Biomedical subjects

Abdelkrim Khadir

Publications and source records attributed to Abdelkrim Khadir.

6 recordsLinked to original sources

PubMed2003

The actin cytoskeleton facilitates complement-mediated activation of cytosolic phospholipase A2.

Cytosolic PLA(2)-alpha (cPLA(2)) and metabolites of arachidonic acid (AA) are key mediators of complement-dependent glomerular epithelial cell (GEC) injury. Assembly of C5b-9 increases cytosolic Ca(2+) concentration and results in transactivation of receptor tyrosine kinases and activation of PLC-gamma 1 and the 1,2-diacylglycerol (DAG)-PKC pathway. Ca(2+) and PKC are essential for membrane association and increased catalytic activity of cPLA(2). This study addresses the role of the actin cytoskeleton in cPLA(2) activation. Depolymerization of F-actin by cytochalasin D or latrunculin B reduced complement-dependent [(3)H]AA release, as well as the complement-induced increase in cPLA(2) activity. These effects were due to inhibition of [(3)H]DAG production and PKC activation, implying interference with PLC. Complement-dependent [(3)H]AA release was also reduced by jasplakinolide, a compound that stabilizes F-actin and organizes actin filaments at the cell periphery, and calyculin A, which induces condensation of actin filaments at the plasma membrane. The latter drugs did not affect [(3)H]DAG production, suggesting their inhibitory actions were downstream of PKC. Neither cytochalasin D, latrunculin B, nor calyculin A affected association of cPLA(2) with microsomal membranes, and cytochalasin D and latrunculin B did not alter the localization of the endoplasmic reticulum. Stable transfection of constitutively active RhoA induced formation of stress fibers, stabilized F-actin, and attenuated the complement-induced increase in [(3)H]AA. Thus in GEC, cPLA(2) activation is dependent, in part, on actin remodeling. By regulating complement-mediated activation of cPLA(2), the actin cytoskeleton may contribute to the pathophysiology of GEC injury.

Actin Cytoskeleton↗

PubMed2003

Extracellular matrix regulates glomerular epithelial cell survival and proliferation.

Glomerular epithelial cell (GEC) injury and apoptosis may contribute to sclerosis in glomerulonephritis. The present study addresses signals that regulate survival of GEC in culture and in the acute puromycin aminonucleoside nephrosis (PAN) model of GEC injury in vivo. Compared with GEC on plastic substratum, adhesion to collagen increased activation of focal adhesion kinase (FAK), c-Src, and ERK and facilitated survival (prevented apoptosis). GEC on plastic exhibited increased caspase-8 and -9 activities, increased expression of the proapoptotic protein, Bax, and decreased the antiapoptotic protein, Bcl-XL, compared with collagen. Stable expression of constitutively active mutants of FAK (CD2-FAK) or MEK (R4F-MEK) activated the ERK pathway and supplanted the requirement of collagen for survival. In contrast, expression of a Ras mutant that activates phosphatidylinositol 3-kinase but blocks ERK activation or pharmacological inhibition of the ERK pathway decreased survival on collagen. Glomeruli isolated from rats with PAN revealed increased beta1-integrin expression, along with increased activation of FAK, c-Src, and ERK, compared with controls. EGF receptor activation was undetectable in PAN. Therefore, adhesion to collagen, resulting in activation of FAK and the Ras-ERK pathway, supports GEC survival. Analogous signals for GEC survival are activated in PAN.

Animals↗

PubMed2003

Renal expression and activity of the germinal center kinase SK2.

Rat fetal kidney mRNA was analyzed by RT-PCR to identify protein kinases. This screening demonstrated expression of a protein kinase consistent with SK2, a group II germinal center kinase and homolog of human Ste20-like kinase (SLK). SK2 mRNA, protein expression, and kinase activity were increased in rat fetal kidney homogenates (embryonic days 17-21) compared with adult controls. In adult kidneys subjected to cross-clamping of the renal artery, followed by reperfusion, SK2 mRNA, protein expression, and kinase activity were increased compared with untreated contralateral controls. By immunohistochemistry, SK2 expression was evident mainly in the cytoplasm of tubular epithelial cells in fetal and adult kidneys. There was also some expression in developing and mature podocytes, but staining of the interstitium was negative. In cultured renal tubular epithelial cells, SK2 kinase activity was increased after incubation with serum, or after exposure to chemical anoxia plus reexposure to glucose. Stable overexpression of SLK reduced cell proliferation and increased apoptosis and exacerbated apoptosis and necrosis after chemical anoxia plus reexposure to glucose. Thus SK2 is a renal epithelial protein kinase whose expression and activity are increased during development and recovery from acute renal failure, where tubular epithelial regeneration may recapitulate developmental processes. The actions of SK2 appear to be antiproliferative and may facilitate cell injury.

Acute Kidney Injury↗

PubMed2003

Solution structure of human BCL-w: modulation of ligand binding by the C-terminal helix.

The structure of human BCL-w, an anti-apoptotic member of the BCL-2 family, was determined by triple-resonance NMR spectroscopy and molecular modeling. Introduction of a single amino acid substitution (P117V) significantly improved the quality of the NMR spectra obtained. The cytosolic domain of BCL-w consists of 8 alpha-helices, which adopt a fold similar to that of BCL-xL, BCL-2, and BAX proteins. Pairwise root meant square deviation values were less than 3 A for backbone atoms of structurally equivalent regions. Interestingly, the C-terminal helix alpha8 of BCL-w folds into the BH3-binding hydrophobic cleft of the protein, in a fashion similar to the C-terminal transmembrane helix of BAX. A peptide corresponding to the BH3 region of the pro-apoptotic protein, BID, could displace helix alpha8 from the BCL-w cleft, resulting in helix unfolding. Deletion of helix alpha8 increased binding affinities of BCL-w for BAK and BID BH3-peptides, indicating that this helix competes for peptide binding to the hydrophobic cleft. These results suggest that although the cytosolic domain of BCL-w exhibits an overall structure similar to that of BCL-xL and BCL-2, the unique organization of its C-terminal helix may modulate BCL-w interactions with pro-apoptotic binding partners.

Amino Acid Sequence↗

PubMed2002

Complement activates the c-Jun N-terminal kinase/stress-activated protein kinase in glomerular epithelial cells.

In the rat passive Heymann nephritis model of membranous nephropathy, complement C5b-9 induces sublethal glomerular epithelial cell (GEC) injury and proteinuria. C5b-9 activates cytosolic phospholipase A(2) (cPLA(2)), and products of cPLA(2)-mediated phospholipid hydrolysis modulate GEC injury and proteinuria. In the present study, we demonstrate that C5b-9 activates c-Jun N-terminal kinase (JNK) in cultured rat GECs and that JNK activity is increased in glomeruli isolated from proteinuric rats with passive Heymann nephritis, as compared with control rats. Stable overexpression of cPLA(2) in GECs amplified complement-induced release of arachidonic acid (AA) and JNK activity, as compared with neo (control) GECs. Activation of JNK was not affected by indomethacin. Incubation of GECs with complement stimulated production of superoxide, and pretreatment with the antioxidants, N-acetylcysteine, glutathione, and alpha-tocopherol as well as with diphenylene iodonium, an inhibitor of the NADPH oxidase, inhibited complement-induced JNK activation. Conversely, H(2)O(2) activated JNK, whereas exogenously added AA stimulated both superoxide production and JNK activity. Overexpression of a dominant-inhibitory JNK mutant or treatment with diphenylene iodonium exacerbated complement-dependent GEC injury. Thus, activation of cPLA(2) and release of AA facilitate complement-induced JNK activation. AA may activate the NADPH oxidase, leading to production of reactive oxygen species, which in turn mediate the activation of JNK. The functional role of JNK activation is to limit or protect GECs from complement attack.

Animals↗

PubMed2002

Complement C5b-9 membrane attack complex increases expression of endoplasmic reticulum stress proteins in glomerular epithelial cells.

In the passive Heymann nephritis (PHN) model of membranous nephropathy, complement C5b-9 induces glomerular epithelial cell (GEC) injury, proteinuria, and activation of cytosolic phospholipase A(2) (cPLA(2)). This study addresses the role of endoplasmic reticulum (ER) stress proteins (bip, grp94) in GEC injury. GEC that overexpress cPLA(2) (produced by transfection) and "neo" GEC (which expresses cPLA(2) at a lower level) were incubated with complement (40 min), and leakage of constitutively expressed bip and grp94 from ER into cytosol was measured to monitor ER injury. Greater leakage of bip and grp94 occurred in complement-treated GEC that overexpress cPLA(2), as compared with neo, implying that cPLA(2) activation perturbed ER membrane integrity. After chronic incubation (4-24 h), C5b-9 increased bip and grp94 mRNAs and proteins, and the increases were dependent on cPLA(2). Expression of bip-antisense mRNA reduced stimulated bip protein expression and enhanced complement-dependent GEC injury. Glomerular bip and grp94 proteins were up-regulated in proteinuric rats with PHN, as compared with normal control. Pretreatment of rats with tunicamycin or adriamycin, which increase ER stress protein expression, reduced proteinuria in PHN. Thus, C5b-9 injures the ER and enhances ER stress protein expression, in part, via activation of cPLA(2). ER stress protein induction is a novel mechanism of protection from complement attack.

Animals↗