exocytic insertion of H(+)-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(+)-ATPase. This raised the possibility that there are separate binding sites within the H3 domain of syntaxin 1A for H(+)-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(+)-ATPase and SNARE proteins in rat kidney homogenate that complexed with GST-syntaxin molecules. Full-length syntaxin isoforms and syntaxin-1ADeltaC [amino acids (aa) 1-264] formed complexes with H(+)-ATPase and SNAP23 and vesicle-associated membrane polypeptide (VAMP). A cassette within the H3 portion was found that bound H(+)-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(+)-ATPase. When this chimerical construct was transected into inner medullary collecting duct cells it inhibited intracellular pH recovery, an index of H(+)-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(+)-ATPase to the apical membrane and confers specificity of syntaxin 1A in the process of H(+)-ATPase exocytosis.