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Dr. Andrey Cybulsky

C5b-9 signaling and mechanisms of cell injury

The complement C5b-9 membrane attack complex is the key mediator of injury in a number of glomerulopathies (including membranous nephropathy), and it is, therefore, important to understand its mechanism of action in renal cells (particularly the glomerular epithelial cell). Although classically regarded as a cytolytic complex, more recently it has become apparent that at limiting concentrations, C5b-9 induces a variety of biochemical changes in target cells, and is itself metabolized by target cells. We have demonstrated that in experimental membranous nephropathy, C5b-9 injures the glomerular epithelial cell (“podocyte”), in association with the activation of phospholipase C and phospholipase (PLA2). Products of these phospholipases can modulate both C5b-9-mediated effects on cell membrane injury, as well as glomerular capillary wall permselectivity. Our studies have been directed at characterizing the mechanism of PLA2 activation by C5b-9; specifically, we examined the isoforms of PLA2 that may be targets for activation by C5b-9, and we have demonstrated that the principal enzyme is cytosolic PLA2 (“cPLA2”). We have overexpressed wild type and mutant forms of cPLA2 in glomerular epithelial cells (by transfection), and have used these cells to characterize the mechanisms of cPLA2 activation, and the potential consequences of cPLA2 activation with regard to prostaglandin production, phospholipid turnover, cytotoxicity, as well as recovery from cell injury. We have also demonstrated that cPLA2 and cyclooxygenase-1 and -2 are activated in experimental membranous nephropathy in vivo, and are functionally important. More recently, we have demonstrated that C5b-9 induces endoplasmic reticulum (ER) stress both in cultured cells and experimental membranous nephropathy in vivo. ER stress is a novel mechanism that cells employ to recover from injury, and offers possibilities for therapeutic targeting in glomerular disease. Our aim is to understand the cellular response to sublethal injury by C5b-9 and to define mechanisms of injury and mechanisms cells use to survive complement attack. This is the best approach to eventually developing specific therapies. (See representative publications) 

 

Extracellular matrix as a determinant of signaling responses in glomerular epithelial cells

The glomerular epithelial cell or “podocyte” plays an important role in maintaining glomerular permselectivity. Our studies have established that adhesion of cultured glomerular epithelial cells to extracellular matrix provides important physiological signals, including inositol lipid turnover (via “outside-in” b1 integrin signaling), and regulation of the epidermal growth factor receptor-Ras-p42/p44 mitogen-activated protein kinase pathway, and proliferation. In vivo, proliferation of highly differentiated glomerular epithelial cells is generally not observed in the normal kidney, but glomerular epithelial cell injury occurs in focal segmental glomerulosclerosis, and may include apoptosis, and proliferation. We propose that extracellular matrix regulates mechanisms that promote glomerular epithelial cell survival, and in the presence of growth factors, enables proliferation. Thus, following glomerular epithelial cell injury in vivo, signals from extracellular matrix, including focal adhesion kinase, Src and extracellular signal-regulated kinase, as well as growth factor receptors, are activated to maintain cell survival (or prevent apoptosis), limit injury and promote healing. Glomerular epithelial cell proliferation may represent an “overcorrective” response, but proliferation occurs in the context of phenotypic dysregulation. Our present studies involve cell culture and in vivo models. The studies are directed at characterizing the extracellular matrix-induced survival mechanisms in glomerular epithelial cells, and focus on defining the role of small GTPases, cytoskeleton, inositol lipids, caspases, and Bcl-2 family proteins. More recently, our studies have also focused on characterizing the functional effects of a mutation in the cytoskeletal protein, a-actinin-4. Such mutations are the basis of certain forms of hereditary focal segmental glomerulosclerosis in humans. (See representative publications) 

Protein kinases in the kidney
Recovery of cells from injury may involve recapitulation of developmental mechanisms. Our studies have demonstrated increased expression and activation of a germinal center kinase, SLK (or rat homolog, SK2) in renal development, and recovery from ischemic acute renal failure. The actions of SLK appear to be anti-proliferative and/or pro-apoptotic. Moreover, SLK is expressed primarily in renal tubular and glomerular epithelial cells, in both developing and adult kidney. The regulation of SLK activity is complex and may involve changes in mRNA stability as well as protein-protein interactions. Signaling by SLK occurs via stress protein kinase pathways. Our present studies employ cell culture and in vivo models, and are directed at further characterizing the regulation, signaling, and functions of SLK in the kidney. The overall aim of these studies is to obtain a better understanding of the role of protein kinases in renal development and mediation of injury. (See representative publications)

 A.V. Cybulsky