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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

Although heat shock protein 72 kDa (HSP72) protects tubular epithelium from a variety of acute insults, its role in chronic renal injury and fibrosis is poorly characterized. In this study, we tested the hypothesis that HSP72 reduces apoptosis and epithelial-to-mesenchymal transition (EMT), important contributors to tubular cell injury in vitro and in vivo. In rats, orally administered geranylgeranylacetone (GGA), an agent that selectively induces HSP72, markedly reduced both apoptosis and cell proliferation in tubular epithelium and decreased both interstitial fibroblast accumulation and collagen I deposition after unilateral ureteric obstruction, a model of chronic renal tubulointerstitial fibrosis and dysfunction. In cultured renal NRK52E cells, exposure to TGF-[beta]1 induced EMT and apoptosis, major causes of renal fibrosis and tubular atrophy, respectively. Exposure to a pancaspase inhibitor (ZVAD-FMK) prevented TGF-[beta]1-induced apoptosis but did not reduce EMT. In contrast, selective HSP72 expression in vitro inhibited EMT caused by TGF-[beta]1 as indicated by preserving the E-cadherln expression level and [alpha]-smooth muscle actin induction. Small interfering RNA directed against HSP72 blocked the cytoprotective effects of HSP72 overexpression on EMT in TGF-[beta]1-exposed cells. Taken together, our data indicate that HSP72 ameliorates renal tubulointerstitial fibrosis in obstructive nephropathy by inhibiting both renal tubular epithelial cell apoptosis and EMT. heat stress protein 72 kDa; epithelial-to-mesenchymal transition, s-smooth muscle actin, E-cadherin, transforming growth factor-[beta]1

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

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

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.

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 site-specific phosphorylation of nucleophosmin (NPM), an essential Bax chaperone, in stress-induced cell death is unknown. We hypothesized that NPM threonine 95 (T95) phosphorylation both signals and promotes cell death. In resting cells, NPM exclusively resides in the nucleus and T95 is nonphosphorylated. In contrast, phosphorylated T95 NPM (pNPM T95) accumulates in the cytosol after metabolic stress, in multiple human cancer cell lines following γ-radiation, and in postischemic human kidney tissue. Based on the T95 phosphorylation consensus sequence, we hypothesized that glycogen synthase kinase-3α (GSK-3α) regulates cytosolic NPM translocation by phosphorylating T95 NPM. In a cell-free system, GSK-3α phosphorylated a synthetic NPM peptide containing T95. In vitro, bidirectional manipulation of GSK-3α activity substantially altered T95 phosphorylation, cytosolic NPM translocation, and cell survival during stress, mechanistically linking these lethal events. Furthermore, GSK-3α inhibition in vivo decreased cytosolic pNPM T95 accumulation in kidney tissue after experimental ischemia. In patients with acute kidney injury, both cytosolic NPM accumulation in proximal tubule cells and NPM-rich intratubular casts were detected in frozen renal biopsy tissue. These observations show, for the first time, that GSK-3α promotes cell death partly by phosphorylating NPM at T95, to promote cytosolic NPM accumulation. T95 NPM is also a rational therapeutic target to ameliorate ischemic renal cell injury and may be a universal injury marker in mammalian cells. [ABSTRACT FROM AUTHOR]

Lader, Alan S., Yihan Wang, G. Robert Jackson, Jr., Steven C. Borkan, and Horacio F. Cantiello. cAMP-activated anion conductance is associated with expression of CFTR in neonatal mouse cardiac myocytes. Am. J. Physiol. Cell Physiol. 278: C436-C450, 2000.--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.sup.-] conditions and symmetrical [Cl.sup.-] in the presence of various counterions, including [Na.sup.+], [Mg.sup.2+], [Cs.sup.+], 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 anticystic 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.sup.-]-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.sup.-]-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.sup.-]-and ATP-permeable pathways in the developing heart. cystic fibrosis; heart; adenosine 5'-triphosphate channels; chloride channels; adenosine 5'-triphosphate release

Prat, Adriana G., C. Casey Cunningham, G. Robert Jackson, Jr., Steven C. Borkan, Yihan Wang, Dennis A. Ausiello, and Horacio F. Cantiello. Actin filament organization is required for proper cAMP-dependent activation of CFTR. Am. J. Physiol. 277 (Cell Physiol. 46): C1160-C1169, 1999.--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.sup.-] currents. In ABP(-) cells expressing CFTR, cAMP was also without effect on [Cl.sup.-] conductance. In contrast, cAMP induced a 10-fold increase in the diphenylamine-2-carboxylate (DPC)-sensitive whole cell [Cl.sup.-] 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.sup.-] channels only in excised patches from ABP(+)/ CFTR(+) cells. Furthermore, filamin alone also induced [Cl.sup.-] 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. ABP-280; cystic fibrosis transmembrane conductance regulator; actin cytoskeleton; adenosine 3',5'-cyclic monophosphate

Protein mimotopes, or blocking peptides, are small therapeutic peptides that prevent protein-protein interactions by selectively mimicking a native binding domain. Inexpensive technology facilitates straightforward design and production of blocking peptides in sufficient quantities to allow preventive and therapeutic trials in both in vitro and in vivo experimental disease models. The kidney is an ideal peptide target, since small molecules undergo rapid filtration and efficient bulk absorption by tubular epithelial cells. Because the half-life of peptides is markedly prolonged in the kidneys compared with the bloodstream, blocking peptides are an attractive tool for treating diverse renal diseases, including ischemia, proteinuric states, such as membranous nephropathy and focal and segmental glomerulosclerosis, and renal cell carcinoma. Therapeutic peptides represent one of the fastest-growing reagent classes for novel drug development in human disease, partly because of their ease of administration, high binding affinity, and minimal off-target effects. This review introduces the concepts of blocking peptide design, production, and administration and highlights the potential use of therapeutic peptides to prevent or treat specific renal diseases. [ABSTRACT FROM AUTHOR]

Proteinuria is a major risk factor for chronic kidney disease progression. Furthermore, exposure of proximal tubular epithelial cells to excess albumin promotes tubular atrophy and fibrosis, key predictors of progressive organ dysfunction. However, the link between proteinuria and tubular damage is unclear. We propose that pathological albumin exposure impairs proximal tubular autophagy, an essential process for recycling damaged organelles and toxic intracellular macromolecules. In both mouse primary proximal tubule and immortalized human kidney cells, albumin exposure decreased the number of autophagosomes, visualized by the autophagosome-specific fluorescent markers monodansylcadaverine and GFP-LC3, respectively. Similarly, renal cortical tissue harvested from proteinuric mice contained reduced numbers of autophagosomes on electron micrographs, and immunoblots showed reduced steady-state LC3-II content. Albumin exposure decreased autophagic flux in vitro in a concentration-dependent manner as assessed by LC3-II accumulation rate in the presence of bafilomycin, an H+-ATPase inhibitor that prevents lysosomal LC3-II degradation. In addition, albumin treatment significantly increased the half-life of radiolabeled long-lived proteins, indicating that the primary mechanism of degradation, autophagy, is dysfunctional. In vitro, mammalian target of rapamycin (mTOR) activation, a potent autophagy inhibitor, suppressed autophagy as a result of intracellular amino acid accumulation from lysosomal albumin degradation. mTOR activation was demonstrated by the increased phosphorylation of its downstream target, S6K, with free amino acid or albumin exposure. We propose that excess albumin uptake and degradation inhibit proximal tubule autophagy via an mTOR-mediated mechanism and contribute to progressive tubular injury. [ABSTRACT FROM AUTHOR]