Stefan Somlo - US grants

genetic mapping; polycystic kidney; family genetics; autosomal dominant trait; disease /disorder proneness /risk; nucleic acid sequence; artificial chromosomes; molecular cloning; human subject; human genetic material tag;

DESCRIPTION (provided by applicant): Our research program centers on achieving a comprehensive molecular understanding of human polycystic liver and kidney disease. Our goal is discovery novel paradigms for effective treatment of patients. We have taken an approach that begins with disease gene discovery through positional cloning to understand the genetic bases for the diseases, followed by functional studies to understand cellular pathogenesis. Under the current grant, we recruited patients with isolated autosomal dominant polycystic liver disease (ADPLD;MIM 174050) and completed a comprehensive clinical characterization of the disease. We established genetic linkage for one locus, PLD1, and discovered the disease gene, PRKCSH. We identified a second locus for ADPLD on chromosome 6 and identified human SEC63 as PLD2. Mutations in these genes account for ~ 25% of ADPLD probands in our cohort and suggest that there is at least one additional locus, PLD3, for ADPLD. PRKCSH encodes the B-subunit of glucosidase II (GIIB), a glucose trimming enzyme involved in protein maturation and quality control in the ER. SEC63 encodes a component of the ER protein translocation machinery that functions in concert with GII to achieve proper topology and folding of integral membrane or secreted glycoproteins. This proposal seeks to further define cellular pathways to cyst formation by discovery of additional genes involved in ADPLD, by determining whether two hits are required for cyst formation in ADPLD, and by defining the relationship between protein maturation in the ER and the normal function of primary cilia in bile duct epithelium. We propose to identify additional genes (e.g., PLD3) responsible for ADPLD by use of a combination of "classical" positional cloning and candidate gene approaches. We will use mouse models to test the hypothesis that cyst formation in ADPLD requires somatic second hits as is the case in ADPKD. We will determine whether Prkcsh and Sec63 mutations result in improper maturation and cilial delivery of the Pkd 1, Pkd2 and Pkhd 1 gene products. These studies will improve our understanding of the cellular and molecular bases of polycystic diseases by using the novel entry points into the pathogenic pathways made possible by our discovery of PRKCSH and SEC63 as disease genes. At the same time, we will improve our understanding of ADPLD as a disease condition and develop insights that will help patients affected by this condition.

family genetics; liver disorder; polycystic kidney; phenotype; gene mutation; genetic mapping; medical complication; clinical research; human subject; molecular cloning;

DESCRIPTION (Adapted from the Applicant's Abstract): "The primary interest of our research is unraveling the pathogenesis of polycystic kidney disease. To this end, they have used positional cloning to identify the second gene for human autosomal dominant polycystic kidney disease (PKD2). The current proposal centers on the hypothesis that genetically altering the murine homologue of the PKD2 gene, Pkd2, will enable us to produce animal models whose phenotypes are faithful to those of human autosomal dominant polycystic kidney disease (ADPKD). They have produced a mouse line with a targeted mutation in which the first coding exon of Pkd2 has been disrupted. Mice heterozygous and homozygous for this allele develop polycystic kidneys and livers that recapitulate the human disease phenotype. The disease develops faster in homozygotes. They propose histopathological and functional characterization of the renal and extra-renal phenotypes of these mutant mouse lines as well as further analysis of the molecular consequences of the gene targeting event. This murine model of ADPKD has some residual polycystin-2 expression in the homozygous state. They propose to create a model in which exons 1, 2, and 3 of Pkd2 have been deleted. These null mice will provide a model system for studying the phenotype. In addition, they propose to introduce naturally occurring premature termination codons found in human families with ADPKD, into Pkd2. They will characterize the ensuing mouse phenotypes in heterozygous and homozygous mice and will characterize the functional consequences, at the level of the protein product, of these truncating mutations. Finally, they plan to study the effects of factors other than germ line mutation in Pkd2 on the occurrence and progression of the renal and extrarenal manifestations in mouse models of ADPKD. They will use marker assisted breeding strategies to produce congenic strains bearing mutations in Pkd2 on different genetic backgrounds. They will investigate the effects of defects in DNA mismatch repair on the progression of ADPKD by breeding Pkd2 mutations onto a MLH1-deficient mouse line."

The primary interest of our research program is unraveling the pathogenesis of human autosomal dominant polycystic kidney disease (ADPKD) with a final goal of developing rational therapeutic interventions to benefit affected patients. To this end, we used homologous recombination-based approaches to inactivate the Pkd 2 gene in mice and produce an adult animal model that recapitulate the human ADPKD phenotype. This animal model has proven the 'two hit' mechanism of cyst formation in PKD2. This work has convinced us that a complete understanding of polycystic kidney disease requires 1) the further development and steady of whole animal model systems and 2) the discovery of additional components in the 'polycystin' pathways. In keeping with this overall approach toward understanding and treating this human disease, our specific aims are: To determine the mechanisms by which polycystin-2 acts to maintain normal renal tubular architecture by producing an animal model in which we have temporal and spatial control of cyst formation. Specifically, we will make a mouse line bearing a floxed exon 3 of the Pkd2 gene and a pair orf inducible-Cre recombinase transgenic lines using the tamoxifen-inducible CreERTM transgene-one under the control of a collecting duct epithelial cell-specific promoter (Ksp- cadherin; cadherin) and another under a general promoter (pCAGGS; CMV-IE enhancer and a modified chicken beta-actin promoter). We will use these models to study the mechanisms of cyst formation in adult animals with ADPKD. In order to analyze structure function relationships and tissue-specific effects of Pkd2 using in vivo model system we will determine the effects of functional re-expression of a Pkd2 EF-hand mutant deficient in binding calcium and of a form of Pkd lacking the putative ER-localization domain. Finally, in order to identify downstream regulators whose expression is effected inactivation of Pkd we will generate well characterized cell lines from adult nephron segments that are genetically identical except for the presence or absence of functional polycystin-2 and profile the gene expression pattern in these cell lines to identify elements whose expression is altered by the loss of polycystin-2 function.

The overall goal of the Yale Interdisciplinary Center for Polycystic Kidney Disease Research is to elucidate the mechanisms by which defects in the polycystin genes result in autosomal dominant polycystic kidney disease (ADPKD) and to understand the factors that modify the expression of the disease phenotype. Studies performed during the first 5 years of this Center Grant have provided the foundation for our present understanding of the importance of PC-1/PC-2 interactions in suppressing cyst formation, established a central role of the cilia in multiple forms of cystic disease, and have promoted novel concepts about how polycystins are processed and traffic in the cell. In the renewal of this award, these results have been utilized to focus the research on the areas of regulated post-translational modification and trafficking of polycystins, as well as their role in ciliary function and signaling. To investigate this hypothesis, Project 1 will define how PC-1 and PC-2 traffic to cilia, and will identify the domains within these proteins that mediate trafficking and determine whether graded interruption of this process can directly promote cystogenesis in animal models. Project 2 will explore the role of signaling by the cleaved C-terminal domain of PC-1, and how this is regulated by PC-2. Project 3 has utilized the power of zebrafish genetic screening to identify a unique ciliary protein that mimics many of the aspects of PKD in the zebrafish model and will explore the role of this protein in normal ciliary function. Project 4 will investigate the role of polycystin signaling in regulating the morphogenic events that mediate tubule formation, and will explore the ability of Ngal to modify these signals and thereby suppress cyst formation in vivo. Project 5 will utilize expertise in calcium channel signaling to define how PC-2 calcium channel activity is regulated in the cilia. These efforts will be supported by the Mouse and Cell Line Core that has an exceptional array of in vivo animal and cell-based models of polycystin function and ADPKD.

The major goal of the Mouse ore is to provide state-of-the-art transgenic and ES cell technology to support the development of authentic mouse models of human autosomal dominant polycystic kidney disease (ADPKD). Support to participating projects will be provided in four areas: 1) ES cell culture and blastocyst injection, 2) Generation of transgenic mice by micro transgenic mice by microinjection of mouse embryos, 3) Maintenance of breeding colonies and performance of genetic crosses, and 4) Genotype analysis. ES cell culture and blastocyst infection will be required for generating mice carrying a foxed Pkd2 allele that can be inactivated by Cre/lox-mediated recombination (Projects 1). Pronuclear microinjection will be required to generate transgenic mice expressing a hormone-inducible form of Cre recombinase using either a ubiquitous or kidney-specific gene promoter (Projects 1). Transgenic mice carrying a green fluorescent protein (GFP) or Pkd2 gene expression in Project 2. Genetic crosses will be performed to produce mice carrying combinations of floxed Pkd2 alleles, a Cre-estrogen receptor deluder gene, and a ROPSA26-Cre reporter gene in which inactivation of Pkd2 an be induced by hormone treatment and mutant cells can be identified by X-Gal staining. Such mice will be used to study the pathogenesis of cyst formation in adult mice (Project 1), the role of Pkd2 in kidney development (Project 2), and the alterations of membrane transport in cyst epithelium (Project 4). Mutant mice will be crossed with an SV40-transgenic mouse (ImmortoMouse) to produce mutant cell lines for studies of polycystin-2 intracellular trafficking (Project 3) and ion channel properties (Project 1). The Mouse Core will provide centralized services for animal husbandry and genotype analysis of the various mutant and transgenic strains.

The inherited polycystic kidney diseases now comprise at least four different disease entities. Three of these are autosomal dominant in pattern of inheritance (ADPKD) and two of these have been mapped to chromosome 16 and 4 respectively. The third form remains unmapped. An autosomal recessive form of the disease (ARPKD) has been mapped to chromosome 6. Finally, in addition to the autosomal dominant polycystic kidney diseases, there is an autosomal dominant polycystic liver disease (ADPLD) which is genetically and clinically distinct from polycystic kidney disease, but in which the pattern of liver involvement is indistinguishable from that seen in the polycystic kidney diseases. The gene for PKD1 on chromosome 16 has been cloned and the gene for PKD2 on chromosome 4 is being cloned in the PIs laboratory. These two genetic loci account for about 99% of autosomal dominant polycystic kidney disease occurring world wide. As a direct consequence of the cloning of these genes, several things will become possible. Among these are direct gene based testing for mutations in affected individuals, and as a corollary of that, an analysis of these mutations in terms of the phenotypic expression seen in affected individuals. In the current study, we plan to use the molecular characterizations of individuals affected with any of the cloned forms of polycystic disease (i.e. those forms for which the genes have been identified) in conjunction with well described clinical characterization of patients as a means of better understanding how specific mutations result in specific clinical manifestations. In the United States there are only one or two substantial collections of PKD patients available for such studies. The most complete collection is comprised primarily of Caucasian population and is apparently over-representative of the PKD1 disease genotype. Our goal is to collect a more ethnically diverse group of PKD patients and in addition to collect a more representative distribution of both PKD1 and PKD2, as well as non-PKD1/non-PKD2 forms of the disease. We will study these families clinically using abdominal ultrasonography, echocardiography and magnetic resonance angiography. Molecular genetic characterizations will be used to identify the mutations in the known genes and/or the genetic loci of the disease gene if they are not in PKD1 or PKD2 in these individuals. Data amassed from these studies will allow us to make prognostic determinations based on genotypes about potential outcomes that will help patients and physicians manage their disease better. In addition, we will begin to understand how different genetic loci and/or other factors influence the progression of the disease and this may enable us to identify a target population who are at high risk for developing renal failure in the course of their disease, and in whom more aggressive intervention may be necessary.

The major goal of the Mouse and Cell Line Core is to provide Center investigators with common set of unique reagents and services to enable them to achieve the highest levels of insight into the problems in the PKD field. A secondary goal of the Core is to make these same reagents available to the research community at large. The Core reagents are and very comprehensive array of PKD-related animal models and well characterized mouse kidney cell lines with Pkd1 and Pkd2 mutations. The service provided by the Core is the expert consultation and collaboration to allow all Center investigators use these reagents to highest effect. The specific aims of the Core are: 1. To make available a comprehensive array of animal models relevant to the study of polycystic kidney and liver disease. 2. To characterize and make available specialized mouse-derived cell lines with fixed and inducible mutations in Pkd1 and Pkd2. 3. To perform the necessary mouse breeding and genotyping to obtain experimental animals. 4. To prepare tissues from experimental animals for imaging-based analysis. 5. To provide expert instruction to Center investigators in the design and study of new animal models. 6. To make mouse and cell line models available to the research community worldwide. The state-of-the-art in mouse models of human disease has moved from simple knockouts to complex combinations of conditional alleles with inducible transgenes. Issues of genetic background must always be borne in mind. New methods of BAG modification and transgenesis offer the ability to address very specific questions. All these approaches require expertise for optimal application. The Core and its PI will provide this service to all Center faculty as well as Center collaborators, thus facilitating and enhancing the respective projects.

We hypothesize that a subset of human mutations in PKD1 and PKD2 result in protein products whose main pathologic defect is improper folding resulting in failure of trafficking into the cilial compartment. This proposal seeks to define critical determinants of polycystin trafficking and to explore the role these have in the pathogenesis of autosomal dominant polycystic kidney disease (ADPKD). We will determine the role of polycystin-1 trafficking in the pathogenesis of ADPKD. The underlying hypotheses of this aim is that a subset of pathogenic amino acid substitution mutations result in aberrant trafficking of PC-1. We will define the role of specific domains and mutations in the localization of PC-1 in the cilia in epithelial cells by examining the consequences of amino acid substitution mutations and cleavage at the GPS site on trafficking of PC-1 to cilia. We will determine the role of the cytosolic C-terminus in the trafficking of full length PC-1. In the second aim, we will identify the determinants of polcysytin-2 trafficking to cilia. We have found that PC-2 contains independent cilia targeting information within its primary sequence that is both necessary and sufficient for cilia targeting. We will use discovery of the responsible motif(s) to elucidate the mechanism by which this and other PKD-related proteins achieve cilial location. Our goal is to identify the necessary and sufficient amino acid motif(s) in PC-2 that direct its localization to cilia. We will isolate proteins interacting with the PC-2 trafficking domain that are part of its ciliary trafficking complex. Finally, we will determine the role of protein trafficking in the clinical features of ADPKD. We will test the hypothesis that disruption of trafficking is a true pathogenic event in ADPKD and that trafficking mutations result in milder clinical features than complete null mutations. We will determine whether naturally occurring disease causing amino acid substitution mutations in PC-2 result in defective trafficking of the protein. We will investigate whether trafficking mutations result in hypomorphic alleles with milder cyst forming capacity than null alleles in true in vivo models due to Pkd1 and Pkd2. This aim will establish in vivo model systems for determining the clinical impact of trafficking mutants and for determining whether therapies directed at trafficking of PC-1 and PC-2 will be effective in the treatment of patients.

DESCRIPTION (provided by applicant): Autosomal dominant polycystic kidney disease (ADPKD) is characterized by progressive enlargement of numerous cysts in affected kidneys that lead to renalfailure in about half of ADPKD patients. The disease burden from ADPKD in U.S. alone is estimated at 600,000 individuals and worldwide it exceeds 12 million. The costs of renal replacement therapy in ADPKD patients in the US exceeds $2 billion annually. The past 15 years have seen substantial progress in our understanding of the pathogenesis of PKD with notable discoveries including the identification of the causative genes, the recognition that somatic second step mutations initiate cyst growth and the discovery that the cilia/basal body complex is the focal point of the pathogenesis of renal cystic diseases in general, and of ADPKD specifically. Despite these major advances, there remain substantial gaps in our knowledge of the molecular pathogenesis of the disease, particular when it comes to the immediate signals downstream of the PC1/PC2 receptor channel complex. These gaps undermine our ability to achieve a comprehensive understanding of ADPKD and directly limit our ability to rationally and effectively target ADPKD for therapy. A critical step for promoting deeper scientific understanding of the factors underlying polycystic kidney diseases and for promoting interest in efforts to develop therapies, is to achieve a compelling and precise definition of the target pathway(s) in ADPKD. The impediments to achieving a comprehensive understanding of ADPKD are two-fold. First, PKD is a disease affecting the development and, more importantly, the maintenance of three dimensional solid organ structure in the kidney and liver. As such, surrogate ex vivo systems based in two or three dimensional cell culture are inadequate for discovery of pathways central to PKD. The relevant readout for the cystic phenotype in such systems, if it exists, is not known. Second, there has been no strategy that has been successful for unknown pathway discovery downstream of the PKD genes. Rather, pathway discovery has been based on examining known pathways, such as those associated with proliferation and planar polarity, as candidates for dysregulation in PKD. We hypothesize that the polycystins act in an as yet undiscovered novel pathway(s) that is most clearly functional in intact organs and the discovery of which requires unbiased, phenotypically-driven forward genetic approaches in whole mammalian organisms. The current proposal puts forth a powerful and novel set of studies to achieve this goal. We propose to use transposon mediated somatic insertional mutagenesis based on a uniquely modified PiggyBac (PB) transposon system to discover activating mutations on a wild type background that result in cyst formation in the mouse kidney. We will do the same for loss-of-function mutations using a background sensitized to cyst formation by mutations in the ADPLD gene, Sec63. Since PB transposition may also yield micro-tumors in the kidney, we will use this system to uncover oncogenic pathways in the kidney as well. Finally, we will use this system to define the therapeutic expectations resulting from selective reactivation of the polycystin pathways in cyst cells. PUBLIC HEALTH RELEVANCE: Autosomal dominant polycystic kidney disease (ADPKD) affects 600,000 individuals in the US adds over $2 billion to health care costs annually. Improved understanding of the molecular basis for this disease is the best means of moving toward treatment. This proposal will use a novel state-of-the-art genetic analysis in mouse kidneys to discover mechanisms underlying ADPKD that have not been previously known and which will improve the prospects for finding effective treatments for the disease.

DESCRIPTION (provided by applicant): Polycystic kidney disease affects 600,000 individuals in the US and is leading cause kidney failure and associated co-morbidities. It is inherited as an autosomal dominant trait and is characterized by the growth of fluid filled cysts that deform and can ultimately destroy the kidney. At the cellular level, somatic second hit events have been identified as initiating events for the growth of cysts. The goal of this proposal is to define the spectrum of mechanisms that determine cyst growth in human polycystic kidney disease. Our hypothesis is that while somatic inactivation of the PKD genes does give rise to cyst growth via cell autonomous mechanisms, non-cell autonomous factors acting locally and at a distance on tubule cells that retain expression of polycystins contribute to polycystic disease progression by either incorporating such cells into cysts or eliminating them so cysts can grow. We provide evidence that cysts in adult onset ADPKD can form by non-cell autonomous pathways. We used inducible conditional gene inactivation confined to the proximal tubule (PT) in adult mice and found that cysts also formed in distal nephron segments that still expressed polycystins. These non-cell autonomous cysts show increased proliferation and activation of the EGF receptor (EGFR) and of MAPK/ERK signaling pathways. In complementary studies, we found that perinatal inactivation of either Pkd gene, known to cause more rapid cyst growth, produce mosaic cysts that include a significant proportion of Pkd+/+ cells in addition to Pkd-/- cells. These data suggest that Pkd1-/- or Pkd2-/- cells not only form cysts but develop activities that impact otherwise normal cells and tubules both locally and at a distance. We will define the in vivo mechanisms of non-cell autonomous cyst formation in ADPKD by establishing the mechanism of disappearance of wild type cells in proximal tubule cysts and determining whether there are differential on wild type cells following inactivation of the Pkd genes in different nephron segments. We will also determine whether adult cysts formed following perinatal Pkd gene inactivation retain wild type cells. At a molecular level, we will define the mechanisms of EGFR and MAPK/ERK activation and their role in non-cell autonomous cyst formation. We will examine the in vivo effects of interfering with EGFR activation on growth of cysts still expressing polcysytins. We will determine whether EGFR activation is mediated by EGF ligand family members, and if so, which one and how is its production related to inactivation of polycystins. We will determine whether inactivation of the specific EGF ligand will abrogate non-cell autonomous cyst growth. In aggregate, these studies will define a more integrated understanding of the novel determinants of cyst progression in ADPKD and will provide data on whether targeting the non-cell autonomous cyst growth in ADPKD will be an effective therapy for improving progression of the disease. PUBLIC HEALTH RELEVANCE: The growth of cysts that destroy the kidney in autosomal dominant polycystic kidney disease has been attributed to cells in the kidney that lose expression of polycystin genes. We have found that not only do these cells form cysts, but they provide signals to other cells that still have polycystin gene expression to also form cysts-a finding that has the potential to identify new targets for drug therapy for a disease which currently has none.

DESCRIPTION (provided by applicant): The functional connection between human polycystic kidney diseases and cilia is based in part on the overlap between genes whose mutation results in kidney cyst formation and genes whose protein products are expressed in the cilia-basal body complex. The occurrence of a common phenotype, kidney cysts, following mutation in either of these classes of genes has led to a conceptual equation of cellular pathways affected by loss of polycystin-1 (PC1) or polycystin-2 (PC2) with those affected following loss of structurally intact cilia. This proposal is based on our novel observation that cyst formation following inactivation o either Pkd1 or Pkd2 is markedly slowed if structurally intact cilia are concomitantly ablated. This effect is present in adult onset and early developmental mouse models of both Pkd1 and Pkd2 and is independent of the genetic mechanism of cilia ablation. The findings provide genetic evidence for a pathway that is inhibited by the PC1/PC2 complex and that requires intact cilia to produce maximal cyst promoting signals in the absence of PC1/PC2-a Cilia Dependent Cyst Activating (CDCA) pathway. The objective of this proposal are to identify the components of the CDCA pathway. To achieve this objective, we will define the specific determinants of cyst progression whose activity following inactivation of polycystins is modulated by the presence or absence of intact cilia. We will use a highly correlated series of in vivo mouse models that subsume all stages of CDCA to investigate known and novel candidate CDCA pathways. We will complement these directed studies with transcriptomic and proteomic discovery approaches. We have identified integrin signaling as a candidate CDCA activity. We will explore the role of polycystins in integrin signaling in cilia and determine which components of this pathway are active in cilia. We will use gene knockdown in cells and conditional knockout models in mice to determine whether integrin signaling is a component of CDCA and if so, we will target it therapeutically to determine preclinical efficacy in ADPKD. The overall program offers two novel discoveries regarding the pathogenesis of ADPKD that each have substantial potential for translation.

PROJECT SUMMARY (See instructions): The past decade has seen quantum advances in the applications of technology to biomedical sciences. Among the more remarkable achievements has been the advent of whole genome sequencing allowing complete knowledge of the genomic DNA structure of innunierable organisms and the associated molecular biologic advances enabling comprehensive manipulation of these newly discovered genomic sequences. With this has come the ability to elevate scientific investigation of kidney disease and function to refined in vivo systems in the mammalian kidney. The validation of basic scientific discoveries in kidney disease now rests with studies in genetically engineered mouse models and naturally occurring human samples. The overarching objective of the Mouse Genetics and Cell Line Core in the Yale George M. O'Brien Kidney Center is to reduce barriers and facilitate application of in vivo mouse-based technologies to the study of kidney disease and to facilitate the extension of the studies to ex vivo cell-based systems derived from engineered mutant mice. The specific aims of the Core are to perform modification of genes of interest in bacterial artificial chromosome (BAC) for use in transgenic mice; to isolate of primary tubule ceNs and conditionally immortalized cell lines from specific nephron segments of mutant mouse strains; to facilitate generation of conditional knockout and knockin gene targeting strategies and constructs and to provide general resources for investigators in mouse genetic applications. This Core will Coordinate with the Renal Physiology and Phenotyping Core to assist investigators couple mouse genetic services with physiological studies. It will coordinate with the Human Genetics and Clinical Research to integrate new human disease gene discoveries with animal and cell line models. There is inherent synergy in the mouse model and cell line components of this Core as it allowing investigators access to the spectrum from defined in vivo models to ex vivo cell line models with identical genetic makeup. The Core now adds a novel mechanism for cell line generation based on technology developed by Core faculty, an improved BAC recombineering methodology and a new service in conditional knockout or knockin allele generation based the BAC technology.

Core B Project Summary/Abstract The past decade has seen quantum advances in the applications of technology to biomedical sciences. Among the more remarkable achievements has been the advent of whole genome sequencing allowing complete knowledge of the genomic DNA structure of innumerable organisms, CRISPR/Cas9 genome editing allowing ready manipulation of these genomes, and the advent of technological advances supporting proteomic analyses of complex biological systems, most notably humans. In order for researchers to achieve major advances in the study of human kidney diseases, it is imperative that we now apply these advances to in vivo mammalian models, in vitro cell systems and directed molecular assessments of human samples. The overarching objective of the Disease Models and Mechanisms Core is to facilitate application of these approaches to benefit and support human kidney disease-based mechanistic studies. In order to achieve this, the Core has developed research expertise in areas specifically targeted to reduce the barriers that hinder users pursuing bedside to bench research (Aims 1, 2 and 3) as well as those who seek to validate bench findings at the bedside (Aims 4 and 5). To support users who are pursuing mechanistic understanding of the recent wealth of human genome sequence information, the first 2 aims provide complementary expertise in manipulating the mouse genome in order to develop orthologous animal models and cell lines that faithfully replicate human disease pathogenesis. The first aim of the Core provides support for using bacterial artificial chromosomes as a means to perform complex modifications of specific genes including insertion of multiple epitope tags, fluorescent tags, point mutations, and deletions, followed by expression in mouse strains. The second aim provides a complimentary approach to genome editing using CRISPR/Cas9 for introducing genetic variants into cell lines and mouse models. The third component of the Core serves to distribute mouse strains and cell lines, for which distribution permission is granted, to users at a minimal cost-recovery price. This includes distribution of the Rosa-DTRfl ?Terminator? mouse for isolation of large quantities of highly purified, non-immortalized renal cell types. The last two components of the Core have been developed to support users who wish to translate bench findings to better understand human kidney disease. In the fourth Aim, users are provided with resources and expertise in performing targeted proteomics of human urine to quantitatively assess >250 urinary proteins. In the final aim, the Core has developed a multiplexed approach to simultaneously analyze the amount and location of up to 42 proteins in human kidney biopsy specimens using Imaging Mass Cytometry. These combined services advance the fields of renal research, enhance the mission of NIDDK and are of value to the broader renal research community.

Abstract Autosomal Dominant Polycystic Kidney Disease (ADPKD) is one of the most common monogenic diseases, affecting >1:1000 individuals worldwide. It is characterized by large fluid-filled renal cysts that remodel, compress and destroy surrounding normal tissue, and that progressively reduce kidney function, leading to end stage renal disease in about 50% of patients by the sixth decade of life. Most ADPKD results from mutations in two genes, PKD1, which encodes the polycystin-1 protein (PC1), and PKD2, which encodes the polycystin-2 protein (PC2). PC1 and PC2 interact with one another and are thought to play a role in cilia signaling. It is generally accepted that the cilium is a central component in the pathways that drive ADPKD pathogenesis. Although their mechanistic connection to the functional PC complex in cilia is unclear, numerous signaling pathways are perturbed in cysts. In the past few years the list of disease-related pathways has grown through new evidence that implicates metabolism as a novel pathway that is profoundly affected in ADPKD and that may both participate in disease pathogenesis and serve as a target for therapeutic development. While it remains to be established whether the newly-identified metabolic derangements that characterize ADPKD are direct drivers of cyst formation, it is clear that the nature and activities of a cell's many and varied intertwined metabolic circuits plays a central role in determining its capacity to invest the energy required in order to participate in the proliferation and active solute and fluid transport that are required for cyst growth. The main goal of this proposal is to provide the research community with novel tools and data sets that will substantially enhance efforts to explore and exploit the metabolic changes that characterize ADPKD. We will produce a uniquely designed and rigorously curated resource based upon novel in vivo models of the cell specific transcriptomic, mitochondrial proteomic and mitochondrial metabolic effects that result from the earliest stages after loss of the PC proteins and that are further informed by the effects of concomitant cilia loss and PC protein reactivation. This program will make use of adult inducible conditional PC knockout mouse models and will employ strategies that will permit conditional isolation of ribosomes (TRAP) and conditional isolation of mitochondria, thus enabling cell-type- specific transcriptomic and mitochondrial proteomic and metabolomic studies. State of the art in vivo metabolic flux studies will be applied to the kidney cortices of these novel genetic mouse models. The results of these analyses will be combined to produce robust biological data sets that will be assembled through application of the requisite informatics mechanisms in order to disseminate these data to the broader research community in near real time. Critically, our in vivo studies are designed to discover the earliest changes that occur after kidney tubules lose polycystin protein expression?at time points well before cysts form. The research team brings together extensive and complementary expertise in ADPKD animal models, PC signaling and biology and in vivo metabolic studies coupled with strong biostatistical and bioinformatics support to produce the proposed resource.

Autosomal dominant polycystic kidney disease (ADPKD) is a common genetic disease that results from mutations in either of two proteins, polcysytin-1 (PC1) or polycystin-2 (PC2). More than two decades have passed since the genes encoding these proteins were discovered and there has been significant progress in understanding the functions of polycystins and their associated disease. Nonetheless, there remain substantial gaps in knowledge and lack of consensus about the precise functions of PC1 or PC2 and the mechanisms of ADPKD. Resolution of these gaps is of great significance given our expectation that optimal therapies for ADPKD are best developed based on the fundamental understanding of polycystin function in the mammalian kidney. Much of the current mechanistic understanding of polycystin function is based on studies of candidate pathways drawn from amongst known cellular mechanisms associated with functions such as differentiation, proliferation, transport and signaling. The lack of coalescence toward an interrelated unifying functional pathway in polycystin biology and the persistence of gaps in understanding of in vivo polycystin function despite extensive investigation suggests that the critical components of the most proximal polycystin signaling cascade have yet to be identified. Indeed, the polycystins were discovered as complex, entirely novel proteins and it stands to reason that they may function in a signaling pathway that is not among those that are currently well understood or studied. We made use of this concept in by applying an unbiased in vivo transcriptomic study using Translating Ribosome Affinity Purification (TRAP) RNASeq. From this, we identified upregulation of cell cycle and down regulation of oxidative phosphorylation as key pathways alterations in vivo. Among these, we found genotype dependent upregulation of a cilia associated transcription factor, Glis2, not previously considered to function in polycystin signaling or ADPKD pathogenesis. We made double mutants of Pkd1 with Glis2 in early onset and adult models and found Glis2 dosage-dependent rescue of cyst formation in both. Based on these findings we propose that Glis2 is a candidate for a downstream effector of PC1 function that is critical for cyst progression in ADPKD. In this study, we will determine the in vivo mechanism of action of Glis2 and establish its effects on cyst cell proliferation, apoptosis and ADPKD due to Pkd2. We will determine whether Glis2 is a target for therapy through both genetic and pharmacotherapeutic studies. We will assess whether in vivo genotype dependent transcriptional changes we have identified are similarly extended to cell culture systems with Pkd mutant genotypes. We will also evaluate the functional properties of Glis2 protein in Pkd mutant cell lines. In aggregate, these studies open a new direction of investigation for polycystin signaling and ADPKD pathogenesis.

Autosomal dominant polycystic kidney disease (ADPKD) results primarily from mutations in PKD1 and PKD2 encoding polcysytin-1 (PC1) and polycystin-2 (PC2), respectively. Since PKD1 and PKD2 were discovered, there has been significant progress in understanding the functions of the polycystins (PCs). Much recent progress has been based on in vivo orthologous gene mouse models which in turn most often rely on controlled inactivation of Pkd1 or Pkd2 using the Cre-loxP system. Our past work has extended beyond Cre-loxP to include modified bacterial artificial chromosome transgenics as well as a model in which second-hit inactivation occurs by a stochastic, Cre recombinase-independent process (Pkd2WS25). While loss of function models offer valuable information, we sought to determine whether ADPKD is actually reversible following Pkd gene reactivation, and if so, at what point in the course of ADPKD is reversal still possible. We developed mouse models that use adult inducible Cre?loxP for the initial Pkd gene inactivation and a separately inducible Flp?FRT system for subsequent reactivation of the same Pkd gene to address these questions. Applying this system to Pkd2, we found that cyst formation is rapidly reversible and that dilated cysts lined by proliferating, squamoid epithelial cells rapidly revert to non-proliferating columnar epithelia with normal appearing nephron lumens, accompanied by markedly decreased total kidney volume and preservation of kidney function. We will now determine the extent to which ADPKD is reversible by extending these studies to Pkd1 inactivation/reactivation and to the Pkd2WS25 mouse which does not require Cre and develops liver cysts as well. We will determine whether there is a minimum fraction of cyst cells that need to be targeted by reactivation to reverse ADPKD and determine the latest disease stage at which ADPKD retains reversibility. We will investigate cellular and molecular alterations operational during resolution of cysts including cell lineage analyses to trace the fates of specific cell types during the repair process, assess alterations in autophagic flux following PC re-expression as a possible modality for the changes in cell shape, and determine whether inflammation and fibrosis reverse with Pkd re-expression. We will attempt to model ADPKD repair in a cell culture-based system. Finally, we will determine the dynamic changes in transcriptionally defined in vivo cell populations during polycystin re-expression and reversal of ADPKD using single cell RNA sequencing (scRNA-seq). This will define the plasticity of the cell populations and the dynamic PC2-expression-dependent changes in transcriptional profiles leading to reversion from the cystic to a more normal nephron state. The reproducible and rapid initial time course of resolution of cysts affords a unique opportunity to monitor transitions within and between cell types in near real time and define on the scale of days the changes that are controlled by PC2 re-expression in the polycystic kidney environment in vivo. All of the findings from this study will be systematically correlated internally to provide an integrated cellular and molecular model for ADPKD repair and to define the capacity and trajectory of kidney repair possible in ADPKD.