Ora A. Weisz - US grants

Many transport functions in the kidney result in movement of ions up an electrochemical gradient and thus require a highly polarized distribution of channels and transporters. This distribution is maintained by selective sorting of proteins, which occurs in acidic intracellular compartments. When acidification of these compartments is disrupted, protein sorting along both the biosynthetic and endocytic pathways is altered. This results in mistargeting of many cell surface proteins, and could have profound effects on epithelial cell function. Defects in acidification has been suggested to play a role in the pathology of diseases as diverse as cystic fibrosis, cancer, and influenza infection. Our goal is to understand the consequences of pH perturbation on traffic through acidified sorting compartments, particularly the trans Golgi network and endosomes. Our approach involves the expression of influenza M2, a protein which modulates intracellular pH of acidic compartments, in polarized Madin-Darby canine kidney cells. Using this molecule, we will dissect the role of acidification in individual steps in protein transport and to determine the mechanism by which pH perturbation disrupts traffic through these compartments.

DESCRIPTION (provided by applicant): Renal cell function requires the maintenance of polarized plasma membrane domains with distinct protein and lipid compositions. This is accomplished in part by the targeted delivery of newly synthesized and recycling membrane proteins to the apical or basolateral surface. Apical sorting signals are extraordinarily diverse and include peptide-, lipid- and glycan-dependent motifs. Interestingly, recent data from our and other laboratories suggest that apical proteins with different targeting signals traffic to the surface in distinct populations of transport carriers. These post-Golgi carriers may traffic directly to the cell surface;however, recent studies suggest that some newly-synthesized proteins transit recycling endosomes en route to the cell surface. Our long term goals are to identify the signals that direct apical delivery of newly- synthesized proteins, and to understand how these signals are interpreted at various stages along the biosynthetic and postendocytic pathways. The aims of this proposal are to identify proteins that regulate distinct pathways to the apical surface in renal epithelial cells, to assess the role of endocytic compartments in the polarized biosynthetic traffic of different classes of apical proteins, and to dissect the mechanism of glycan-dependent sorting along the biosynthetic and postendocytic pathways. The results of our studies will refine our understanding of how transport cues on physiologically relevant molecules are interpreted at distinct intracellular sites to enable proper sorting. PROJECT NARRATIVE The primary function of the kidney is the reabsorption of water, ions, and metabolites from the forming urine to the bloodstream. The surfaces of kidney cells are subdivided into different domains that face the urine and bloodstream and which contain distinct proteins and lipids;and this asymmetric distribution of surface components is essential for proper kidney function. Our goal is to understand how kidney cells create and maintain these distinct surface domains. The results of our research will provide critical basic information that can be applied to the design of potential therapies to combat kidney-related diseases including renal carcinoma, acute renal failure, and hypertension.

[unreadable] DESCRIPTION (provided by applicant): The local generation of distinct phosphatidylinositol (PI) lipid species has been implicated in the regulation of numerous membrane trafficking events and in the control of cytoskeletal dynamics. The compartmentalized synthesis of noninterchangeable pools of PIs by PI-metabolizing enzymes is critical to the cell's ability to control these multiple PI-dependent functions independently. We are interested in the role of phosphatidylinositol kinases and inositol polyphosphate phosphatases in polarized membrane traffic in renal epithelial cells, and have found that apical and basolateral biosynthetic pathways are differentially sensitive to overexpression of the PI metabolizing enzymes PI-4 kinase beta and PI-5 kinase alpha. Moreover, the observation that Lowe Syndrome (a disease with severe renal manifestations) is caused by molecular defects in a Golgi-tocalized PI-catabolizing enzyme suggests a role for inositol polyphosphate 5-phosphatase activity in biosynthetic transport. In this proposal, we will correlate the effects of expressing individual PI-metabolizing enzymes on transport with actual changes in PI lipid composition across the Golgi complex and test hypotheses to explain the mechanisms by which distinct PI species regulate membrane traffic. These experiments will significantly enhance our understanding of how localized PI synthesis operates at a molecular level to regulate polarized biosynthetic traffic in normal and disease states. [unreadable] [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): The local generation of distinct phosphatidylinositol (PI) lipid species has been implicated in the regulation of numerous membrane trafficking events and in the control of cytoskeletal dynamics. The compartmentalized synthesis of noninterchangeable pools of PIs by PI-metabolizing enzymes is critical to the cell's ability to control these multiple PI-dependent functions independently. We are interested in the role of phosphatidylinositol kinases and inositol polyphosphate phosphatases in polarized membrane traffic in renal epithelial cells, and have found that apical and basolateral biosynthetic pathways are differentially sensitive to overexpression of the PI metabolizing enzymes PI-4 kinase beta and PI-5 kinase alpha. Moreover, the observation that Lowe Syndrome (a disease with severe renal manifestations) is caused by molecular defects in a Golgi-tocalized PI-catabolizing enzyme suggests a role for inositol polyphosphate 5-phosphatase activity in biosynthetic transport. In this proposal, we will correlate the effects of expressing individual PI-metabolizing enzymes on transport with actual changes in PI lipid composition across the Golgi complex and test hypotheses to explain the mechanisms by which distinct PI species regulate membrane traffic. These experiments will significantly enhance our understanding of how localized PI synthesis operates at a molecular level to regulate polarized biosynthetic traffic in normal and disease states. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): The maintenance of polarized plasma membrane domains with distinct protein and lipid compositions is critical for efficient renal cell function. The distribution of receptors and ion transporters in these cells is regulated in part by the rate at which these proteins are internalized from the apical and basolateral cell surfaces. The mechanisms that underlie cargo recruitment to clathrin coated pits where this process is initiated are poorly understood. Localized synthesis of phosphatidylinositol 4,5-bisphosphate (PIP2) at endocytic sites plays multiple roles in endocytosis, and modulation of surface PIP2 levels in nonpolarized cells can differentially affect the internalization of distinct proteins. The central hypothesis of this proposal is that apical and basolateral PIP2 metabolism in renal epithelial cells is independently regulated to selectively modulate clathrin-dependent endocytosis from these domains. Consistent with this idea, that the three isoforms of PI5-kinase (the enzymes that generate cell surface PIP2) are differentially localized in polarized renal epithelial cells. The specific aims of our proposal are to determine whether apical and basolateral pools of PIP2 are independently modulated in renal epithelial cells;to examine how changes in PIP2 levels upon agonist stimulation of G protein coupled receptors affect endocytosis from polarized membrane domains;and to determine how the apically localized PI5-kinase mPI5KI1 regulates apical endocytosis. The results of our studies have important implications for our understanding of how polarized cells compartmentalize PI synthesis and catabolism and for how this process is affected during normal signaling and in renal disease. PUBLIC HEALTH RELEVANCE: The surface of kidney cells is segregated into distinct domains with unique protein and lipid compositions that face the urine and the bloodstream. This asymmetric distribution of surface components is essential for kidney cells to properly reabsorb water, ions, and metabolites from the forming urine. These processes are controlled by receptors and ion transporters whose delivery to and removal from the cell surface is regulated by numerous cellular factors. Our goal is to understand how kidney cells differentially regulate the removal of these proteins from the surface, and how these mechanisms are selectively adjusted during the response to specific physiological signals. The results of our research will provide new information about how the trafficking of kidney proteins is regulated under normal conditions and under conditions of physiological stress.

DESCRIPTION (provided by applicant): Kidney cell function requires the maintenance of polarized plasma membrane domains with distinct protein and lipid compositions, and defects in proper targeting of proteins results in disease. Despite the critical role of cell polarity in maintaining kidney function, we know little about the mechanisms that selectively guide proteins to the apical and basolateral cell surfaces. Our long term goal is to understand how proteins are segregated and targeted to the apical surface of kidney cells with the objective of manipulating protein sorting to treat disease. Apical sorting signals are diverse and multiple biosynthetic routes exist to the apical plasma membrane. Here we propose a unifying model for apical protein sorting in which cargo clustering within sorting compartments provides the avidity necessary to recruit membrane and cytoplasmic proteins required for surface delivery. We will test this model using a protein with an essential role in kidney function that is targeted to the apical surface in a glycan dependent manner. Our proposed studies utilize cutting edge biophysical, biochemical, and proteomic approaches and take full advantage of the unique facilities and broad expertise in renal epithelial cell biology at the University of Pittsburgh. Ths work will significantly enhance our understanding of how apical sorting and polarity are maintained in the kidney and illuminate new strategies to manipulate protein traffic for therapeutic advantage.

DESCRIPTION (provided by applicant): The proximal tubule (PT) of the kidney is the primary site for reabsorption of ions, solutes, and filtered low molecular weight (LMW) proteins. PT cells acutely modulate ion transport capacity in respond to changes in fluid shear stress (FSS) that accompany alterations in glomerular filtration rate. This proposal is focused on understanding whether PT cells also adjust the capacity of megalin-and cubilin- mediated endocytosis of LMW proteins in response to altered demand. Defective uptake of these proteins leads to tubular proteinuria, which can eventually lead to renal failure. We have discovered that apical endocytosis of the megalin/cubilin ligand albumin as well as fluid phase markers is markedly increased upon exposure of PT cells to FSS. Moreover, primary cilia are required for this response. The aims of this proposal are to (1) determine how changes in FSS are transduced into effects on apical endocytosis and (2) determine whether defective modulation of flow-dependent endocytosis contributes to LMW proteinuria observed in animal models for Lowe Syndrome, an X- linked disorder that may involve defects in ciliogenesis. We have assembled an outstanding team of investigators with essential expertise to carry out this broad range of studies. The results of our experiments will provide key information about a newly discovered pathway that plays an essential role in maintaining kidney function.

? DESCRIPTION (provided by applicant): Microperfusion of dissected tubules is an established technique that can provide information about cellular function within isolated nephron segments in the absence of confounding feedback variables. This information cannot be obtained using other approaches, including intravital microscopy. Recent advances in transduction approaches to express heterologous constructs as well as to efficiently knock down endogenous proteins in kidney and bladder have expanded the opportunity to use live cell approaches, including FRET, to investigate physiologic regulation of membrane trafficking and signaling pathways in real time in isolated tubules. This request is for funds to purchase a perfusion imaging system to enable high speed, high resolution imaging of microperfused kidney tubules and blood vessels as well as other tissues and cells. The system consists of a single photon Nikon A1R confocal microscope with a resonant scanner, spectral detector and FRET module, dual micromanipulators, and an environmental chamber. The system will be housed within the P30-funded George M. O'Brien Pittsburgh Center for Kidney Research at the University of Pittsburgh School of Medicine. The mandate of this center is to facilitate multidisciplinary research, trainin and information transfer related to kidney physiology, cell biology, pharmacology, and pathophysiology. The Center includes cores for Kidney Imaging, Cellular Physiology, Single Nephron and Metabolomics, and Model Organisms, and serves a large group of NIH-funded investigators at the University of Pittsburgh and at other institutions. Our center lacks a dedicated microscope for live imaging of perfused tubules, and the availability of the proposed system will significantly enhance the services we are able to provide to center users. Relevance- The proposed system will provide a crucial tool for the research of numerous biomedical investigators studying the cellular basis of disease. In particular, studies of isolated tubules and vessels conducted with this system are uniquely powerful for elucidating signal transduction processes associated with normal cell function and disease processes.

Abstract A major function of cells linking the proximal tubule (PT) of the kidney is to recover proteins that escape the glomerular filtration barrier to maintain a protein-free urine. Apical endocytosis in PT cells is acutely modulated by changes in fluid shear stress (FSS), presumably to enable efficient protein uptake over normal variations in glomerular filtration rate (GFR). The large multiligand receptors megalin and cubilin/amnionless (CUBAM) are expressed at the apical surface of PT cells and mediate the internalization of >50 different plasma proteins. Despite the critical role of the PT in reclaiming proteins from the ultrafiltrate, we know surprisingly little about how the cells lining this segment accommodate variations in filtered load to maintain a protein free urine. Fundamental issues, including how the robust PT apical endocytic pathway is developed and maintained, the individual role of megalin and CUBAM receptors in albumin uptake, how PT cells respond to changes in albumin concentration or tubular flow rate, and the fate of internalized albumin remain controversial. We have developed a new cell culture model that recapitulates morphological and functional features of PT cells in vivo necessary for efficient and rapidly modulated apical endocytosis of albumin. We will use these cells in conjunction with studies in mouse models to address the following questions about how PT cells respond to normal and pathologic variations in flow and filtered protein load: 1) How do PT cells develop and maintain a high capacity apical endocytic pathway? 2) How do megalin and CUBAM contribute to albumin uptake under normal and nephrotic conditions? 3) How does the PT respond endocytically to acute changes in GFR? and 4) How does endocytosis contribute to cytotoxic responses of PT cells during albumin overload?

Abstract Tubular proteinuria resulting from impaired endocytic uptake of filtered proteins by the kidney proximal tubule (PT) is a common feature of early kidney dysfunction that poses a significant risk for development of end-stage renal disease. This proposal aims to understand the mechanistic basis of Dent disease, a progressive genetic disorder characterized by tubular proteinuria that is caused by mutations in the 2Cl-/H+ exchanger ClC-5. Enhanced degradation of megalin, a multiligand co-receptor that binds to filtered proteins, is thought to underlie the tubular proteinuria in Dent disease, however, the step in trafficking that is affected is unknown, and the contribution of pH vs. Cl- homeostasis to the disease phenotype is disputed. We will utilize a combination of genetic, morphological, mathematical modeling, and biochemical approaches to accomplish the following aims: (1) identify the step(s) in membrane traffic that are impaired in ClC-5 knockdown PT cells; (2) determine the molecular mechanism that links loss of ClC-5 to reduced megalin expression; and (3) identify therapeutic targets for restoring megalin expression and function in a mouse model of Dent disease.