William E. Smoyer - US grants

Nephrotic syndrome (NS), a common kidney disease in children, is characterized by retraction (effacement) of the distal "foot" processes of glomerular epithelial cells (GEC) that surround glomerular capillaries. Foot processes are an essential part of the kidney's filtration barrier, and their structure is regulated primarily by actin microfilaments which are abundant in foot processes. Actin and the GEC itself are thought to be anchored the glomerular basement membrane by a complex of actin-associated proteins and beta1 integrin adhesion molecules. Effacement of GEC foot processes has been linked to altered distributions of GEC actin microfilaments and increased glomerular expression and phosphorylation of hsp27, a heat shock protein reported to regulate actin polymerization. We hypothesize that: 1) Specific changes in the expression and/or interaction of actin microfilaments, the actin-associated proteins vinculin, talin, and alpha-actin, and beta1 integrin adhesion molecules are critical for the development of GEC foot process effacement during nephrotic syndrome, and 2) These changes are mediated by altered expression of GEC hsp27. To test these hypotheses we plan to determine; 1) If specific changes in the expression or interaction of actin, the actin-associated proteins vinculin, talin and alpha-actinin, and beta1 integrins in GECs are necessary for the development of foot process effacement and NS, and to determine the relationship between these alterations and variable degrees of GEC foot process effacement, and 2) If induced changes in the expression of glomerular hsp27 result in development of, or protection from, GEC foot process effacement and NS. Using both in vivo (PAN nephrosis in rats) and in vitro (protamine treatment of isolated glomeruli) models of GEC foot process effacement, we will analyze changes in F- and G-actin, actin-associated proteins (vinculin, talin, alpha- actinin), and beta1 integrins in glomeruli using semiquantitative confocal microscopy, Northern, and Western blots, and immunoprecipitation studies. The relationship between changes in the expression and interaction of these molecules and the extent of GEC foot process effacement at multiple stages of disease will be determined using electron microscopy. The effect of hsp27 induction on foot process effacement will be studied both in vitro (heat shock and hsp27 transfections of glomeruli) and in vivo (whole body hyperthermia and inducible sense and antisense hsp27 cDNA transgenic mice). Identification of specific molecular alterations in the regulation of GEC structure which are critical for the development of GEC foot process effacement and NS would permit the development of more specific and less toxic therapies for NS.

Nephrotic syndrome is one of the most common forms of kidney disease in children. It is characterized by massive leakage of protein across the kidney's filtration barrier and dramatic structural changes in podocytes, which in part comprise the barrier. These changes include retraction (effacement) of the actin-rich podocyte foot processes with disruption of their actin filaments, and can be attenuated by treatment with reactive oxygen molecule scavengers, suggesting a link between NS and oxidant injury to podocytes. We recently detected a reported regulator of actin polymerization, heat shock protein 27 (hsp27), in normal podocytes, and reported induction of hsp27 in glomeruli during NS. We hypothesize that hsp27 has an important role in mediating the podocyte structural changes which occur in NS, via regulation of actin filament dynamics. We also hypothesize that hsp27 has an important role in the podocyte response to oxidant stress, and that the therapies commonly used to treat NS act by protecting podocytes from oxidant-induced injury via alterations in hsp27 expression and/or phosphorylation. To test these hypotheses we will: 1) Determine if induced changes in podocyte hsp27 expression and/or phosphorylation protect against NS, 2) Identify glomerular hsp27-binding proteins and measure changes in the interaction between hsp27 and the identified proteins during NS, and 3) Measure the protective effects of induced alterations in podocyte hsp27 on the podocyte stress response, and compare these effects to those resulting from podocyte treatment with corticosteroids, cyclosporine A, and cyclophosphamid (common treatments for NS). We will use both in vivo (PAN nephrosis in rats) and in vitro (PAN and protamine treatment of cultured "differentiated" podocytes) models of NS to determine if induction of hsp27 in vivo (whole animal hyperthermia, hsp27 transgenic animals) or in vitro (hsp27 sense/antisense/phosphorylation mutant stable transfections) protects podocytes against foot process effacement and NS. A yeast two hybrid library from rat kidney glomeruli will be used to identify, and define hsp27-binding proteins, and alterations in their interactions with hsp27 during NS will be determined by biochemical analyses. Cultured "differentiated" podocytes transfected with hsp27 sense/antisense/phosphorylation mutants will be treated with stressors with specific biological relevance to NS (oxidant stress, actin filament disruption, heat shock) and the cellular stress response (survival, actin filament structure, induction of hsps and antioxidants) compared to that after treatment with drugs used for NS. Identification of a biologically important role for hsp27 in regulating podocyte structure in NS would permit the development of more highly targeted and less toxic therapies for this very common form of kidney disease.

DESCRIPTION (provided by applicant): The Pediatric Nephrology Training Program at the University of Michigan is designed to recruit the best post-doctoral trainees and to provide them with high quality research training in one of two major tracks, Basic Science or Clinical Investigation and Epidemiology in preparation for successful academic careers in Pediatric Nephrology. The Training Program will provide strong clinical Pediatric Nephrology training for M.D. trainees, combined with 2 or more years of intensive postdoctoral research training for M.D. and Ph.D. trainees using an individualized and closely-mentored research training program designed to best fit each trainee's skills and interests. In order to provide outstanding mentorship for the trainees in these two major research areas, the Training Program will be actively supported by 24 faculty from 7 different departments at the University of Michigan, all of whom have extensive research and mentoring experience within their area of expertise. The Training Program will also include an extensive didactic component tailored to each trainee's research interests. Trainees in the Basic Science track will complete a three-month Postgraduate Research Training Program designed to expose trainees to a variety of state-of-the-art techniques, as well as to enhance their skills in performing hypothesis-driven and appropriately controlled research studies. Trainees in the Clinical Investigation and Epidemiology track will choose between a focus on Outcomes and Epidemiology or Clinical Research. These trainees will all complete a course of study leading to either a Master of Public Health in Biostatistics and Epidemiology degree or a Master of Science in Clinical Research Design and Statistical Analysis degree. In addition to their formal curriculum, the trainees will attend weekly clinical and research seminars and monthly research conferences. Mentored research will be supported by the resources of the Medical School, the Department of Pediatrics, the Division of Pediatric Nephrology, the Division of General Pediatrics, the Division of Nephrology in the Department of Internal Medicine, the Kidney Epidemiology and Cost Center and the Clinical Research Training Core of the Center for the Advancement of Clinical Research. To optimize each trainee's potential for development of a successful academic career and to ensure Pediatric Nephrology Board eligibility, trainees will also be required to submit abstracts to national meetings, submit at least one first-authored research manuscript, and apply for individual grant funding. Previous graduates of our Training Program have an excellent record of developing successful academic careers in Pediatric Nephrology. Expansion and enhancement of our Training Program will help to alleviate the critical shortage of successful Pediatric Nephrologists and Pediatric Physician-Scientists.

DESCRIPTION (provided by applicant): Nephrotic syndrome (NS) is a common kidney disease, but the molecular mechanisms underlying the disease remain unclear for most cases. We previously reported that the small heat shock protein, hsp27, a known regulator of actin polymerization, is highly expressed in glomerular podocytes (the cells most affected in NS), that its expression and phosphorylation are induced in podocytes in experimental NS, and that hsp27 overexpression can protect cultured podocytes from injury. To clarify the mechanism of this protection, we screened a glomerular yeast two-hybrid library for hsp27-binding proteins, and found hic-5, a known focal adhesion and steroid receptor co-activator protein. We confirmed hic-5 as a true hsp27 binding protein by coimmunoprecipitation, partially mapped the interaction domains, and demonstrated that interaction of hic-5 with hsp27 can alter hsp27 function. Recently, we also found that glucocorticoids, the primary treatment for NS, can act directly on podocytes to protect and enhance recovery from PAN-induced injury, in striking contrast to the dominant paradigm that glucocorticoids exert their therapeutic effect in NS by suppressing production of a soluble disease mediator by circulating lymphocytes. We therefore hypothesize that the interaction between hsp27 and the multifunctional binding protein, hic-5, plays a critical role in regulating both podocyte injury during NS and podocyte recovery following glucocorticoid therapy, via hic-5's known roles in: 1) Focal adhesion dynamics, and 2) Glucocorticoid receptor coactivation. To test these hypotheses we will determine the molecular states of hic-5 and hsp27 required for their protein-protein interaction, and the effects of alterations in these states on their intracellular localization in podocytes. We will also determine if induced alterations in the expression, phosphorylation, or hsp27- binding of hic-5 can regulate: 1) Podocyte focal adhesion protein composition, rate and extent of formation, and function before and after podocyte injury, and 2) Glucocorticoid-induced activation of podocyte gene transcription, as well as protection and enhanced recovery of podocytes from injury. Podocytes will be infected with adenovirus containing full-length and phosphorylation- and/or truncation-mutant hic-5 and hsp27 constructs, and detailed analyses performed of the effects of altered hic-5-hsp27 interaction on the: 1) Intracellular localization of hsp27 and hic-5, 2) Focal adhesion composition, formation rate, and function, and 3) Glucocorticoid-induced protection and enhanced recovery from PAN induced injury and actin filament disruption by latrunculin A and cytochalasin D. Correlative in vivo studies will include analyses of hic-5 and hsp27 co-localization in podocytes, as well as protection against disease by dexamethasone, in PAN-induced nephrotic syndrome in rats. Identification of a biologically important role for the interaction between hic-5 and hsp27 in regulating podocyte structure and the therapeutic effect of glucocorticoids in NS would improve our understanding of the molecular mechanism(s) underlying the development of NS, and permit the development of more highly targeted and less toxic therapies for this very common kidney disease.

DESCRIPTION (provided by applicant): Nephrotic syndrome (NS) is a common kidney disease, but the molecular mechanisms underlying the disease remain unclear for the vast majority of cases. We previously reported that the small heat shock protein, hsp27, a known regulator of actin polymerization, is highly expressed in glomerular podocytes (the cells most affected in NS), and that glomerular hsp27 expression and phosphorylation are significantly induced in experimental NS. We also reported that hsp27 is able to dramatically regulate (i.e. hsp27 overexpression protects, while reduced expression sensitizes) the podocyte response to PAN-induced cellular injury and actin cytoskeletal disruption. More importantly, we recently confirmed that glomerular hsp27 expression is induced in both multiple animal models of NS, as well as in human NS, suggesting that induction of glomerular hsp27 represents a generalized podocyte stress response. Since hsp27 has not been confirmed to bind actin directly in vivo, we attempted to clarify the mechanism of hsp27-mediated regulation of podocyte structure by screening a glomerular yeast two-hybrid library, and identified two novel hsp27 binding proteins: 1) Hic-5, a known focal adhesion protein and paxillin homologue, and 2) Arpda, a known member of the Arp2/3 actin polymerization initiation complex. We confirmed hic-5 as a true hsp27 binding protein by co-IP, mapped its interaction domains with hsp27, and showed that hic-5 can inhibit hsp27-induced thermo-protection in an interaction-dependent manner. We also confirmed both ArpCIa and ArpClb as true hsp27 binding proteins by co-IP and quantitative FRET analyses. Based on the above, we hypothesize that hsp27 plays a critical role in the regulation of podocyte structure and response to injury, and that these actions are mediated via its novel binding proteins, hic-5 (a paxillin homologue with a role in focal adhesion dynamics), and ArpC1 (a member of the Arp2/3 actin initiation complex). To test this hypothesis we will complete development of podocyte-specific hspbl (hsp25) gene-targeted mice and perform phenotypic analyses to definitively determine the role of hsp25 in podocyte biology. We will also determine if induced alterations in hic-5 expression or interaction with hsp27 can regulate podocyte: 1) Focal adhesion formation rate, composition, and function (i.e. adhesion), and 2) Response to injury. Lastly, we will determine if induced alterations in ArpC1 expression or interaction with hsp27 can regulate podocyte: 1) Arp2/3 complex function (i.e. initiation of actin polymerization) and composition, and 2) Response to injury. Confirmation of hsp27's importance in the regulation of podocyte structure, and identification of its molecular mechanism(s) of action in podocytes, could not only improve our understanding of the molecular mechanism(s) underlying the development of NS, but also permit the development of more highly targeted and/or less toxic therapies for this very common kidney disease.

DESCRIPTION (provided by applicant): Primary nephrotic syndrome is the dominant cause of acquired kidney disease in children, with significant morbidity and mortality from the disease and its treatment, and a substantial risk of kidney failure. Yet, there is limited information on te natural history of primary glomerular disease in children, risk factors for poor outcomes and differential response to therapy. There is clearly a need for better therapies given the side effects and limited response to current therapies. Our long-term goal is to define specific molecular pathways that mediate cell injury in order to develop more targeted, effective, and less toxic therapies for glomerular disease. The overall objective of this proposal is to successfully recruit 600 children with four glomerular diseases (Minimal Change Nephrotic Syndrome [MCD], Focal Segmental Glomerulosclerosis [FSGS], IgA Nephropathy [IgAN], and Idiopathic Membranous Nephropathy[IMN]) into a longitudinal cohort study that will include integrated proteomic and metabolic analyses of highly phenotyped biological samples to identify predictive biomarkers for pediatric glomerular disease. Our central hypothesis is that integrated proteomic and metabolic analyses from clinically phenotyped sequential biological samples will identify novel biomarkers that can predict both clinical outcomes and therapeutic responses for pediatric glomelular disease, and identify molecular targets for future therapies. Thus, the rationale for this proposal is that recruitment and clinical phenotyping of a very large cohort of children with glomerular disease, combined with innovative pilot studies integrating proteomic and metabolomic analyses from sequential biological samples, will identify novel biomarkers that can predict both clinical outcomes and therapeutic responses, as well as identify molecular targets for future therapies. To test our central hypothesis, we propose the following Specific Aims: 1) To successfully recruit 600 children with MCNS, FSGS, IgAN, and IMN into a longitudinal cohort study that includes sequential detailed clinical phenotyping, combined with standardized collection, processing, and storage of serum, plasma and urine samples. 2) To define protocols that integrate proteomic and metabolic analyses of highly phenotyped biological samples in order to identify biomarkers able to predict clinical outcomes and therapeutic responses for pediatric glomerular disease. Metabolomics and proteomics will identify biomarkers that provide information on disease prognosis and predicted response to therapy, which will support the design and implementation of clinical trials. Biomarker identification will guide the development of more targeted, effective, and less toxic therapies for one of the most common kidney diseases in the US.

DESCRIPTION (provided by applicant): Glomerular disease is the third leading cause of end stage renal disease in the US, with its related health care costs estimated at $4.1 billion annually. Nephrotic syndrome (NS), characterized by podocyte injury, is one of the most common forms of glomerular disease. Importantly, progressive podocyte injury and loss are also known to be critical determinants of glomerular disease progression. Since the signaling pathways in podocytes most critical for regulation of injury are not yet known, there is an urgent need to better understand which pathways are most able to regulate podocyte injury and recovery to enable the development of more targeted and effective therapies for NS. Our long-term goal is to define specific molecular signaling pathways able to regulate podocyte injury in NS to develop more targeted and less toxic therapies for NS. The overall objective of this proposal is to determine the ability of the glucocorticoid receptor (GR), peroxisome proliferator-activated receptor g (PPARg), and MAPK signaling pathways to regulate podocyte injury, and to exploit this knowledge to develop more effective novel therapies for NS. Based on this, we hypothesize that specific manipulation of the GR and PPARg nuclear receptor pathways and MAPK pathways, and cross-talk among them, will reduce podocyte injury during NS. The rationale for the proposed studies is that specific manipulation of GR-, PPARg-, and MAPK-mediated pathways or cross-talk among them can ameliorate glomerular injury in NS, and will enable the development of more effective novel approaches to treat NS in the future. To test our hypothesis, we propose the following Specific Aims: 1) To determine if manipulation of critical components of GR and PPARg nuclear receptor signaling can enhance podocyte protection from injury, 2) To determine the extent and biologic significance of cross-talk among the GC-, TZD-, and MAPK-mediated signaling pathways during podocyte injury, and 3) To determine if manipulation of GC-, TZD-, and MAPK-mediated signaling can ameliorate glomerular injury in animal models of NS. These studies will identify specific potential targets for future drug therapy in NS, and potentially many other glomerular diseases where podocyte injury plays a central role. Validation of these podocyte signaling components as potential drug targets will guide the development of more targeted, more effective, and less toxic therapies for one of the most common kidney diseases in the US.

Abstract/Project Summary Glomerular disease is the third leading cause of end stage kidney disease in the US, with its related health care costs estimated at $4.1 billion annually. Immunosuppressive (IS) drugs are the primary therapies for most glomerular diseases, but ~20-50% of patients fail to achieve a remission. Unfortunately, in the absence of biomarkers to predict treatment responsiveness, many patients receive prolonged yet ineffective IS therapy, leaving them at high risk for both toxic side effects and disease progression. Since the prognostic factors and specific molecular pathways that are the most critical regulators of the various glomerular diseases are not yet known, there is an urgent need to develop strategies to prevent drug-induced toxicity, and to identify more targeted and effective treatments for glomerular disease. Our long-term goal is to identify prognostic biomarkers and molecular pathways that regulate glomerular injury, and use this knowledge to develop predictive biomarkers and more targeted treatments for glomerular disease. The specific objective of this proposal is to identify diagnostic and predictive biomarkers and novel molecular pathways/targets for each of the four CureGN glomerular diseases by integrating state-of-the-art proteomic and metabolomic analyses of serial plasma samples from pediatric glomerular disease patients. Based on this, we hypothesize that integrated proteomic and metabolomic analyses of serial clinically and histologically phenotyped pediatric plasma samples will identify novel biomarkers that predict treatment responsiveness, as well as molecular targets for targeted glomerular disease treatments. CureGN is a prospective observational study to clinically phenotype and collect serial blood and urine samples from 2,400 children and adults with four of the most common types of glomerular disease: MCNS, FSGS, MN, and IgAN. The rationale for this proposal is that integrating proteomic and metabolomic analyses of serial clinically and histologically phenotyped pediatric plasma samples will enable identification and validation of novel biomarkers that can predict glomerular disease treatment responsiveness, as well as novel molecular targets for future treatments. To test our hypothesis, we propose the following Specific Aims: 1) To compare sequential pediatric plasma proteomic and metabolomic signatures to identify predictive biomarkers, and to differentiate molecular pathways that are distinctive or common among all four CureGN glomerular diseases, 2) To integrate proteomic and metabolomic data to identify combined predictive biomarkers, and to differentiate molecular pathways that are distinctive or common among all four CureGN glomerular diseases, and 3) To validate candidate biomarkers in independent CureGN pediatric samples. These studies will apply a state-of-the-art integrated proteomic and metabolomic systems biology approach to a large cohort of phenotyped pediatric plasma samples to identify and validate novel biomarkers that can predict treatment responsiveness, and identify new molecular targets for potential future glomerular disease treatments.

Abstract Primary glomerular diseases including minimal change disease (MCD), focal segmental glomerulosclerosis (FSGS), immunoglobulin A nephropathy (IGAN), and membranous nephropathy (MN) account for 10% of end-stage renal disease and are associated with significant morbidity and mortality in both adults and children. Despite the significant disease burden associated with these heterogeneous disorders, disease pathogenesis, natural history, predictors of clinical outcomes and predictors of response to therapy remain incompletely defined. To address these gaps in knowledge, the CureGN consortium assembled a large consortium of 70 clinical study sites, nephrologists, scientists with diverse expertise, affected patients and advocacy groups, the biopharmaceutical industry, federal funding agencies, and regulatory agencies. CureGN is conducting a prospective, longitudinal observational study, successfully enrolling and retaining a race and ethnicity-balanced cohort of nearly 2400 adult and pediatric participants with IgAN, FSGS, MN and MCD. We propose to maintain and enhance the CureGN Consortium infrastructure and its ancillary study program to accelerate patient-relevant glomerular disease research. We will continue our core prospective, longitudinal observational study of glomerular disease patients, enrolling additional subjects by a recruit-to-replace strategy to maintain an active cohort of 2400, while banking and curating high quality clinical data and biomaterials. This foundational work is being conducted by a refined study administrative structure and a well-functioning collaboration between the Data Coordinating Center (at Arbor Research Collaborative for Health and the University of Michigan) and four Participating Clinical Centers (managed at the University of Pennsylvania, Columbia University, University of North Carolina, and the Midwest Pediatric Consortium). CureGN sits at the nexus of multiple domains that are collaborating in overcoming the barriers to improving care for glomerular disease patients. By its now experienced scientific working groups, its publications committee, and its ancillary study infrastructure, by enabling modern analytical approaches in multiple relatable knowledge domains, by administering a research opportunity pool in conjunction with NephCure Kidney International, by continuing refinement of study instruments, by enhancing career development activities, and by convening patient and public-private collaborations for validating and implementing precision medicine-based therapies, the resource created by CureGN should accelerate improving care of glomerular disease patients.