Steven C. Borkan - US grants

The mechanism(s) by which proteins are translocated across membranes remains a central focus of research in modern cell biology. The cell membrane is thought to act as a selective protein barrier and with few exceptions, exogenous proteins are either excluded from the cell or enter a 'default pathway' that rapidly culminates with lysosomal degradation. Entry of an exogenous protein into the cell's interior is a rare event and thus far, appears restricted to a few bacterial toxins with unique targeting sequences that manage to escape lysosomal degradation. In contrast to any previous description of exogenous protein translocation, we recently found an abundant, 15.5 kDa protein in the rat proximal tubule that is a proteolytic cleavage product of alpha2mu-microglobulin (A2), a 19 kDa protein synthesized predominantly by the liver. A2 is a member of a protein superfamily (including retinol binding protein) thought to function as transport proteins for hydrolyphobic ligands such as long chain fatty acids. Accumulation of this hepatic protein within the proximal tubule of the kidney may be the first physiologically significant example of 'retrograde protein transport' in which a naturally synthesized, exogenous protein is accumulated in the cytosol of another cell type. We suggest that following glomerular filtration, the proximal tubule cell metabolizes A2 by a novel pathway, ultimately accumulating large amount of a proteolytic cleavage product (A2-fragment) in the cytosol. To evaluate this hypothesis, we propose to: (1) describe the process of A2 uptake and translocation by the proximal epithelial cell; (2) determine whether this process is mediated by endocytosis (receptor-mediated, fluid-phase, or adsorptive endocytosis) or simple diffusion; (3) evaluate whether binding of A2 to a hydrophobic ligand is required for, or modifies A2 uptake; (4) identify the cellular compartment(s) responsible for processing A2 so that cytosolic accumulation, rather than degradation, of A2 occurs and (5) determine whether unique structural features determine how A2 is processed by the proximal tubule cell. These studies could provide new insights regarding a previously unknown pathway for moving proteins from outside to inside the cell. This information could have relevance for targeted delivery of various agents or drugs to the kidney. In addition, A2 binds fatty acids in vitro and act as a 'fatty acid binding like-protein' in vivo. As a transport protein, A2 could modulate fatty acid oxidation rates during both normal and pathophysiologic states such as rental ischemia.

Stresses such as ischemia or ATP depletion induce the synthesis of cytoprotective heat stress proteins (HSP). Of the inducible cytoprotectants, HSP 72 is the most abundant and well-characterized. HSP 72 is a molecular chaperone that removes and repairs damaged cell proteins. In non-renal cells and tissues, selective over-expression of HSP 72 decreases subsequent injury from ischemia or ATP depletion. In these models, the cell functions protected by HSP 72 have not been identified. In the renal epithelial cell, ischemia in vivo or ATP depletion in vitro induce HSPs, cause collapse of the actin cytoskeleton, loss of tight junction integrity, impair mitochondrial ATP production and trigger apoptosis. We have previously shown that prior heat stress, sufficient to upregulate HSP 72, preserves these cell functions and improves renal cell survival after ATP depletion. The central hypothesis of this project is that HSP 72 is a major renal cytoprotectant. We suggest that by binding to proteins that regulate: (a) the cytoskeleton, (b) cell-cell contact sites, (c) native DNA, (d) mitochondrial function and (e) the apoptotic pathway, HSP 72 decreases cell injury and inhibits apoptosis caused by ATP depletion. This hypothesis is supported by our preliminary studies in which the selective induction (or suppression) of HSP 72 alone alters survival in ATP depleted renal epithelial cells. In an in vitro model of ATP depletion that resembles ischemia/reperfusion in vivo, we will: (1) identify cell functions protected by HSP 72; (2) identify some of the intracellular proteins that interact with HSP 72; (3) determine the role of protein binding vs. refolding in mediating cytoprotection by HSP 72 and (4) evaluate intracellular signal(s) that can induce HSP 72 without requiring heat stress. We have developed the molecular, immunologic and biochemical tools to manipulate HSP 72 content in renal epithelial cells and to identify proteins that bind to it. Human HSP 72 deletion mutants with well-described defects in protein binding or re-folding will be used to determine the mechanism by which this molecular chaperone protects target proteins in ATP depleted cells. To evaluate some of the intracellular signals that induce HSP 72, we have developed a novel, permeabilized cell system in which intracellular pH, calcium and adenine nucleotide content can be individually manipulated. These studies will examine some of the pathways that permit HSP 72 to protect the renal epithelial cell and may ultimately provide a means for increasing cellular resistance to ischemic injury, a common cause of acute renal failure.

DESCRIPTION (provided by applicant): Renal ischemia in vivo and ATP depletion in vitro cause renal epithelial cells to die by apoptosis. Apoptosis is caused by mitochondrial membrane injury that is regulated by well-characterized members of the BCL2 protein family. We have established that ATP depletion in renal cells increases bax (pro-apoptotic), decreases bcl2 (anti-apoptotic) and causes apoptosis. Concomitant with this pro-apoptotic change in the bcl2:bax ratio, cytochrome c and apoptosis inducing factor (AIF) translocate from mitochondria into the cytosol and state III mitochondrial respiration is reduced. Cytosolic cytochrome c activates caspases, whereas AIF enters the nucleus, activates endonucleases and causes DNA degradation. Prior heat stress, sufficient to induce hsp72, a molecular chaperone, stabilizes the bcl2:bax ratio, decreases cytochrome c and AIF leak, reduces caspase activation and DNA degradation, completely preserves organeile function and enhances long term survival after ATP depletion. The selective over-expression of hsp72 mimics the protective effects of prior heat stress on apoptosis and renal cell survival. The fact that hsp72 binds bcl2 (but not bax), cytochrome c and AIF suggests that hsp72 itself is an anti-apoptotic protein. We hypothesize that hsp72 decreases primary injury to the mitochondrial membrane caused by bax and inhibits the secondary, pro-apoptotic effects of leaked cytochrome c and AIF on caspase activation and DNA degradation, respectively. In isolated organelles and intact ceils, this study will: (1) determine whether cytoprotection by hsp72 against bax-mediated mitochondrial membrane injury requires bcl2; (2) evaluate the ability of hsp72 to rescue bcl2 function, prevent caspase activation and/or to inhibit AIF-mediated DNA degradation and (3) identify the specific hsp72 domain(s) responsible for its anti-apoptotic actions. To achieve these AIMS, hsp72, bax and bcl2 content will be manipulated in renal epithelial cells using adenoviral infection with established vectors including wild type human hsp72, antisense and known hsp72 deletion mutants with specific functional defects. These studies will identify the anti-apoptotic mechanism(s) of hsp72. These insights can prompt the development of preemptive strategies for preventing renal cell injury during ischemic insults as well as other forms of renal injury in which apoptosis contributes to organ failure.

Project Summary/Abstract Ischemic acute kidney injury (AKI) is a common and often a life-threatening consequence of reduced blood flow to this critical organ causes major morbidity and mortality, and lacks an effective treatment. For the first time, we have detected identical biochemical events in both ischemic murine and human kidneys that mediate renal cell death leading to AKI. In this proposal, we study the role of nucleophosmin (NPM), a protein present in all mammalian cells that is converted from ?friend? to a ?foe? during ischemic stress. During renal ischemia, NPM undergoes biochemical changes that promote its toxicity in renal epithelial cells, a major contributor to organ failure during AKI. These steps include: NPM translocation from the nuclear to the cytosolic compartment, NPM de-oligomerization, interaction with Bax, a major cause of outer mitochondrial membrane injury, and renal cell death by both apoptosis and necrosis, the two forms of cell death consistently detected in ischemic human kidneys. Preliminary data using mass spectroscopy have identified 5 phosphorylation changes in murine and human proximal tubule cells, kidney tissue, and urine that regulate NPM toxicity during renal ischemia. In three AIMS, we will link post-translational phosphorylation events with NPM toxicity, identify the role of NPM in human renal disease using banked kidney biopsy tissue, test NPM phospho-mutant proteins that replicate toxic modifications, and develop novel peptide reagents for ameliorating NPM toxicity. Once identified in vitro, we will test the most effective peptides and pharmaceuticals to treat ischemic AKI in vivo. This in vitro testing minimizes the number of animals required to perform our proposed studies that will examine the biologic effects of gender in AKI diagnosis and treatment. We have developed a novel urinary NPM assay that will be used to as an early AKI biomarker and also for measuring the therapeutic efficacy of our treatment. Assessment of total and site-specific phosphorylated NPM in urine and tissue will trigger early treatment, and may ultimately permit us to predict the severity of AKI and its recovery. Detection of NPM in human kidney biopsy tissue will identify potential AKI causes (e.g., ischemia or nephrotoxin-induced AKI) that are amenable to anti-NPM therapeutics. Since NPM is identically regulated during ischemia in mice and humans, it is likely that effective interventions in mice will translate to human clinical trials.