Rick G. Schnellmann - US grants

The fact that the kidney receives 25 percent of the cardiac output and has specialized concentrating and transporting abilities makes it unique in its susceptibility to chemical-induced toxicity. Halogenated hydrocarbons (HH) are a large group of chemicals known to produce nephrotoxicity. Because HHs are used widely as drugs, pesticides and herbicides, humans and animals are constantly exposed to these environmental pollutants. While several studies have related the metabolism of HHs to their toxicity in vivo and have superficially characterized the renal damage, they have not addressed the cellular mechanism(s) of toxicity. The main objective of the proposed research is to determine how the nephrotoxic metabolites of two model HH (hexachlorobutadiene and bromobenzene) alter proximal tubular cellular physiology to produce toxicity. Since studies from this laboratory have shown that kidney proximal tubular mitochondria are one of the earliest intracellular targets of toxicity, this project will concentrate on determining the mechanism by which these compounds produce mitochondrial dysfunction. Using a well-defined suspension of rabbit renal proximal tubules and isolated mitochondria, experiments are designed 1) to determine the relative role of mono- and di-substituted glutathione conjugates of 2-bromohydroquinone (BHQ) in producing toxicity (mitochondrial) and relate their metabolism to cysteine, BHQ and the cysteine conjugates of BHQ at the organelle (mitochondria) level; 3) to determine whether these compounds cause mitochondrial dysfunction by alkylating or oxidizing the site of toxicity; 4) to identify the "reactive species" and the amino acids altered by these compounds; and 5) to determine the subcellular site(s) of bioactivation of these compounds. By understanding how these chemicals induce toxicity, it may be possible to 1) alter their structure to maintain their usefulness but not their toxicity, 2) develop antidotal treatments, and 3) gain a better understanding of normal cellular function.

Toxicological, physiological, and biochemical information gained from tissue culture studies must be interpreted with caution since cells in culture are different from the parent cell type found in vivo. The loss of specific differentiated functions and the presence of abnormal karyotypes, biochemical characteristics, and hormonal responses are common features of established cell lines. Although some of these limitations may be overcome by using cells in primary culture, primary cultures quickly switch from aerobic to glycolytic metabolism and eventually dedifferentiate. The loss of aerobic metabolism and differentiated functions alters the relationships and interdependence of cellular processes which limits extrapolations between in vitro and in vivo studies. Since tho dedifferentiation that occurs during primary culture may be a consequence of changes in cellular energy metabolism and the extracellular environment, the first goal of this proposal is to examine the role of aerobic metabolism and the extracellular environment in maintaining differentiated function. Using primary cultures of renal proximal tubule cells, standard culture conditions will be modified to maintain aerobic metabolism (Specific Aim 1) and to more closely reproduce the extracellular environment of the proximal tubule cell in vivo (Specific Aim 2). The model systems used for these studies will be proximal tubule cells grown on Millicell culture inserts (SHAKE and MICROPERFUSED). Using diverse markers such as morphology, hormonal responsiveness, and functionally specific enzyme markers and transporter systems, the degree of differentiated function maintained in these systems will be compared against freshly isolated proximal tubules and primary cultured proximal tubule cells grown using standard culture conditions (highest vs. lowest metabolic and differentiated state, respectively). The second goal of this proposal is to investigate the mechanism of aryl-glutathione conjugate-induced nephrotoxicity in the newly developed culture system which maintains metabolic and differentiated functions similar to the proximal tubule cell in vivo (Specific Aim 3). This proposal will investigate some of the current issues regarding maintenance of differentiated functions in primary culture and develop a superior culture system for more direct and relevant comparisons between toxicology, physiology, and biochemistry studies in vitro and studies in vivo. Additionally, information gained from these studies can be applied to other organ types and animal species to reduce the number of vertebrate animals used in research.

DESCRIPTION: (Adapted from the Investigator's Abstract) The long-term goal of this research is to identify the cascade of events that lead to cell injury and death in renal proximal tubules (RPT), and to identify and examine the mechanism of action of known cytoprotective agents. The role of Ca2+ in cell injury and death has been studied and debated. Calpains, non-lysosomal calcium-activated cysteine proteases, have been implicated in cell injury also. Calpain inhibitor 1 decreased RPT cell death produced by bromohydroquinone, antimycin A, T-butylhydroperoxide, and tetrafluoroethly-L-cysteine, suggesting that calpains plan an important role in cell injury produced by diverse toxicants. The plasma membrane Ca2+ channel blocker nifedipine, calpain inhibitor 1 and the extracellular Ca2+ chelator EGTA acted in the late phase of cell injury and blocked influx of extracellular Ca2+ calpain activity, Cl- influx and cell death in rabbit RPT exposed to a mitochondrial toxicant (antimycin A). The late phase of cell injury is important to study since a variety of cytoprotectants (ex. Glycine, muscimol, neurosteroids, nifedipine, Cl- channel blockers) act in the late phase of cell injury to prevent cell death/lysis. Furthermore, experiments have shown that many of these cytoprotectants allow RPT to regain mitochondrial function and active Na+ transport following the removal of the cellular insult, strongly suggesting that these compounds are true cytoprotectants. Thus, the investigators propose that during the late phase of cell injury, influx of Ca2+ through a nifedipine-sensitive channel and calpain activation play a critical role in RPT cell death/lysis. The specific aims of this application are: Specific aim 1: Use biochemical and fluorescence techniques, and immunoblot analysis to define the role of cytosolic free Ca2+, extracellular Ca2+ influx and calpains in the late phase of RPT cell injury and death. Specific aim II: Use patch clamp techniques to determine and characterize the signaling pathway for Ca2+ entry through a nifedipine-sensitive channel in the late phase of RPT cell injury. Specific aim III: Use results from specific aims I and II to determine the mechanisms of action of a diverse group of cytoprotectants that act in the late phase of cell injury. Completion of these specific aims will add significant new information to our understanding of cell injury and death, particularly to those events that occur in the late phase. Determining the mechanisms of action of a number of known cytoprotectants from diverse chemical classes may ultimately result in the identification of new pharmacological agents that prevent cell death and organ failure.

DESCRIPTION: (Adapted from the Investigator's Abstract) Cl- channels play a critical role in transepithelial Cl transport, cell volume regulation and cell injury. However, the identity, characterization, localization and pharmacological properties of Cl channels remain limited, particularly in renal proximal tubules (RPT). The long-term goal of this research is to identify and characterize novel renal Cl channels in RPT and determine their roles in RPT physiology and pathology. The neuronal glycine receptor (GlyR) and neuronal GABA-A receptor (GABAR) are heteroligomeric, ligand-gated Cl channels. The initial novel finding was the identification of proteins corresponding to the beta-subunits of the GlyR and GABAR in human, rabbit and rat RPT using immunoblot, immunohistochemical, and molecular studies. Adding to the intrigue was the polarized distribution of the beta subunits; the GlyR beta subunit was localized to the basolateral membrane and the GABAR beta subunit was localized to the brush boarder membrane. GlyR alpha subunits were not detected. Since the only known function of GlyR and GABAR beta subunits is the lining of Cl channels, we propose that the RPT express novel Cl channels composed of selective subunits of the GlyR and GABAR. The first step in addressing this hypothesis will be to: Specific Aim I: Develop and use a GlyR beta subunit selective antibody to identify and localize proteins associated with the GlyR beta subunit in human, rabbit and rat kidney, and thereby begin to determine their specific assemblage into complexes, Specific Aim II. Use a GABAR beta subunit selective antibody to identify and localize additional subunits of the GABAR complex in rat kidney, Specific Aim III. Use GABAR subunit selective antibodies and PCR based strategies to identify and localize GABAR subunits in human kidney. Completion of these specific aims will result in the identification and localization of GlyR and GABAR subunits in the kidney of humans and model animals. Identification of these subunit complexes will lead to their electrophysiological characterization and may ultimately result in the development of new pharmacological agents that modulate Cl transport under physiological and pathological conditions.

The long term goal of this research is to understand how the kidney recovers from a xenobiotic insult though cell repair and regeneration. Numerous xenobiotics such as halocarbons and oxidants produce nephrotoxicity through renal proximal tubule cellular (RPTC) injury and death. The kidney can recover from renal failure through repair and regeneration of the non-injured and sublethally-injured RPTC; though the mechanisms of repair and regeneration are poorly understood. Recently, in vitro models of cell repair and regeneration using primary cultures of rabbit RPTC have been developed, and differences observed in the ability of injured RPTC to repair cellular functions and regenerate following exposure to the nephrotoxic halocarbon dichlorovinyl-L- cysteine (DCVC) and the oxidant t-butylhydroperoxide (TBHP). Extracellular matrix (ECM)-integrin interactions play a critical role in cell attachment and in triggering/transducing signals for cell proliferation and functions. Collagens are important ECM proteins and preliminary studies demonstrate that TBHP alters collagen Type IV mRNA expression in RPTC. The central hypothesis of this proposal is that repair of physiological functions and regeneration of RPTC following sublethal injury is dependent on the re-establishment of collagen production and collagen binding integrin levels and localization on the plasma membrane. Specific Aim I will determine the role of collagens I and IV in the promotion of RPTC physiological functions. Specific AIM II will determine the role of collagens and collagen binding integrins in the promotion of proliferation and repair of membrane polarity and physiological functions in RPTC following TBHP exposure. Specific AIM III will determine the role of collagens and collagen binding integrins in the lack of RPTC repair and regeneration following DCVC exposure. Completion of these aims will result in a better understanding of the mechanisms involved in renal cell regeneration and the repair of physiological functions following toxicant exposure.

DESCRIPTION (adapted from the application) The importance of the kidney as a target organ for toxic injury has been appreciated for decades, as has its status as a high frequency site for the induction of tumors in carcinogenicity bioassays and human cancers. Recently, numerous advances have been made in our understanding of the mechanisms of action of nephrotoxicants and nephrocarcinogens at the cellular, biochemical and molecular levels. These advances along with emerging issues related to clinical outcome following toxic exposures and the impact on the kidney of specific renal toxicants will be highlighted in this meeting. The purpose of this Contemporary Concepts on the Mechanisms of Nephrotoxicity and Nephrocarcinogenicity meeting is to provide a forum for toxicologists, physiologists, pharmacologists, nephrologists, and their trainees to exchange information and promote research into the mechanisms of nephrotoxicity and nephrocarcinogenicity. Exchange of information in these fields will lead to a better understanding of both clinical renal disease and its treatment. The format of the conference will be a series of state-of-the-art lectures on renal xenobiotic transport and biotransformation, mechanisms of epithelial cell injury and death, understanding renal repair and regeneration, molecular mechanisms of nephrocarcinogenicity, and drug-induced renal dysfunction from a clinical perspective. In addition, the conference will feature a plenary lecture, discussion groups with audience participation and poster sessions. The meeting will be held at the Harbor View Hotel, Martha's Vineyard, MA, on April 15-18, 2000.

DESCRIPTION (provided by applicant): Reactive oxygen species are formed in the kidney directly or indirectly following toxicant exposures and other injuries. Oncosis (necrotic cell death) is a form of cell death characterized by ATP depletion, disruption of Ca2+ homeostasis, organelle and cellular swelling, and gross breakdown of the plasma membrane, and is one outcome of oxidant exposure. Consequently, oxidant-induced oncosis has been implicated in the pathogenesis of various acute nephropathies and nephrotoxic states. Phospholipase A2 (PLA2) isoforms have been proposed to play different roles in oxidant/toxicant-induced renal cell injury and oncosis, including acting as a phospholipid repair enzyme and playing a protective role during oxidant/toxicant injury. We have observed that inhibition of a Ca2+ independent PLA2 (iPLA2) prior to exposure to a number of diverse oxidants or cisplatin (a model nephrotoxicant) markedly potentiated oxidant- and cisplatin-induced renal proximal tubular cell (RPTC) oncosis. Further, the majority of iPLA2 activity and protein in RPTC was identified in the endoplasmic reticulum. These results suggest that an endoplasmic reticulum iPLA2 (m-iPLA2) may act as a phospholipid repair enzyme and attenuate cell injury and oncosis. The hypothesis to be tested by the proposed studies is that a novel m-iPLA2 plays a protective role and prevents oxidant/toxicant-induced RPTC oncosis. The specific aims designed to test this hypothesis are: 1) Determine the selectivity of RPTC m-iPLA2 for phospholipids and their oxidized products, 2) Determine the role of m-iPLA2 in RPTC oncosis mediated by oxidants/toxicants, and 3) Identify the m-iPLA2 in RPTC. These integrated aims will result in the systematic examination of an important medical and scientific issue, the role of PLA2 in oxidant injury. While numerous studies have suggested that PLA2 contributes to cell injury, little effort has been expended to address the issue of a phospholipid repair mechanism. Successful completion of the proposed studies will result in this issue being addressed.

[unreadable] DESCRIPTION (provided by applicant): The long-term goal of this project is to elucidate the events that cause mitochondrial dysfunction in ischemia/reperfusion- and toxicant-induced acute renal failure (ARF), and to identify a therapeutic approach that prevents the mitochondrial dysfunction and reduces ARF. The role of mitochondrial dysfunction and disruption of Ca 2+ homeostasis in renal cell injury and death has been demonstrated in numerous models of ARF and nephrotoxicity. The importance of calpains (Ca2+-activated neutral cysteine proteases) in renal proximal tubule cellular (RPTC) injury and death produced by hypoxia/reoxygenation and toxicants has been shown using calpain inhibitors. In particular, two dissimilar calpain inhibitors not only blocked hypoxia/reoxygenation RPTC death, but also blocked the mitochondrial dysfunction and promoted the recovery of respiration during reoxygenation. These results strongly support a key role for calpains in mitochondrial dysfunction. The above experiments showing calpain inhibitor protection of mitochondrial function in RPTC, suggest that mitochondria may contain a calpain. In a number of diverse preliminary experiments using isolated renal cortical mitochondria (RCM) we have obtained additional evidence of a novel mitochondrial calpain that is responsible for mitochondrial dysfunction. These data resulted in the hypothesis that mitochondrial Ca 2+- uptake leads to the activation of a mitochondrial calpain, which causes the mitochondrial dysfunction and ultimately results in RPTC death and ARF. The specific aims of this application are: Specific Aim I: Identify and characterize the mitochondrial calpain and examine its regulation in isolated RCM and RPTC. Specific Aim II: Elucidate the mechanism of mitochondrial calpain-mediated mitochondrial dysfunction in RPTC and isolated RCM, and identify the mitochondrial protein targets of mitochondrial calpain. Specific Aim III: Determine the effectiveness of currently described calpain inhibitors on mitochondrial calpain and develop new specific inhibitors of mitochondrial calpain using novel, non-natural amino acid analogues and determine their effectiveness in RPTC and isolated RCM. Specific Aim IV: Determine the efficacy of current and/or developed calpain inhibitors in an in vivo model of mitochondrial dysfunction and ARF. Completion of these Specific Aims will add significantly to our basic understanding of cell injury and death, particularly events mediating mitochondrial dysfunction. Further, we will identify a mitochondrial calpain and develop novel calpain inhibitors, including those that are mitochondrial calpain specific. Ultimately, these studies may lead to the development of therapeutic agents that improve clinical outcomes in patients with ARF. [unreadable] [unreadable]

DESCRIPTION (provided by applicant) Environmental Stress Signaling and Cellular Consequences (ESSCC) result from exposure to environmental xenobiotics, drugs, oxidants, radiation, ischemia/reperfusion, and nutritional imbalances. Despite the diversity of environmental stresses, there is a commonality in the responses of numerous cell types to these insults. Because environmentally and/or xenobiotically mediated diseases and the majority of other diseases incorporate cell injury, death, carcinogenesis, and a diminished capacity for cellular repair and regeneration in their pathology, the need for scientists trained in ESSCC is critical. The objective of the ESSCC Training Program is to train new scientists to address mechanisms of ESSCC and to translate findings into the development of interventions or novel therapeutics that prevent or diminish cell injury, death, and carcinogenesis, and/or promote repair and regeneration. In addition, new scientists entering this area of research will be able to use recently developed genomic, proteomic, and bioinformatic technologies to elucidate mechanisms of ESSCC. The trainees will come from various backgrounds that encompass chemical, physical, and biomedical sciences and will be integrated into the interdepartmental didactic and research ESSCC Training Program. It is anticipated that the training afforded by the ESSCC Program will result in new scientists that address the consequences of exposures to environmental xenobiotics. The primary principle uniting the mentors of the ESSCC Training Program is the universal role of environmental stress signaling and cellular consequences in environmentally and xenobiotically mediated diseases and other diverse diseases, and the belief that cells from different organ systems exhibit many common responses to diverse insults and stresses. It is viewed that advances in understanding ESSCC and training future scientists in ESSCC can be accomplished more quickly through integration of ESSCC efforts of scientists across classic departments and disciplines.

Cell death induced by oxidative stress occurs during ischemia/reperfusion, leading to failure of different organs such as the heart, brain, liver, and/or kidneys. Furthermore, oxidative stress is often the mediator of drug- and toxicant-induced cell death. Our previous studies in renal cells suggested that inhibition of calcium- independent phospholipase A2 (iPLA2) with the iPLA2 inhibitor bromoenol lactone (BEL) potentiated lipid peroxidation and necrotic cell death induced by oxidants.These results led us to the hypothesis that iPLA2 acts to repair or prevent lipid peroxidation induced by oxidants. We have identified IPLA2 gamma (Group VIB) in renal cells and localized it to the endoplasmic reticulum (ER). Exciting new preliminary studies have revealed that iPLA2 gamma also is present in the mitochondria of renal cells. The localization of a protein to both the ER and mitochondria is rare and limited to few other proteins. It should be noted that the major sources of reactive oxygen species in cells, ER and mitochondria, coincide with IPLA2 gamma localization. Our current hypothesis is that iPLA2 gamma protects cells from oxidative stress by preserving endoplasmic reticulum and mitochondrial membrane integrity and function. We propose to investigate this hypothesis through the following Specific Aims: Specific Aim 1. Determine the mechanism by which iPLA2 gamma is targeted to the ER and mitochondria. Specific Aim 2. Determine the consequences of "over-expression"and "knock-down" of iPLA2 gamma on ER and mitochondrial functions and necrotic cell death following oxidative stress. Specific Aim 3. Determine the mechanism(s) by which iPLA gamma protects ER and mitochondria during oxidative stress. Successful completion of these aims will increase our limited knowledge on the targeting of proteins to multiple cellular locations and elucidate the function of iPLA2 gamma, a potential natural defense enzyme against oxidative stress in the mitochondria and ER. Ultimately, these studies may lead to new therapeutic and pharmacological approaches to increase cell and organ survival in numerous pathologic situations.

DESCRIPTION (provided by applicant): The goal of this project is to identify therapeutics that accelerate the recovery from acute organ failure. Cell injury and death induced by ischemia/reperfusion (I/R), drugs, toxicants, and trauma lead to failure of many organs, including the kidney. Mitochondrial dysfunction is a common consequence of these insults and a major mechanism of cell injury and death. The majority of research in the field of acute kidney injury (AKI) has focused on the events that initiate renal dysfunction, but therapeutic agents are lacking. This suggests that more successful therapies require the examination of new targets and a focus on accelerating recovery from AKI. We recently determined that mice subjected to bilateral renal I/R induced AKI and rats subjected to myoglobinuric AKI had elevated serum creatinine 24 h after injury which partially recovered over six days post- injury. Mitochondrial electron transport chain and ATP synthesis proteins were depleted 24 h after injury and did not recover over six days, revealing persistent disruption of mitochondrial function after AKI. Consequently, we propose that therapeutics that increase mitochondrial biogenesis (MB) will promote recovery from AKI. As part of our drug discovery program to identify drugs that induce MB we identified formoterol, a specific long- acting b2-adrenergic receptor agonist (b2-AR), was a potent and efficacious inducer of MB in renal proximal tubular cells (RPTC). Formoterol is a FDA-approved drug used to treat asthma. Additional studies revealed that formoterol induced MB in the kidneys of mice at a low dose. However, the signaling pathway(s) responsible for formoterol-induced MB has not been elucidated. Finally, preliminary studies demonstrated that mice treated with formoterol 24 h after I/R, when renal dysfunction is established, accelerated recovery of [mitochondrial and] renal function. We hypothesize that formoterol induces MB through the b2-AR and that formoterol accelerates recovery of [mitochondrial and] renal function following I/R in mice. To address this hypothesis we propose the following Specific Aims: 1) Elucidate the signaling pathway(s) of formoterol- induced MB in RPTC [and following oxidant injury in RPTC], 2) Determine efficacy, potency and mechanism of formoterol-induced MB in mice, and 3) Elucidate the effects of formoterol on the recovery of mitochondrial and renal function following renal I/R-induced AKI in vivo. Successful completion of these experiments will advance the field by 1) identifying the precise signaling pathway that induces MB through b2-AR, 2) demonstrating that stimulating the recovery of mitochondrial function results in the acceleration of recovery of renal function, and 3) providing a rapidly clinically translatable treatment for AKI.

DESCRIPTION (provided by applicant): The long-term goal of this project is to identify and validate biomarkers of mitochondrial dysfunction due to environmental stressors. Diverse acute insults from surgery, trauma, ischemia/reperfusion (I/R) and drug and environmental chemical toxicity lead to mitochondrial dysfunction and result in cell injury and death in many organs/tissues (e.g. heart, lung, brain, liver and kidney). Furthermore, mitochondrial dysfunction can contribute to cell injury through increased production of reactive oxygen and nitrogen species. Mitochondrial dysfunction is also a component of many chronic diseases such as metabolic syndrome, diabetes, neurodegenerative diseases, and aging. Consequently, there is a great need for non-invasive biomarkers of mitochondrial dysfunction. We hypothesize that urinary mitochondrial DNA (mtDNA) and urinary protein levels of mitochondrial ATP synthase (ATPS) subunits are sensitive and specific markers of mitochondrial dysfunction in acute kidney injury (AKI). Our preliminary studies support this hypothesis by demonstrating increased urinary mtDNA and ATPS in mice subjected to I/R induced AKI when renal mitochondrial dysfunction was present. These preliminary studies provide strong evidence in support of our hypothesis. The following Specific Aims will be examined: 1) Using a mouse model with different degrees of I/R induced AKI, elucidate urinary changes in mtDNA, mitochondrial ATPS subunits and other mitochondrial proteins; integrate these changes with renal mitochondrial dysfunction over time; and compare and contrast the changes in these endpoints with general urinary AKI biomarkers. These studies will result in new urinary markers of mitochondrial dysfunction in animals. Comparison of mitochondrial DNA, protein and function over a range of times and grades of injury will permit better understanding of the timing and mechanisms of injury and recovery. Finally, these biomarkers can be tested in humans and translated into laboratory and clinical practice.

The goal of this project is to validate a lead drug that will accelerate the recovery of glomerular function after injury. Glomerular function is highly dependent on specialized cells known as podocytes, which are the critical components of glomerular filtration system and whose loss results in progressive renal failure. While podocyte injury is a common denominator in many glomerular diseases including focal and segmental glomerulosclerosis (FSGS), specific drugs that can restore injury-induced loss of podocyte structure and function remain unknown. As part of our therapeutic target identification program in podocytes, we performed mRNA profiling of cultured podocytes that were injured with adriamycin. The bioinformatics analysis revealed induction of genes related to mitochondrial function. Mutations in mitochondrial genes are known to result in mitochondrial dysfunction and FSGS, and have been implicated in the loss of podocyte function. Podocytes are terminally differentiated cells that have lost mitotic activity, and typically do not proliferate after injury. It is known that mitochondria play a critical role in maintaining podocyte energy homeostasis. We propose that podocytes can recover from injury by increasing MB (mitochondrial biogenesis) and therapeutics that increases MB will promote recovery from glomerular injury. To investigate this hypothesis, we tested the effect of a lead drug that is known to be a potent and efficacious inducer of MB, on the recovery of glomerular injury in mice. Our preliminary studies show that this drug is a potent inducer of MB in podocytes. Importantly, using a mouse model of podocyte injury that mimics FSGS phenotype, we demonstrate that the oral or intraperitoneal administration of this drug in mice, 6h after the injury, when glomerular dysfunction is established, accelerated the recovery of glomerular function, significantly reduced proteinuria and restored glomerular structure. Thus we hypothesize that this therapeutic treatment accelerates the recovery of glomerular function following injury through induction of podocyte MB. To address this hypothesis, we propose the following specific aims: Specific Aim 1: Determine the efficacy and potency of this drug in inducing MB and function in mice glomeruli following injury. Specific Aim 2: Determine the efficacy and potency of this drug in accelerating recovery from injury in various mouse models of glomerulopathy that mimic human FSGS. This will also test the spectrum of glomerular diseases that can be targeted with this drug. Specific Aim 3: Since this drug associates with albumin, which is heavily excreted in nephrotic syndrome patients, we will measure the pharmacokinetic data for this agent in nephrotic mice during the course of induced disease to determine the effect of albuminuria on its half-life and metabolism. Successful completion of these experiments will establish this lead drug as an efficacious treatment for glomerular diseases particularly FSGS that specifically targets podocyte dysfunction.