Andrey V. Cybulsky

[38] Experimental glomerulonephritis

Publisher Summary This chapter discusses the analysis of experimental glomerulonephritis. There exist several animal models that closely resemble various forms of human glomerulonephritis. The resemblance includes, in most instances, morphological, immunohistological, and pathophysiological similarities. The chapter also describes different methods for producing and studying three models that cover the spectrum of fixed and planted antigens and various mediator systems. Mediators of the glomerular injury induced by such immune deposits vary widely in different models. They include the complement (C) system acting either through its terminal, membranolytic pathway or via the inflammatory action of leukocytes; antibody-directed influx of leukocytes; and antibody alone. Host species, the nature and dose of antibody, and the distribution of antigen influence which mediators will operate in the various models studied to date. The redistribution of such glomerular epithelial cell antigens follows antibody binding; subsequent shedding of the resulting complexes is thought to account for the observed subepithelial electron densities.

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 A2 (cPLA2). This study addresses the role of endoplasmic reticulum (ER) stress proteins (bip, grp94) in GEC injury. GEC that overexpress cPLA2 (produced by transfection) and “neo” GEC (which expresses cPLA2at 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 cPLA2, as compared with neo, implying that cPLA2 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 cPLA2. 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 cPLA2. ER stress protein induction is a novel mechanism of protection from complement attack.

The intersecting roles of endoplasmic reticulum stress, ubiquitin–proteasome system, and autophagy in the pathogenesis of proteinuric kidney disease

Protein misfolding in the endoplasmic reticulum (ER) leads to ER stress. The unfolded protein response and ER-associated degradation (ERAD) interact in a coordinated manner with the ubiquitin-proteasome system and autophagy to alleviate protein misfolding or its consequences. The intersecting actions of these processes are evident in normal podocyte physiology, and in proteinuric glomerular diseases, including experimental membranous nephropathy, focal segmental sclerosis, and diabetic nephropathy. There is some evidence for the induction of ER stress, changes in the ubiquitin-proteasome system, and presence of autophagy in human glomerulopathies. Various therapeutic approaches to the unfolded protein response, ERAD, and the ubiquitin-proteasome system have corrected experimental glomerular diseases involving protein misfolding, and could potentially be developed as therapies in humans.

Complement C5b-9-mediated arachidonic acid metabolism in glomerular epithelial cells : role of cyclooxygenase-1 and -2.

In the passive Heymann nephritis (PHN) model of membranous nephropathy, complement C5b-9 induces glomerular epithelial cell (GEC) injury and proteinuria, which is partially mediated by eicosanoids. This study addresses the role of cyclooxygenase (COX)-1 and -2 in C5b-9-mediated eicosanoid production in GEC. Unstimulated rat GEC in culture primarily express COX-1. When stimulated with sublytic C5b-9, COX-2 was significantly up-regulated, whereas COX-1 was not affected. Compared with control, complement-treated GEC produced 32% more prostaglandin (PG) E(2) in the presence of exogenous substrate, and the increase was abolished with the COX-2-selective inhibitor, NS-398. Release of arachidonic acid from GEC phospholipids via C5b-9-induced activation of cytosolic phospholipase A(2) was associated with a marked stimulation of PGE(2) production, which was inhibited by 60% with NS-398. The results in cultured GEC were extended to GEC injury in vivo by examining COX-1 and -2 expression in PHN. Glomeruli from rats with PHN expressed significantly more COX-1 and COX-2, as compared with normal rats. PGE(2) production in glomeruli of rats with PHN was about twofold greater than in control glomeruli, and the increase was partially inhibited with NS-398. Thus, in GEC in culture and in vivo, C5b-9-induced eicosanoid production is regulated by both isoforms of COX. The inducible COX-2 may be an important novel mediator of C5b-9-induced glomerular injury.

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 A2 (cPLA2), and products of cPLA2-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 cPLA2 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 α-tocopherol as well as with diphenylene iodonium, an inhibitor of the NADPH oxidase, inhibited complement-induced JNK activation. Conversely, H2O2 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 cPLA2 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.

Induction of Apoptosis by the Ste20-like Kinase SLK, a Germinal Center Kinase That Activates Apoptosis Signal-regulating Kinase and p38

Expression and activity of the germinal center kinase, Ste20-like kinase (SLK), are increased during kidney development and recovery from ischemic acute renal failure. In this study, we characterize the activation and functional role of SLK. SLK underwent dimerization via the C-terminal domain, and dimerization enhanced SLK activity. In contrast, the C-terminal domain of SLK did not dimerize with a related kinase, Mst1, and did not affect Mst1 activity. Phosphorylation/dephosphorylation of SLK were not associated with changes in kinase activity. SLK induced phosphorylation of apoptosis signal-regulating kinase-1 (ASK1) and increased ASK1 activity, indicating that ASK1 is a substrate of SLK. Moreover, SLK stimulated phosphorylation of p38 mitogen-activated protein kinase via ASK1, but not c-Jun N-terminal kinase nor extracellular signal-regulated kinase. Chemical anoxia and recovery during re-exposure to glucose (ischemia-reperfusion injury in cell culture) stimulated SLK activity. Overexpression of SLK enhanced anoxia/recovery-induced apoptosis, release of cytochrome c, and activities of caspase-8 and -9, and apoptosis was reduced significantly with p38 and caspase-9 inhibitors. Induction of the endoplasmic reticulum stress response by anoxia/recovery or tunicamycin (monitored by induction of Bip or Grp94 expression, phosphorylation of eukaryotic translation initiation factor 2α subunit, expression of CHOP, and activation of caspase-12) was attenuated in cells that overexpress SLK. Thus, SLK is an anoxia/recovery-dependent kinase that is activated via homodimerization and that signals via ASK1 and p38 to promote apoptosis. Attenuation of the protective aspects of the endoplasmic reticulum stress response by SLK may contribute to its proapoptotic effect.

Glomerular epithelial cell injury associated with mutant α-actinin-4

Focal segmental glomerulosclerosis (FSGS) may be associated with glomerular epithelial cell (GEC; podocyte) apoptosis due to acquired injury or mutations in alpha-actinin-4. This study addresses how FSGS-associated mutant alpha-actinin-4 may induce GEC injury, focusing on endoplasmic reticulum (ER) stress and metabolism of mutant alpha-actinin-4 via the ubiquitin-proteasome system. In a model of experimental FSGS induced by expression of an alpha-actinin-4 K256E transgene in podocytes, we show induction of ER stress, including upregulation of ER chaperones (bip, grp94), phosphorylation of the eukaryotic translation initiation factor-2alpha subunit, and induction of the proapoptotic gene C/EBP homologous protein-10 (CHOP). To address mechanisms of ER stress, we studied signaling in cultured GEC and COS cells expressing alpha-actinin-4 K256E. Previously, we showed that expression of this alpha-actinin-4 mutant in GEC increased apoptosis. In the present study, we show that alpha-actinin-4 K256E upregulates grp94 and CHOP expression in COS cells and significantly exacerbates induction of bip and CHOP in GEC in the presence of tunicamycin. ER stress was associated with aggregation and ubiquitination of alpha-actinin-4 K256E and impairment of the ubiquitin-proteasome system. In addition, alpha-actinin-4 K256E exacerbated apoptosis in the context of mild proteasome inhibition. Thus alpha-actinin-4 K256E triggers several metabolic abnormalities, which may lead to GEC injury and glomerulosclerosis.

Cyclooxygenases-1 and 2 couple to cytosolic but not group IIA phospholipase A2 in COS-1 cells

Phospholipases A2 (PLA2) and cyclooxygenases (COX) are important enzymes responsible for production of potent lipid mediators, including prostaglandins (PG) and thromboxane A2. We investigated coupling between PLA2 and COX isoforms by using transient transfection in COS-1 cells. Untransfected cells, incubated with or without phorbol ester + the Ca2+ ionophore ionomycin, generated trivial amounts of PGE2. In cells co-transfected with cytosolic PLA2 (cPLA2) and COX-1 or COX-2, phorbol ester + ionomycin markedly stimulated PGE2 production. There was no preferential coupling of cPLA2 to either of the COX isoforms. In contrast, group IIA secretory PLA2 (sPLA2) co-transfected with COX-1 or COX-2 did not lead to an increase in PGE2 production, despite high levels of sPLA2 enzymatic activity. Transfection of cPLA2 did not affect basal free arachidonic acid (AA) levels. Phorbol ester + ionomycin stimulated release of AA in cPLA2-transfected COS-1 cells, but not in untransfected cells, whereas sPLA2 transfection (without stimulation) led to high basal free AA. Thus, AA released by cPLA2 is accessible to both COX isoforms for metabolism to PG, whereas AA released by sPLA2 is not metabolized by COX.

Role of apoptosis signal-regulating kinase 1 in complement-mediated glomerular epithelial cell injury

In the rat passive Heymann nephritis (PHN) model of membranous nephropathy, complement C5b-9 activates protein kinases in glomerular epithelial cells (GEC), and induces sublethal GEC injury and proteinuria. Complement induces production of reactive oxygen species (ROS) via the NAPDH oxidase, and stimulates phosphorylation of c-Jun N-terminal kinase (JNK) and p38 kinase in a ROS-dependent manner. In the present study, we demonstrate that apoptosis signal-regulating kinase 1 (ASK1) was activated in glomeruli of rats with PHN, and that incubation of GEC in culture with antibody and sublytic C5b-9 stimulated ASK1 activity. The latter was, in part, mediated via the NADPH oxidase and ROS. Sublytic complement induced JNK and p38 phosphorylation, which was amplified in GEC that stably overexpress ASK1, as compared with Neo (control) GEC. Complement-induced lysis was enhanced in GEC that overexpress ASK1, as compared with Neo, and was attenuated in GEC that overexpress a dominant negative ASK1 mutant. Inhibition of p38, but not JNK, attenuated complement lysis in GEC that overexpress ASK1, but not in Neo GEC. In Neo GEC, generation of ROS restricted complement-mediated GEC injury but the protective effect of ROS was lost when ASK1 was overexpressed. We propose that the level of ASK1 expression determines the functional effect of p38 activation, i.e. when ASK1 is overexpressed, p38 activation is amplified, and C5b-9 assembly leads to GEC injury via ASK1 and p38. The present study thus defines a novel role for ASK1 as a mediator of C5b-9-dependent cell injury.

Cytosolic phospholipase A2-alpha associates with plasma membrane, endoplasmic reticulum and nuclear membrane in glomerular epithelial cells.

Eicosanoids mediate complement-dependent glomerular epithelial injury in experimental membranous nephropathy. The release of arachidonic acid from phospholipids by cytosolic phospholipase A(2) (cPLA(2)) is the rate-limiting step in eicosanoid synthesis. The present study examines the association of cPLA(2) with membranes of organelles. Glomerular epithelial cells were disrupted by homogenization in Ca(2+)-free buffer; organelles were separated by gradient centrifugation. The distribution of cPLA(2) and organelles was analysed by immunoblotting with antibodies against cPLA(2) and organelle markers, or by enzyme assay. In cells incubated with or without the Ca(2+) ionophore ionomycin plus PMA, cPLA(2) co-localized with plasma membrane, endoplasmic reticulum and nuclei, but not with mitochondria or Golgi. A greater amount of cPLA(2) was associated with membranes in stimulated cells, but membrane-associated cPLA(2) was readily detectable under resting conditions. The pattern of association of cPLA(2) with membrane in cells treated with antibody and complement was similar to that in cells stimulated with ionomycin plus PMA; however, complement did not enhance the membrane association of cPLA(2) protein. To determine the functional role of membrane association of cPLA(2), phospholipids were labelled with [(3)H]arachidonic acid. Cells were then incubated with or without antibody and complement and were fractionated. Complement induced a loss of radioactivity from the plasma membrane, endoplasmic reticulum and nuclei, but not from the mitochondrial fraction. Thus the release of arachidonic acid by cPLA(2) is due to the hydrolysis of phospholipids at multiple subcellular membrane sites, including the endoplasmic reticulum, plasma membrane and nucleus.