A novel cellular survival factor--the B2 subunit of vacuolar H+-ATPase inhibits apoptosis.

The ubiquitous vacuolar H(+)-ATPase, a multisubunit proton pump, is essential for intraorganellar acidification. Disruption of its function leads to disturbances of organelle function and cell death. Here, we report that overexpression of the B2 subunit of the H(+)-ATPase inhibits apoptosis. This antiapoptotic effect is not mediated by an increase in H(+)-ATPase activity but through activation of the Ras-mitogen-activated protein kinase (MAPK)-signaling pathway that results in the serine phosphorylation of Bad at residues 112 and 155. Increased Bad phosphorylation reduces its translocation to mitochondria, limits the release of mitochondrial cytochrome c and apoptosis-inducing factor and increases the resistance of the B2 overexpressing cells to apoptosis. Screening experiments of kinase inhibitors, including inhibitors of cAMP-activated protein kinase, protein kinase C, protein kinase B, (MAPK/extracellular signal-regulated (ERK) kinase) MEK and Ste-MEK1(13), a cell permeable ERK activation inhibitor peptide, revealed that the B2 subunit of H(+)-ATPase acts upstream of MEK activation in the MEK/ERK pathway to ameliorate apoptosis.

Hsp72 expression enhances survival in adenosine triphosphate-depleted renal epithelial cells.

Although prior heat stress (HS) inhibits apoptosis in adenosine phosphate (ATP)-depleted renal epithelial cells (REC), the specific stress protein(s) responsible for cytoprotection have not been identified. The present study evaluated the hypothesis that Hsp72, the major inducible member of the Hsp70 family, protects REC against ATP depletion injury. In the presence of isopropyl-beta-D-thiogalactoside (IPTG), a stable line of transfected opossum kidney cells was induced to overexpress human Hsp72 tagged with the flag epitope. Transfected cells from 2 clones that expressed Hsp72 at a level comparable with wild-type cells were subjected to transient heat stress (43 degrees C for 1 hour). To assess the cytoprotective effect of Hsp72, transfected cells were subjected to transient ATP depletion followed by recovery in the presence vs the absence of IPTG. ATP depletion resulted in nuclear chromatin condensation without cell membrane injury (ie, minimal leak of lactate dehydrogenase) and activation of caspase-3, confirming that apoptosis is the major cause of cell death. In both clones cell survival 1-3 days after ATP depletion was significantly improved in the presence of IPTG. Selective overexpression of Hsp72 reproduced nearly 60% of the protective effect on the survival afforded by prior heat stress. In transfected cells subjected to ATP depletion, Hsp72 overexpression significantly inhibited caspase activation. In native renal cells brief ATP depletion markedly induced the expression of native Hsp72, a finding identical to that observed after renal ischemia in vivo. These studies are the first to directly show that Hsp72 per se mediates acquired resistance to ischemic injury in REC.

Heat stress prevents mitochondrial injury in ATP-depleted renal epithelial cells.

The events that precipitate cell death and the stress proteins responsible for cytoprotection during ATP depletion remain elusive. We hypothesize that exposure to metabolic inhibitors damages mitochondria, allowing proapoptotic proteins to leak into the cytosol, and suggest that heat stress-induced hsp72 accumulation prevents mitochondrial membrane injury. To test these hypotheses, renal epithelial cells were transiently ATP depleted with sodium cyanide and 2-deoxy-D-glucose in the absence of medium dextrose. Recovery from ATP depletion was associated with the release into the cytosol of cytochrome c and apoptosis-inducing factor (AIF), proapoptotic proteins that localize to the intermitochondrial membrane space. Concomitant with mitochondrial cytochrome c leak, a seven- to eightfold increase in caspase 3 activity was observed. In controls, state III mitochondrial respiration was reduced by 30% after transient exposure to metabolic inhibitors. Prior heat stress preserved mitochondrial ATP production and significantly reduced both cytochrome c release and caspase 3 activation. Despite less cytochrome c release, prior heat stress increased binding between cytochrome c and hsp72. The present study demonstrates that mitochondrial injury accompanies exposure to metabolic inhibitors. By reducing outer mitochondrial membrane injury and by complexing with cytochrome c, hsp72 could inhibit caspase activation and subsequent apoptosis.

c-Src and HSP72 interact in ATP-depleted renal epithelial cells.

Disruption of cell contact sites during ischemia contributes to the loss of organ function in acute renal failure. Because prior heat stress protects cell contact sites in ATP-depleted renal epithelial cells in vitro, we hypothesized that heat shock protein 72 (HSP72), the major inducible cytoprotectant in mammalian cells, interacts with protein kinases that regulate cell-cell and cell-matrix interactions. ATP depletion increased the content of Tyr(416) Src, the activated form of this kinase. c-Src activation was associated with an increase in the tyrosine phosphorylation state of beta-catenin, paxillin, and vinculin, three c-Src substrate proteins that localize to and regulate cell contact sites. Prior heat stress inhibited c-Src activation and decreased the degree of tyrosine phosphorylation of all three Src substrates during ATP depletion and/or early recovery. HSP72 coimmunoprecipitated with c-Src only in cells subjected to heat stress. ATP depletion markedly increased the interaction between HSP72 and c-Src, supporting the hypothesis that HSP72 regulates Src kinase activity. These results suggest that alterations in the tyrosine phosphorylation state of proteins located at the cell-cell and cell-matrix interface mediate, at least in part, the functional state of these structures during ATP depletion and may be modulated by interactions between HSP72 and c-Src.

Hepatic alpha 2 mu-globulin localizes to the cytosol of rat proximal tubule cells.

BACKGROUND: Alpha 2 mu-Globulin (A2), an 18.6 kD protein of hepatic origin, accumulates in the proximal tubule as an abundant, 15.5 kD cleavage product termed "A2-fragment" (A2-f). A2-f facilitates proximal tubule fatty acid oxidation, presumably by binding hydrophobic ligands. This requires some A2-f to enter the cytosol of the renal epithelial cell (REC). The localization of A2/A2-f in the proximal tubule cell was evaluated in this study. METHODS: Immunoblot analysis of renal cortical homogenates separated by differential centrifugation and quantitative immunoelectron microscopy (IEM) was performed to localize A2/A2-f using an affinity-purified antibody that detects both proteins. To evaluate A2 as a physiologically relevant ligand, the accumulation of A2-f in the female rat kidney (normally devoid of A2-f) was examined after the induction of hepatic A2 synthesis. Ligand binding, uptake, and degradation assays were used to assess A2 processing by RECs in vitro. RESULTS: Although A2 and A2-f were detected in the "lysosomal" fraction, only A2-f was found in the soluble protein fraction. IEM confirmed the presence of significant signal in the vesicular and lysosomal as well as the cytosolic compartments. In contrast, both beta 2 mu globulin (B2) and cathepsin B were restricted to endosomes. In the female rat, induction of hepatic A2 production resulted in A2-f accumulation in the renal cortex. In RECs in culture, uptake of A2 and B2 demonstrated nonsaturable, nondisplacable surface binding and similar uptake rates. Compared with B2, A2 was markedly resistant to degradation. CONCLUSIONS: A fraction of A2 escapes lysosomal degradation, permitting A2-f to accumulate in the cytosol of the proximal tubule epithelial cell. A2 may represent an unusual example of a physiologic protein capable of accumulating in a distant cell type.

cAMP-activated anion conductance is associated with expression of CFTR in neonatal mouse cardiac myocytes.

In this study, patch-clamp techniques were applied to cultured neonatal mouse cardiac myocytes (NMCM) to assess the contribution of cAMP stimulation to the anion permeability in this cell model. Addition of either isoproterenol or a cocktail to raise intracellular cAMP increased the whole cell currents of NMCM. The cAMP-dependent conductance was largely anionic, as determined under asymmetrical (low intracellular) Cl(-) conditions and symmetrical Cl(-) in the presence of various counterions, including Na(+), Mg(2+), Cs(+), and N-methyl-D-glucamine. Furthermore, the cAMP-stimulated conductance was also permeable to ATP. The cAMP-activated currents were inhibited by diphenylamine-2-carboxylate, glibenclamide, and an anti-cystic fibrosis transmembrane conductance regulator (CFTR) monoclonal antibody. The anti-CFTR monoclonal antibody failed, however, to inhibit an osmotically activated anion conductance, indicating that CFTR is not linked to osmotically stimulated currents in this cell model. Immunodetection studies of both neonatal mouse heart tissue and cultured NMCM revealed that CFTR is expressed in these preparations. The implication of CFTR in the cAMP-stimulated Cl(-)- and ATP-permeable conductance was further verified with NMCM of CFTR knockout mice [cftr(-/-)] in which cAMP stimulation was without effect on the whole cell currents. In addition, stimulation with protein kinase A and ATP induced Cl(-)-permeable single-channel activity in excised, inside-out patches from control, but not cftr(-/-) NMCM. The data in this report indicate that cAMP stimulation of NMCM activates an anion-permeable conductance with functional properties similar to those expected for CFTR, thus suggesting that CFTR may be responsible for the cAMP-activated conductance. CFTR may thus contribute to the permeation and/or regulation of Cl(-)- and ATP-permeable pathways in the developing heart.

Actin filament organization is required for proper cAMP-dependent activation of CFTR.

Previous studies have indicated a role of the actin cytoskeleton in the regulation of the cystic fibrosis transmembrane conductance regulator (CFTR) ion channel. However, the exact molecular nature of this regulation is still largely unknown. In this report human epithelial CFTR was expressed in human melanoma cells genetically devoid of the filamin homologue actin-cross-linking protein ABP-280 [ABP(-)]. cAMP stimulation of ABP(-) cells or cells genetically rescued with ABP-280 cDNA [ABP(+)] was without effect on whole cell Cl(-) currents. In ABP(-) cells expressing CFTR, cAMP was also without effect on Cl(-) conductance. In contrast, cAMP induced a 10-fold increase in the diphenylamine-2-carboxylate (DPC)-sensitive whole cell Cl(-) currents of ABP(+)/CFTR(+) cells. Further, in cells expressing both CFTR and a truncated form of ABP-280 unable to cross-link actin filaments, cAMP was also without effect on CFTR activation. Dialysis of ABP-280 or filamin through the patch pipette, however, resulted in a DPC-inhibitable increase in the whole cell currents of ABP(-)/CFTR(+) cells. At the single-channel level, protein kinase A plus ATP activated single Cl(-) channels only in excised patches from ABP(+)/CFTR(+) cells. Furthermore, filamin alone also induced Cl(-) channel activity in excised patches of ABP(-)/CFTR(+) cells. The present data indicate that an organized actin cytoskeleton is required for cAMP-dependent activation of CFTR.

Prior heat stress inhibits apoptosis in adenosine triphosphate-depleted renal tubular cells.

BACKGROUND: This study tested the following hypotheses: (a) renal tubular epithelial cells subjected to transient adenosine triphosphate (ATP) depletion undergo apoptosis, and (b) induction of heat stress proteins (HSPs) inhibits cell death following ATP depletion, possibly by interacting with anti-apoptotic signal proteins. METHODS: To simulate ischemia in vivo, cells derived from opossum kidney proximal tubule (OK) were subjected to ATP depletion (5 mM cyanide, 5 mM 2-deoxy-D-glucose, and 0 mM glucose) for 1 to 1. 5 hours, followed by recovery (10 mM glucose without cyanide). The presence of apoptosis was assessed by morphological and biochemical criteria. The effect of prior heat stress or caspase inhibition on apoptosis and cell survival were assessed. RESULTS: In the ATP-depleted cell, both Hoechst dye and electron microscopy revealed morphological features that are typical of apoptosis. On an agarose gel, a "ladder pattern" typical of endonucleosomal DNA degradation was observed. Prior heat stress reduced the number of apoptotic-appearing cells, significantly decreased DNA fragmentation, and improved cell survival compared with controls (73.0 +/- 1% vs. 53.0 +/- 1.5%; P < 0.05). Two different caspase inhibitors also improved survival, suggesting that apoptosis is a cause of cell death in this model. Compared with ATP-depleted controls, prior heat stress inhibited the pro-apoptotic changes in the ratio of Bcl2 to BAX, proteins known to regulate the apoptotic set point in renal cells. HSP 72, a known cytoprotectant, co-immunoprecipitated with Bcl2, an anti-apoptotic protein. Prior heat stress markedly increased the interaction between HSP 72 and Bcl2. CONCLUSIONS: Transient ATP depletion causes apoptosis in tubular epithelial cells. Prior HS inhibits apoptosis and improves survival in these cells. Novel interactions between HSP 72 and Bcl2 may be responsible, at least in part, for the protection afforded by prior heat stress against ATP depletion injury.

Electrodiffusional ATP movement through the cystic fibrosis transmembrane conductance regulator.

Expression of the cystic fibrosis transmembrane conductance regulator (CFTR), and of at least one other member of the ATP-binding cassette family of transport proteins, P-glycoprotein, is associated with the electrodiffusional movement of the nucleotide ATP. Evidence directly implicating CFTR expression with ATP channel activity, however, is still missing. Here it is reported that reconstitution into a lipid bilayer of highly purified CFTR of human epithelial origin enables the permeation of both Cl- and ATP. Similar to previously reported data for in vivo ATP current of CFTR-expressing cells, the reconstituted channels displayed competition between Cl- and ATP and had multiple conductance states in the presence of Cl- and ATP. Purified CFTR-mediated ATP currents were activated by protein kinase A and ATP (1 mM) from the "intracellular" side of the molecule and were inhibited by diphenylamine-2-carboxylate, glibenclamide, and anti-CFTR antibodies. The absence of CFTR-mediated electrodiffusional ATP movement may thus be a relevant component of the pleiotropic cystic fibrosis phenotype.

Heat stress ameliorates ATP depletion-induced sublethal injury in mouse proximal tubule cells.

The role of prior heat stress (HS) in ameliorating changes in the actin cytoskeleton and the loss of tight junction integrity that accompany ATP depletion was examined. Mouse proximal tubule cells in primary culture were exposed to sodium cyanide (CN) in the absence of dextrose for 1 h, a maneuver that produced equivalent degrees of ATP depletion in control and in HS cells. After ATP depletion, actin stress fibers were completely disrupted in control cells. In contrast, HS cells with elevated HSP-72 content showed preservation of stress fibers after CN exposure. ATP depletion in control and HS cells produced similar and reversible depletion of the G-actin pool without altering total actin content. Integrity of the tight junction was assessed by transepithelial electrical resistance (TER) and unidirectional flux of lucifer yellow (LY, mol wt 482). After CN alone, the nadir in TER was lower than that of HS + CN cells (51.6 +/- 2.5 vs. 96.2 +/- 3.2 omega x cm2, respectively; P < 0.05). After 30-min recovery, TER of HS + CN recovered to control values (277 +/- 7.2 vs. 227 +/- 6.6 omega x cm2; P > 0.05), whereas CN did not (165 +/- 7.3 vs. 227 +/- 6.6 omega x cm2; P < 0.05). Changes in LY flux paralleled those in TER. HS is associated with preservation of the actin cytoskeleton and improved integrity of the tight junction after sublethal ATP depletion injury. These protective effects may contribute to the preservation of epithelial cell polarity and function following an ischemic insult.

Hepatic alpha 2 mu-globulin: a potential metabolic role in the rat proximal tubule.

In the present study, we provide immunohistochemical and immunologic evidence to localize an abundant, 15.5-kDa protein to the soluble protein fraction of the proximal tubule. This 15.5-kDa protein binds fatty acids in vitro and has identity with amino acids 10-117 of alpha 2 mu-globulin (A2 fragment), a 19-kDa protein synthesized predominantly in the male liver. With reverse transcription-polymerase chain reaction, mRNA for A2 was detected in male liver but not in the male kidney. De novo accumulation of the 15.5-kDa protein was observed in the renal cortex of female rats given intravenous injections of purified 19-kDa protein (A2), suggesting intrarenal processing of the larger protein. The potential role of this protein in the proximal tubule, a site that utilizes fatty acids as an important metabolic substrate, was determined in isolated proximal tubule segments. Fatty acid and glucose oxidation rates were measured in three experimental models in which the 15.5-kDa protein was virtually absent: 1) uninephrectomized male rats treated with deoxycorticosterone acetate and salt, 2) male rats subjected to bilateral adrenalectomy, and 3) normal female rats. In the absence of the 15.5-kDa protein, fatty acid oxidation rates decreased by 30-55%, whereas glucose oxidation significantly increased in all three models. In female renal cortex, depletion of the 15.5-kDa protein was associated with a rise in heart fatty acid binding protein, an alternative intracellular transporter of fatty acids. These data support the hypothesis that a proteolytic cleavage product of hepatic alpha 2 mu-globulin may facilitate the oxidation of oleate, a hydrophobic ligand, in the proximal tubule.

Prior heat stress enhances survival of renal epithelial cells after ATP depletion.

The 72-kDa heat stress protein (HSP-72) is an inducible cytoprotectant protein. Although transient renal ischemia in vivo induces HSP-72, it is not known whether prior heat stress protects renal epithelial cells from injury mediated by ATP depletion. To evaluate this hypothesis, opossum kidney (OK) cells were exposed to sodium cyanide and 2-deoxy-D-glucose in the absence of medium glucose, a maneuver that reduced cell ATP content to < 10% of the control value within 10 min and decreased cell survival. One day after 2 h of ATP depletion, OK cells previously exposed to heat stress (to induce accumulation of HSP-72) exhibited marked improvement in survival (a > 4-fold increase in total DNA), less uptake of vital dye, and less release of lactate dehydrogenase (LDH) than cells subjected to ATP depletion alone (23.0 +/- 1.6 vs. 34.1 +/- 1.2% of total LDH, respectively). Enhanced clonogenicity post-heat stress was completely prevented by cycloheximide and positively correlated with the steady-state content of HSP-72. In the recovery period after ATP depletion, cell ATP content, maximum mitochondrial ATP production rate, and total LDH activity were all significantly higher in cells with abundant HSP-72. Although the protective effects associated with heat stress are likely to be multifactoral, preserved cell metabolism and higher ATP content could enhance cellular repair processes after ATP depletion.

Heat stress protein-associated cytoprotection of inner medullary collecting duct cells from rat kidney.

Although heat stress proteins (HSPs) mediate thermotolerance, the cellular targets of thermal injury and mechanisms of acquired cytoprotection are unknown. To describe the metabolic effects of hyperthermia and the potential mechanisms of thermotolerance, the following were measured in inner medullary collecting duct cells after a 43 degrees C and/or a 50 degrees C thermal insult: 1) state III mitochondrial respiration (SIII MR), 2) glycolytic rate, 3) lactate dehydrogenase activity, 4) membrane permeability, and 5) HSP 72 content. Compared with controls incubated at 37 degrees C, cells heated to 50 degrees C showed a 30 and 50% reduction in glycolysis and SIII MR, respectively. After heating to 50 degrees C, the cell membrane remained intact and immunoreactive HSP 72 was not detected. In contrast, heating to 43 degrees C induced accumulation of HSP 72 and transiently increased both SIII MR and glycolysis. In addition, prior exposure to 43 degrees C completely prevented the fall in SIII MR and glycolysis anticipated with a subsequent 50 degrees C insult. Cytoprotection gradually diminished over several days and correlated with the disappearance of HSP 72. Preservation of oxidative and anaerobic metabolism associated with HSPs may be important in developing resistance to thermal injury.

Transient ischemia or heat stress induces a cytoprotectant protein in rat kidney.

Sublethal heat exposure induces the production of heat stress protein (HSP) 72 kDa, a reported cytoprotectant, in several tissues including the rat kidney. However, the localization and time course of HSP 72 accumulation in the kidney in response to heat or other cell stresses such as transient ischemia have not been described. In anesthetized rats exposed to either 42 +/- 0.5 degrees C (heat stress) or 37 degrees C (sham) for 15 min, accumulation of HSP 72 in kidney homogenates, detected by immunoblot analysis using a specific monoclonal anti-HSP 72 antibody, peaked 4-6 h after heat stress and persisted for 10 days. HSP 72 appeared rapidly in renal papilla within 1 h and in medulla and cortex within 4 h after heat stress. No HSP 72 was detected in tissues from sham heat stress. HSP 72 was also detected within 3 h of at least 15 min of renal ischemia in situ. Accumulation was maximal after 60 min of ischemia and persisted for 5 days, whereas no HSP 72 was detected after 90 min of ischemia or in the contralateral nonischemic kidney at any time point. This study demonstrates that transient ischemia, like heat stress, results in the rapid cytosolic accumulation of HSP 72, a known cytoprotectant that may be important in mediating cell repair or increasing resistance to subsequent injury.

Strategies to improve teaching in the ambulatory medicine setting.

Expansion of resident training in ambulatory medicine has created new challenges for faculty preceptors. Outpatient teaching is hampered by inadequate time and a reliance on methods of instruction that are more useful for the inpatient setting. Effective outpatient teaching requires an understanding of the objectives of ambulatory medical training and improved facility with teaching methods that accommodate the brief, impromptu nature of ambulatory teaching. In a hypothetical outpatient teaching encounter, the interactions between the patient, resident, and attending physician are dissected to reveal missed opportunities to teach and to explore alternative approaches to the educational process. These approaches include promoting the resident's role as the primary provider, developing a limited teaching agenda for each teaching encounter, focusing on the learner rather than on the diagnostic dilemma posed by the patient, and using questions, role modeling and observation with feedback to promote learning.

Role of oxygen free radical species in in vitro models of proximal tubular ischemia.

Lipid peroxidation (LP) of cell membranes by oxygen free radical species (OFRS) during reflow may be a mechanism for progressive proximal tubule injury after ischemia. We examined the relationship between tubule function and an endproduct of LP, malondialdehyde (MDA), in two in vitro models using a suspension of rat proximal tubule segments (PTS). Exposure of PTS to tert-butyl hydroperoxide (tBHP), a potent oxidant, induced dose-dependent decrements in the PTS respiration (QO2) but was associated with a progressive increase in MDA. The relationship between increasing doses of tBHP, QO2, and MDA content, as well as that between MDA and QO2 were highly correlated (r greater than or equal to 0.89, n = 6). Dithiothreitol (1 mM), a sulfhydryl reagent, prevented the tBHP-induced changes in both QO2 and LP. In the second model, 45-min O2 deprivation followed by 30 min of reoxygenation produced similar decrements in QO2 as did 0.75 mM tBHP but without a rise in MDA (n = 9). Mitochondria isolated from ischemic PTS had a 49 +/- 8.2% decrement in state III respiration (P less than 0.006, n = 4) but their MDA content was unchanged. Furthermore, allopurinol, superoxide dismutase, and catalase, agents that reduce tissue OFRS, did not attenuate ischemic tubular injury (n = 9). These results suggest that OFRS do not mediate transient O2 deprivation injury to the proximal tubule.