Hepato/renal fibrocystic disease (HRFD) is a cohort of genetic mutations that lead to a number of different manifestations the most common of which is the formation of cystic structures in kidney and liver. The most common forms of HRFD are autosomal dominant (AD) and autosomal recessive (AR) polycystic kidney disease (PKD) in which there are mutations in Pkd1 or Pkd2 (ADPKD) or in Pkhd1 (ARPKD). There are a number of other forms of PKD including Meckel Syndrome (MKS), Nephronophthisis (NPHP), Joubert Syndrome (JBTS), Bardet-Biedl Syndrome (BBS). The finding that protein products of these genes reside, at least in part, in cilia has led to the concept of HRFD as a ciliopathic disorder. There has been a concerted effort to understand the mechanisms involved in cystogenesis. Although a number of potential signaling mechanisms have been implicated in cyst formation, which signaling pathways are crucial to cystic development has remained elusive. This has hampered development of effective therapeutic agents.

To understand HRFD and to test the effects of therapeutic compounds requires the ability to assess physiological readouts in both in vivo and in vitro systems. Core C has developed a unique resource that utilizes an integrated physiology-proteomics-genomics approach to mechanisms that lead to HRFD. This approach now includes the use of RNAseq and proteomics including expert consultation on data analysis and interpretation as powerful tools to understand HRFD.

Specific Aims 

Aim 1: To characterize mouse models of HRFD in vivo. The development of inducible Cre/lox systems has provided the means of knocking out HRFD genes in the adult animal. It is therefore possible to determine the initiation and assess the progression of HRFD disease and to evaluate the efficacy of therapeutic targets identified through efforts of Core D. Multiple HRFD mouse models from Core B and from the investigator base will be evaluated for physiological changes by Core C using a battery of innovative techniques generally not readily available in individual laboratories. Ambulatory blood pressure will be measured with telemetry sequential measurements of GFR will be performed by transcutaneous using FITC-sinistrin, cystic progression will be monitored over time by MRI, and renal damage/disease progression sequentially evaluated by proteomics of relevant urinary biomarkers. These studies will provide valuable information needed for assessing the therapeutic potential of lead compounds identified in Core D. Other services will include tissue processing, histology, immunofluorescence, immunocytochemistry, and gene array analysis. Acute studies can also be performed using multiphoton confocal imaging to assess cilia tagged (13) or biosensor (e.g. GCaMP Ca2+ and others, Core B) mice. For instance, it will be possible to test the acute effects of drugs or agents identified in Core D on cilia or second messenger systems in vivo.

Aim 2: To establish mTERT and hTERT immortalized cell lines. To directly study altered cell function/signaling in HRFD requires the generation of cell lines from various HRFD mouse models as well as cell lines derived from humans with HFRD. We have previously produced renal epithelial lines with Telomerase Reverse Transcriptase (TERT) immortalization in mice using mTERT and we are in the process of making cell lines from patients with ARPKD and ADPKD using hTERT. We have also been successful in isolating cholangiocytes. Primary cell lines are grown from kidney, immortalized using TERT technologies, and then FAC sorted with ligands to obtain pure renal epithelial cells of specific origin, such as collecting duct and proximal tubule. Cell lines will also be established from the recent cilia tagged and biosensor mice. For human cell lines, genetic analysis will be performed to identify the specific gene mutation through Core A that has led to HRFD in this patient. Core C will serve as a central repository for all cells lines made by the Center and/by the investigator base. Core C will distribute these lines upon request.

Aim 3: Physiological and molecular characterization of HFRD cell lines. Targeted approaches will be used to study immortalized cell lines and to examine in detail the effects of drugs developed in Core D. Using flow through or closed loop systems that are in an environmental chamber that is mounted to a confocal microscope, cells can be exposed to various levels of flow over periods of time and cell signaling pathways and cell proliferation will be assessed using biosensor cells, fluorescence probes, and then subject to time-point specific gene array, proteomic analysis or RNAseq. In addition Core C will utilize 3D gel matrix systems, which allows for the formation and evaluation of cystic growth in vitro. Specific channels can be studied using patch clamp and overall transport can be assessed using tubular perfusion in combination with fluorescence techniques. Core C will provide the investigative base with powerful new tools to understand the mechanisms that cause HRFD and to test the efficacy of novel new therapeutic compounds.