Dale John Benos - US grants

The proposed research is to elucidate the molecular basis of the facilitated sodium entry mechanism which exists in the outer (or apical) membrane of all tight epithelial tissues. This sodium entry step is an essential component of the overall active transport system responsible for the net movement of salt and water across these structures. This entry process is passive, and is sensitive to inhibition by the diuretic drug, amiloride. The analysis of the mechanism of Na entry encompasses determinations of cation selectivities, the effects of chemical group-specific reagents and other pharmacological substances on Na entry and amiloride efficiency, a determination of site number from binding studies of a photosensitive amiloride analogue (bromoamiloride), and the preparation of membrane vesicles containing functional Na-entry sites. A comparative approach, utilizing the isolated amphibian skin and bladder preparations as well as the isolated rabbit colon, will be taken. These studies will contribute to our knowledge of the kinetic and biochemical properties of epithelial transport systems and increase our comprehension of the mode of action of amiloride and other diuretic compounds.

DESCRIPTION (provided by applicant): Sodium reabsorbing epithelia, such as renal, distal, and collecting tubules, have as their major function the control of whole-body sodium balance. These epithelia contain apical membrane Na + channels that are inhibited by the diuretic amiloride. It is at the level of these channels that the feedback control mechanisms necessary for the maintenance of Na + homeostasis occur. The long-term goal of this project remains to elucidate at the molecular level the mechanisms responsible for the regulation of ion flow through these conductive entry pathways. During the previous grant period four novel observations were made that form the basis of this continuation application. First, we discovered that Ca 2v was involved in the effect on conductance following the interaction of actin with ENaC. Second, a short, 14-aa segment in the C-terminal of alpha-ENaC was identified as being crucial for actin's functional interaction with ENaC. Third, we have identified new functional and physical interactions between ENaC, syntaxin, and other novel cytoplasmic regulatory elements. Fourth, we have utilized the baculovirus system to produce milligram quantities of pure, functionally intact alpha-ENaC. Therefore, we propose to 1) test the hypothesis that t-SNARES (e.g., syntaxin 1A) and annexins directly modulate ENaC function; and 2) test the hypothesis that actin directly binds to ENaC, thereby inducing a conformational change resulting in changes in channel conductance and cation selectivity. We will identify the site of physical contact between ENaC and syntaxin, actin, annexins, and other cytoskeletal linking elements such as ezrin. Proteins that regulate the activity of syntaxin, such as SNAP 23/25 and munc-18, will also be examined for their functional and physical influences on syntaxin-ENaC interactions. We will also crystallize a-ENaC with the goal of providing a detailed molecular picture of this subunit. These results will offer new insights into the nature and regulation of amiloride-sensitive Na+ channels, the ways that these channels interact with and are modulated by the cytoskeleton, and provide the first near atomic-level detail of these important ion channels. Thus, understanding the molecular basis for ENaC regulation, conduction, and selectivity will provide unique opportunities for therapeutic interventions in an ever-increasing plethora of ENaC-related diseases.

The overall objective of the proposed research is to elucidate the physiological transport mechanisms involved in the swelling of the blastocyst, an important prerequisite for implantation. Studies will be done on maximally (rabbit) and minimaly (mouse) expanding types of blastocyst. There are five specific aims, all directed towards investigating the molecular mechanisms involved in ion and water transport across the trophectodermal epithelium. Specific aims 1-3 involve the development of a voltage clamp/perfusion system, the study of potassium ion fluxes, and measurement of the distribution and activity of Na/K-ATPase pumps within the trophectoderm. Specific aim four concerns studies designed to determine whether exogenous agents, particularly those likely to arise in the mother, can affect transport across the epithelium. Particular emphasis will be placed upon elucidating the physiological control mechanisms involved in the on/off signaling of various ion transport systems. Fifth intracellular concentrations of physiologically important ions like Na+, K+, Cl+, phosphate, and calcium will be made using electron probe microanalysis. These studies will contribute to our knowledge of the kinetic and biochemical properties of developing ion transport systems in preimplantation mammalian embryos, and increase our comprehension of the physiology of early pregnancy.

New information obtained during the past five years has pointed to an abnormality in secretory chloride channel regulation as the basic defect in cystic fibrosis (CF). The apical membrane-located chloride channel represents the rate-limiting step for Cl- secretion and, thus, influences the composition of the secretory products of the airways, pancreas, and sweat glands, the major target organs in CF. Although much important knowledge has been learned about the phenomenology of epithelial secretory Cl- channel kinetics and regulation by cAMP-and Ca++-dependent pathways, virtually nothing is known about the structural details of the protein(s) comprising the anion channel complex. The biochemical identification of secretory Cl- channels has been hampered by the lack of suitable probe molecules. The central goal of this Cystic Fibrosis Research Center application is to isolate and characterize this secretory C1- channel protein complex and any associated regulatory components so that a molecular basis for CF can be specified. The development of this proposal revolves around the generation of antibodies that appear to interact specifically with the secretory Cl- channel-containing vesicles. Four interdisciplinary, interrelated projects are proposed: 1) isolation of secretory Cl- channels using antibody probes: 2) cloning and expression of the secretory Cl- channel; 3) immunocytochemical localization and intracellular trafficking of Cl- channels; and 4) disulfonic stilbenes as probes of reconstituted Cl- channels. Thus, the secretory Cl- channel will be studied at both the molecular and physiological levels to [provide a comprehensive framework for understanding the mechanisms of anion conduction and regulation. By concentrating our efforts on the molecular and cell biological aspects of C1- channel structure and movement, new information will be generated that will undoubtedly lead to novel strategies for pharmacological intervention to overcome the regulatory deficiencies occurring in CF.

DESCRIPTION (provided by applicant): Continuation of an institutional training program in Cellular Physiology is proposed for postdoctoral fellows from the Basic Health Sciences or subsequent to residency programs in Nephrology, Anesthesiology, Pulmonary Medicine, or Internal Medicine. The goal is to train potential academic investigators in cell membrane physiology, biochemistry, and molecular biology. Basic laboratory training requires a full-time commitment of 2-3 years under the guidance of an established scientist with training directed at the utilization and application of modern electrophysiological, biochemical, and cell and molecular biological tools to problems relating to epithelial transport, ion channels, growth and differentiation, and various pathophysiological states such as cystic fibrosis and renal failure. All participating faculty have their primary or secondary appointments in Physiology and Biophysics. The intimate relationship between the mentor and the trainee will foster a thorough understanding of the scientific method, informal seminars and discussions of planned experiments and current results are important components of the program. All preceptors have a clear commitment to training new investigators and possess ample laboratory space and external funding for training. Trainees will be expected to master at least one major investigative technique, complete at least one major research project, and present their work at a national meeting. Emphasis will be placed upon developing critically thinking scientists who are well-versed in the potential uses, advantages, and disadvantages of each technique. The existence of over 70 separate research centers (such as the Comprehensive Cancer Center, the Center for AIDS Research, the Nuclear Imaging Research Center, and the Center for Biophysical Sciences and Technology) on the UAB campus amplify the academic and research opportunities, creating an excellent environment for this training program. Fellows completing the program will be competitive candidates for academic research careers.

Central nervous system involvement often occurs in individuals infected with human immunodeficiency virus type I (HIV-1). The most common clinical syndrome, characterized by cognitive, motor, and behavioral disturbances, is the acquired immunodeficiency syndrome (AIDS) Dementia Complex (ADC), and is unique to HIV-1 infection. However, the pathogenesis of this syndrome is poorly understood. The long-range goal of this project is to elucidate the molecular and cellular processes responsible for ADC. Previous work from our laboratories focused directly on virological aspects of ADC, reporting for the first time (i) the complete nucleotide sequence, genome organization, and biological phenotype of virus cloned directly from uncultured human brain, (ii) the identification of the HIV- 1 envelope glycoprotein as a critical determinant for macrophage tropism (and consequently "neurotropism"), (iii) a limited degree of viral genome heterogeneity and defectiveness within the CNS compartment, and (iv) a significant correlation between HIV-1 viral load, gene expression, localization within the CNS, and clinical severity of ADC. The results of these studies thus implicated macrophage-tropic strains of HIV-1 in the pathogenesis of ADC, and strongly suggested indirect mechanisms not involving direct infection of principal functional elements of the CNS, namely, the neurons, oligodendrocytes and astrocytes. Evidence is now accumulating that neurons and astrocytes may be functionally compromised by exposure to viral components or cellular factors released from HIV-1 infected macrophages/microglia. In this project, we are proposing to examine the role of astrocytes in the pathogenesis of ADC by employing complementary expertises in HIV biology, neuroimmunology, and cell physiology. Using this multidisciplinary approach, we will test the hypothesis that exposure to HIV-1 virions,individual viral gene products, cytokines, or other soluble products of infected macrophages/microglia directly alters normal astrocyte function ultimately leading to neuronal cell injury. The specific aims are: 1) to determine if virions, HIV-1 envelope glycoproteins (gp160, 41), or HIV-1 regulatory proteins (tat) alter astrocyte cytokine expression, in particular that of TNF-alpha, IL-1, and IL-6; and 2) to determine if these viral products, cytokines, or other products released from infected macrophages/microglia alter human astrocyte exchange (antiport) system, Na+-glutamate cotransport system, and/or ion channels, specifically K+ channels. The emphasis of this project is to study these functions in human astrocyte populations using rat astrocytes for comparison when appropriate, and to use biologically relevant and molecularly well characterized HIV-1 viral strains known to be associated with ADC. These studies thus represent a novel approach for evaluating the cellular and physiological basis of neuronal dysfunction in ADC by examining directly, in well-defined biological assays, the effect of HIV-1 and HIV-1 induced- cytokines on the function of the astrocyte, a critically important supporting cell within the CNS.

DESCRIPTION (Adapted from applicant's abstract): Central nervous system involvement often occurs in individuals infected with human immunodeficiency virus type I (HIV-1). The most common clinical syndrome, characterized by cognitive, motor, and behavioral disturbances, is the acquired immunodeficiency syndrome (AIDS) Dementia Complex (ADC), and is unique to HIV-1 infection. However, the pathogenesis of this syndrome is poorly understood. The long-range goal of this project is to elucidate the molecular and cellular processes responsible for ADC. Previous work by the investigators' laboratories has implicated indirect mechanisms as being responsible for the etiology of ADC, rather than direct infection of principal functional elements of the CNS, namely, the neurons, astrocytes, and oligodendrocytes. The PI presents evidence that neurons, astrocytes, and oligodendrocytes may be functionally compromised by exposure to viral components or cytokines released from HIV-1 infected macrophages/microglia. The proposed studies will examine the role of astrocytes and oligodendrocytes in the pathogenesis of ADC by employing complimentary expertise in HIV biology, neuroimmunology, and cell physiology. Using this multidisciplinary approach, the investigators will test the hypothesis that exposure to cytokines, individual viral gene products, HIV-1 virions, or other soluble products of infected macrophages/microglia, directly alters normal astrocyte and oligodendrocyte function, ultimately leading to neuronal cell injury and myelin damage. The three specific aims are: 1) to test the hypothesis that cytokine-induced astrocyte Na+/H+ exchanger and pH-sensitive K+ channel activities are mediated by novel isoforms of the amiloride-sensitive Na+/H+ exchanger apamin-sensitive K+ channel; 2) to test the hypothesis that alterations in normal astrocytic Na+/H+ exchange, glutamate transport, and K+ conductance produced by cytokines change the extracellular milieu of neurons in vivo, and thus contribute directly to Ca2+ induced neurotoxicity; and 3) to test the hypothesis that proinflammatory cytokine-induced ICAM-1 expression by oligodendrocytes contributes directly to changes in oligodendrocyte function. The emphasis of this project is to study these functions in human astrocytes and oligodendrocytes populations, using rat cells for comparison when appropriate. These studies thus represent a novel approach for evaluating the cellular and physiological basis of neuronal dysfunction in ADC by examining directly the effect of HIV-1 induced cytokines and HIV-1 itself on the function of the astrocyte, a critically important supporting cell within the CNS, and the oligodendrocyte, the myelin-producing cell of the CNS.

Recent experimental evidence suggests that amiloride-sensitive epithelial Na+ channels are present in the mammalian lung, including the apical membrane of the alveolar type II pneumocyte. Active Na+ transport across the adult alveolar epithelium is under hormonal control, and is important in regulating alveolar fluid balance under normal physiological and pathological conditions. The biochemical and molecular characteristics of these important ion channels, the mechanisms involved in hormonal modulation of these channels, and their involvement in pathophysiological processes, such as hyperoxic lung injury and Adult Respiratory Distress Syndrome (ARDS), are not known. We propose to test the hypotheses that mammalian alveolar type II cells (ATII) contain epithelial Na+ channels and that the activity of these channels are regulated by post- translational modifications including phosphorylation. Specifically, we propose to: 1) test the hypothesis that mammalian ATII cells contain low amiloride affinity Na+ channels by biochemically isolating and purifying this protein to homogeneity; 2) test directly the hypothesis that the protein purified from ATII cells forms amiloride affinity Na+ channels by biochemically isolating and purifying this protein to homogeneity; 2) test directly the hypothesis that the protein purified from ATII cells forms amiloride-sensitive cation channels by reconstituting the purified protein into planar lipid bilayers; 3) identify and characterize full length cDNA's corresponding to polypeptides comprising the ATII Na+ channel in order to verify that the protein isolated indeed functions as an ion channel; and 4) examine the hypothesis that phosphorylation reactions and mineralocorticoid hormones influence Na+ channel expression at the transcriptional or translational levels. An important element of this study is that the biochemical, physiological, and molecular biological characteristics of these channels in ATII cells will be elucidated for the first time, and that new probes for this important protein will be generated. This research is important both from the basic science and clinical aspects. Because active Na+ transport plays a very important role in maintaining alveoli free of fluid, especially under pathological conditions in which the pulmonary surfactant system has been compromised and surface tension increases, the information obtained from this work may eventually have important implications in the treatment of hyperoxic lung injury and ARDS. The results obtained from this study will offer new insights as to the nature of ATII Na+ channels, the way they are modified by hormones, and will help establish new rational approaches by which lung injury may be alleviated.

The main goal of the proposed research is to understand the mechanisms and regulation of ion permeation through C1- channels other than the cystic fibrosis transmembrane conductance regulatory (CFTR) present in secretory epithelia. These alternative C1- channels may be useful and important targets for pharmacological therapy in cystic fibrosis (CF). Our laboratory has successfully isolated and cloned a protein from bovine trachea that behaves as a Ca2+ -sensitive C1- channel (CaCC), and has semi-purified and reconstituted an outwardly-rectified C1- channel (ORCC). This application has four specific aims: (1) to test the hypothesis that the translated bovine tracheal cDNA forms an anion channel of identical characteristics to the native protein, that the 38 kDa subunit of the native tracheal CaCC protein is the result of post- translational processing of the cloned 100 kDa CaCC cDNA product, and to determine the biochemical properties of both native and cloned CaCCs. The functional properties of the proteins will also be characterized following reconstitution into planar lipid bilayers or transfection into eukaryotic cells; (2) to identify a full-length cDNA corresponding to the human CaCC homolog and to characterize the translated protein. The molecular structure and function of the human homolog of the bovine CaCC will be determined by screening of appropriate human epithelial cDNA libraries; (3) to purify a protein that behaves as an ORCC from bovine tracheal apical membrane vesicles and to identify and characterize the full-length cDNA that encodes this protein. Candidate proteins will be used to raise polyclonal antibodies that will be used to screen a bovine tracheal cDNA expression library. The ultimate goal is to isolate a full-length cDNA that encodes an ORCC and to characterize the translated protein with the aim of understanding is potential interaction with CFTR and/or other ion channels; (4) to determine if heterologous intestinal specific expression of the CaCC can overcome the lethal intestinal obstruction found in the CF knockout mouse model. We will test the hypothesis that tissue specific expression of the bovine CaCC in the intestine will prevent the lethal consequences of intestinal obstruction by ameliorating the adverse effects of impaired chloride secretion in the intestine. These studies will further our knowledge of the physiological, biochemical, and molecular properties of these important C1- transport pathways and increase our understanding of fluid secretion across airway and intestinal epithelial so that potential avenues of alternate therapy in CF can be devised and evaluated.

In the later stages of HIV infection, patients suffer motor and cognitive problems known as the AIDS dementia complex (ADC). Autopsy reports in HIV patients reveal areas of diffuse myelin pallor and astrocytosis. This injury is somewhat unique in that neurons and astrocytes are usually not infected with the virus despite their participation in the pathology. Therefor, we have begun to look at indirect mechanisms for this injuries. Our working hypothesis is that HIV-related proteins alter the function of glial cells in the brain such that there is a release of more HIV proteins as well as cytokines. These proteins and cytokines then increase Na+/H+ exchange in the astrocyte resulting in an increase in intracellular pH (pHi), outward K+ conductance, and a decrease in glutamate influx. The increase in extracellular K+ and glutamate would result in the excitotoxicity of the surrounding neuros. In a healthy individual, astrocytes play a major role in the homeostatic mechanisms of the brain by regulating ion transport and regulation. Previous experiments in the primary rat astrocytes demonstrate that with a challenge of gp120, an envelope glycoprotein of HIV, there is an amiloride-sensitive intracellular alkalinization and an increase in a pH sensitive K+ channel conductance. In the human astrocyte, we see the increase in K+ conductance and a decrease in the uptake of the neurotransmitter glutamate. We would propose that this alteration in astrocyte activity would then be responsible for pathophysiology of ADC. [unreadable]The importance of this study can only be fully appreciated if our hypothesis is investigated in an intact system. Therefore, we have begun to look at pH in the brains of HIV patients. The pH is determined by the chemical shift of inorganic phosphate (Pi) vs. phosphocreatine (PCr) of the 31P NMR spectra acquired in human brains using NMR spectroscopic imaging at the field of 4.1T. Our preliminary studies of asymptomatic HIV+ patients show some of the 9 patients have a slightly increase in pH within the cerebellum. Further studies need to be done on this small pH changes and to look at the metabolite concentration of these patients with stringent statistical analysis. We are proposing to have these patients undergo a series of neuropsychological evaluation to correlate these changes in pH to a measured dementia state.

Sodium reabsorbing epithelia, such as renal distal and collecting tubules, have as their major function the regulation of whole-body sodium balance. These epithelia contain sodium channels localized in their luminal or apical membranes that are inhibited by the diuretic drug amiloride. It is at the level of these channels that the primary feedback control mechanisms necessary for the maintenance for sodium homeostasis occur. Recently, an epithelial sodium channel (termed ENaC) has been cloned. This channel, at a minimum, consists of at least three structurally related subunits (termed alpha-ENaC, beta-ENaC, and gamma-ENaC). The primary and predicted secondary structures of these ENaCs have been described, but the assignment of specific functional roles to domains within each ENaC subunit has just begun. The long term goal of this project is to understand the functional roles of specific amino acid domains within ENaC. We hypothesize that the cation selectivity filter and channel pore regions of ENaC are spatially distinct. Moreover, we hypothesize that there are specific glutamic acid residues in the pre-H2M2 region of each ENaC subunit that participates in calcium binding/inhibition of the channel. We also hypothesize that there are specific negatively charged amino acids (aspartic acid and glutamic acid) located within the M2 region of each ENaC subunit that constitute part of the conduction pathway, or pore of the channel. This application has two specific aims: (1) to test the hypothesis that two specific negatively charged amino acids, located 4 and 12 amino acids upstream from the beginning of the second large hydrophobic domain (H2M2) within the selectivity filter of alpha, beta, and gamma-rENaC, are the sites involved in Ca2+ block of both alpha and alphabetagamma-rENaC; and (2) to test the hypothesis that specific hydrophilic residues (E568, E571, and D575) within the M2 region of each ENaC subunit are essential for the conduction properties of the pore. Important aspects of this study are that unique biochemical, physiological, and molecular biological characteristics of ENaCs will be elucidated; regions of protein sequence that participate in specific channel functions will be identified; and new insights into the gross molecular architecture of a functional ENaC will be developed. These results will offer new insights into the nature of amiloride-sensitive sodium channels, in that important structural regions of the channel involved in cation selectivity, calcium interactions with the channel, and conductions through the pore will be defined.

Central nervous system (CNS) involvement often occurs in individuals infected with human immunodeficiency virus type-1 (HIV-1). The most common clinical syndrome characterized by cognitive, motor, and behavioral disturbances is the acquired immunodeficiency syndrome (AIDS) dementia complex (ADC) or HIV- associated dementia (HAD), and is unique to HIV-1 infection. Although anti-retroviral agents (RT and protease inhibitors) are being used in HIV-infected individuals, it is not yet clear how these agents will affect HAD or if these drugs can even penetrate the brain. Thus, a major problem facing HAD patients is the that drugs used to combat systemic viral infection may not influence the CNS, a potential reservoir for virus. Because the physiological status of the brain in AIDS patients cannot be readily sampled, there is a critical need for the development of non-invasive techniques to detect and monitor the extent of HIV- associated cognitive/motor disorders. In this application, we intend to translate basic science findings obtained in the previous grant period to the human, and perform clinical cognitive studies on HIV-infected patients. We will develop non- invasive methodologies, based on 31P nuclear magnetic resonance (NMR) spectroscopy, eventually to investigate how pharmacological and/or immunological manipulations can affect the pathological and psychomotor abnormalities in humans infected with HIV-1. We will also correlate such brain metabolic changes with the degree of dementia in HAD patients. This will be accomplished through he neuropsychological assessment of participants. Thus, a sophisticated array of experimental approaches will be used to define molecular mechanisms underlying the pathophysiology of HAD, which ultimately will be critical for the development and assessment of new therapeutic strategies. There is one specific aim: 1) to test the hypothesis that cerebrospinal fluid (CSF) viral load, CD4+ cells, and/or cytokine content correlates with increases in pH in various regions of the brain, specifically the basal ganglia and cerebellum. In addition, neuropsychological testing will be performed on all subjects enrolled in this study in an effort to a) assess subject neurocognitive/motor status and b) to link the clinical developmental stage of dementia with CSF viral load and brain pH.

DESCRIPTION (provided by applicant): Gliomas are primary brain tumors that arise from differentiated glial cells through a poorly understood process of malignant transformation. Brain tumors display a complex biology because of their remarkable degree of antigenic heterogeneity, variable mutations in their genome, and their propensity for invasion into normal brain tissue. In studying gliomas obtained from patients that were diagnosed with tumors of varying degrees of malignancy, we observed the expression of a voltage-independent, amiloride-inhibitable, inward Na+ conductance that was not present in normal human glial cells or in low-grade tumors. We hypothesize that high-grade glioma cells show functional up-regulation of this characteristic Na + conductance. Glioma cell migration, cell proliferation, and cell volume regulation are all compromised if this conductance pathway is blocked with amiloride or by a peptide isolated from a spider venom. Thus, the channel membrane proteins that underlie this conductance are potentially unique, therapeutic targets. The research proposed in this application has one objective of characterizing thoroughly the ability of one subunit (ASIC2) of this amiloride-sensitive Na+ conductance pathway to traffic through the cellular biosynthetic pathway. In addition, we hypothesize that this same subunit (ASIC2) is transcriptionally regulated. Thus, in malignant brain tumors, ASIC2 either is not expressed or is retained intracellularly. There are two Specific Aims: 1) to test the hypothesis that lack of plasma membrane expression of ASIC2 in a subset of high-grade tumor cells is a consequence of endoplasmic reticulum retention due to channel misfolding; and 2) to test the hypothesis that in the majority of high-grade gliomas ASIC2 gene expression is transcriptionally regulated by factors specific to the brain tumor microenvironment. We anticipate that this work will provide new fundamental insights into the molecular mechanisms involved in the regulation of amiloride-sensitive Na + channels in brain tumors. Moreover, this work will provide important clues as to the role of these channels in the pathogenesis and life cycle of glioma cells.

AIDS /HIV neuropathy; method development; magnetic resonance imaging; cerebrospinal fluid; cognition; nervous system disorder diagnosis; HIV infections; brain imaging /visualization /scanning; noninvasive diagnosis; acidity /alkalinity; virus load; cerebellum; basal ganglia; patient oriented research; human subject; clinical research; neuropsychological tests;

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DESCRIPTION (provided by applicant): Continuation of an institutional Training Program in Kidney-Related Diseases (formally Cellular Physiology) is proposed for postdoctoral fellows from the Basic Health Sciences or subsequent to residency programs in Nephrology or Internal Medicine. The goal of this program is to train potential academic investigators in four distinct kidney-related thematic areas: (i) renal physiology and pathophysiology, (ii) epithelial biology related specifically to polycystic kidney disease, (iii) vascular biology related to kidney disease, and (iv) clinical and translational research in kidney disease. These areas will provide comprehensive training in both basic and clinical aspects of kidney-related research. Basic laboratory training requires a full-time commitment of 2-3 years under the guidance of an established scientist with training directed at the utilization and application of modern electrophysiological, biochemical, and cell and molecular biological tools. The training program also provides opportunities for clinical and translational research in kidney diseases and will require a full-time commitment of 2-3 years. The intimate relationship between the mentor and the trainee will foster a thorough understanding of the scientific method. Formal and informal seminars and discussions of planned experiments and current results are important components of the program. All preceptors have a clear commitment to training new investigators and possess ample laboratory space and external funding for training. Trainees will be expected to master at least one major investigative technique, complete at least one major research project, and present their work at a national meeting. Emphasis will be placed upon developing critically thinking scientists who are well-versed in the potential uses, advantages, and disadvantages of each technique. The existence of over 100 separate research centers on the UAB campus amplify the academic and research opportunities, creating an excellent multidisciplinary environment for this training program. Fellows completing the program will be competitive candidates for academic research careers.