Thomas Weimbs, Ph.D. - US grants

DESCRIPTION (provided by applicant): Autosomal dominant polycystic kidney disease (ADPKD) is one of the most common genetic diseases worldwide. Mutations in the PKD1 or PKD2 genes lead to phenotypic changes in affected renal epithelial cells that include increased proliferation and apoptosis, defects in protein trafficking and changes in transepithelial transport functions. This leads to the formation of large renal cysts, the destruction of the normal renal tissue and finally renal failure. Since the functions of the PKD1/2 gene products, polycystin-1 and polycystin-2, are currently unknown, the rational design of strategies for therapeutic intervention has been difficult. This research proposal seeks to close the gap in our knowledge on what lies between PKD1 gene mutations and the epithelial phenotype that finally leads to cyst formation. We have developed an in vitro cell culture system in the polarized renal epithelial cell line MDCK for the tetracycline-regulated expression of polycystin-1 domains that are proposed to act as dominant-negative inhibitors of the endogenous protein. Preliminary results show that this leads to the induction of several of the typical phenotypes of authentic cystic epithelial cells in ADPKD such as increased proliferation, apoptosis and apical mistargeting of the EGF receptor. The molecular mechanisms that lie downstream of the polycystin-1 defect and are responsible for these phenotypes will be identified and their relationship to each other defined. Proteins that interact with the dominant-negative polycystin-1 domains to mediate these mechanisms will be identified. Of particular importance is the investigation of defects in intracellular membrane trafficking that have been proposed to be responsible for several of the final phenotypes of cystic epithelial cells. Preliminary results from this laboratory suggest that specific changes in the SNARE membrane fusion machinery are involved in membrane trafficking defects ADPKD. The role of these SNARE changes in the acquisition of the cystic epithelial phenotype, and how they are caused by polycystin-1 disruption will be investigated. These studies will help to define the normal function of polycystin-1 and the downstream effects that result from its disruption.

[unreadable] DESCRIPTION (provided by applicant): The majority of human cell types are polarized, i.e. they exhibit asymmetry, which is essential to their function. This includes epithelial cells that form barriers between the outside world and the underlying basement membrane and connective tissue, and make up most major organs. Establishment and maintenance of epithelialcell polarity depends on the precise targeting of proteins to the apical and basolateral plasma membrane domains usingvesicular transport pathways. Understanding the mechanismsthat underlie polarized trafficking is of fundamental importanceto understand functionand dysfunction of polarized cells. [unreadable] [unreadable] Vesicle transport pathways depend on the SNARE machinery for the final membrane fusion step. Syntaxins are the most central SNARE component as they directly interact with almost all other components. We have found that syntaxins 3 and 4 are specifically localized at the apical and basolateral domain, respectively, where they govern the fusion of incoming transport vesicles. [unreadable] [unreadable] The central hypothesis of this proposal is that syntaxins define the sites of vesicle exocytosis and that the apical and basolateral separation of syntaxins 3 and 4 is necessary for epithelial cell polarity. To test this, the targeting signals of syntaxins 3/4 will be identified, and the pathways that they follow en route to the apical or basolateral plasma membrane will be defined. Syntaxins 3/4 will be re-localized by mutagenesis of their targeting signals and by generation of chimeric molecules, and the consequences on specific membrane trafficking pathways and on cell polarity will be measured. The fusion sites of post-Golgi transport carriers containing GFP-tagged apical and basolateral markers will be monitored and we will ask whether they specifically co-localize with pre-assembled syntaxin 3/4 clusters. [unreadable] [unreadable] Collectively, this project is designed to provide answers to the following fundamental questions. (1) How are syntaxins 3/4 targeted in polarized MDCK epithelial cells? (2) Is the mutually exclusive localization of syntaxins 3/4 required for polarized cargo transport in epithelial cells and hence for the formation/maintenance of cell polarity? (3) Is the fidelity of polarized trafficking encoded in the specificity of v/t-SNARE interactions? (4) Do stable syntaxin clusters exist on the plasma membrane, and are they the sites of carrier fusion? (5) Do syntaxin 3/4 clusters specifically fuse only 'their' cargo carriers? (6) Is the localization of a syntaxin alone enough to establish a fusion site and create a surface domain?

[unreadable] DESCRIPTION (provided by applicant): The survival rates for patients diagnosed with renal cell carcinoma (RCC) -the most common type of kidney cancer- are extremely low. A major reason is that early-stage RCC is usually asymptomatic leading to a high frequency of patients that present already with metastatic disease. No clinically relevant marker is available that would allow the detection of early-stage RCC. Ideally, a screening assay for RCC should be non-invasive, and should be possible to be developed into a cost-effective, high-throughput assay. An RCC-specific urinary marker may fulfill these criteria. Our preliminary results indicate that urine from RCC patients contains increased levels of matrix metalloproteinase (MMP) activity which causes the degradation of normally excreted extracellular matrix (ECM) proteins. In a preliminary analysis, the detection of the absence of urinary ECM proteins has allowed the detection of RCC with a sensitivity of 95% (21/22) and specificity of 95% (21/22). Importantly, all early-stage cases were correctly identified. The over-all goal of this project is to develop a rapid screening assay based on the detection of urinary MMPs or MMP activity, and to test its clinical usefulness for the detection of RCC. First, the excreted MMP(s) will be identified using specific antibodies or by affinitychromatography and sequencing. Second, a micro-titer plate screening assay will be developed based on the degradation of fluorogenic substrates and/or on the immunological detection of MMPs. Third, using larger RCC patient and control populations, the sensitivity and specificity of the developed screening assay(s) will be determined. [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): Autosomal-dominant (ADPKD) is the most common life-threatening genetic disease. Tuberous Sclerosis Complex (TSC) is 10-times less common than ADPKD. Both diseases are characterized by renal cysts. No treatment is currently available to slow the onset or progression of either disease. We have recently found that polycystin-1 (PC1), the protein affected in ADPKD, interacts with tuberin, the protein affected in TSC, and regulates the activity of the kinase mTOR. mTOR plays an important role in cell growth and proliferation and is known to be inhibited by tuberin. We propose a model in which the C-terminal cytoplasmic tail of PC1 functions to induce the formation of a complex between tuberin and mTOR and other regulatory proteins. The function of this complex is to suppress mTOR activity in normal renal epithelial cells. Our results suggest that mTOR inhibition is regulated by apical fluid flow. We found that mTOR is inappropriately active in the kidneys of ADPKD patients and in mice with polycystic kidneys. Treatment of polycystic kidney mice with the mTOR inhibitor rapamycin results in the dramatic reduction of renal and cyst sizes and in the preservation of kidney function. This strongly suggests that aberrant mTOR activation in ADPKD is critical for renal cyst growth. We now propose to study in detail the mechanism of the suppression of mTOR activity by PC1. In Aim 1, we will investigate the interaction between PC1 and mTOR and identify additional regulatory factors and the subcellular localization of the complex. We will test whether this complex is disrupted in renal cysts. In Aim 2, we will test how fluid flow regulates mTOR activity. In Aim 3, using samples from ADPKD kidneys and a mouse model with a conditionally inactivated PKD1 gene we will characterize which of the known downstream effectors and upstream regulators of mTOR are affected in the disease. In Aim 4, in preparation for possible future clinical trials, we will investigate the effects of rapamycin on the PKD1 mouse model. We will assess dosage and treatment regimens and investigate the effects on proliferation and apoptosis of cystic epithelial cells. These investigations will be important to guide possible future clinical trials. Rapamycin is already a clinically approved drug, used for long-term treatment for immunosuppression in renal transplant patients. Based on our data, we are cautiously optimistic that rapamycin has promise to become the first available treatment to slow the disease progression in ADPKD. Furthermore, results from this research will enhance our understanding of the mechanisms underlying the renal involvement in TSC. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Abstract Autosomal-dominant (ADPKD) is the most common life-threatening genetic disease. We have recently found that polycystin-1 (PC1), the protein affected in ADPKD, functions in the regulation of STAT6 activity by ciliary mechanotransduction in renal epithelial cells. PC1 undergoes flow-dependent proteolytic cleavage which releases its cytoplasmic tail from the membrane, followed by nuclear translocation. The PC1 tail binds to STAT6 and the transcriptional co-activator P100, and stimulates STAT6-dependent gene expression. STAT6 translocates from primary cilia to nuclei upon cessation of fluid flow. The nuclear PC1 tail is highly expressed in cyst-lining epithelial cells in ADPKD. Expression of the PC1 tail stimulates proliferation in MDCK cells and results in renal cyst-formation in zebrafish embryos. Furthermore, MDCK cells respond to interleukin-4 (IL4) and IL13 similar to immune cells in that they activate STAT6, and up-regulate the expression if IL4/13 receptor chains. These results strongly suggest that PC1 functions to silence STAT6 activity in the normal healthy kidney. Cessation of lumenal fluid flow, e.g. due to renal injury, triggers PC1 cleavage, STAT6 activation and a proliferative response. We hypothesize that lack of functional PC1 in ADPKD leads to constitutive STAT6 activity and an aberrant proliferative "repair" response leading to cyst growth. Our preliminary results show that a clinically approved drug, known to inhibit STAT6, strongly inhibits renal growth and preserves renal function in a polycystic mouse model. We now propose to study in detail the mechanism of the regulation of STAT6 activity by PC1 and to test our hypotheses regarding the role of this novel signaling pathway in renal injury repair and ADPKD. In Aim 1, we will characterize the regulation of STAT6 in MDCK cells expressing a STAT6-responsive GFP reporter. The effects of the state of differentiation, PC1 tail expression, apical fluid flow and IL4/13 will be investigated. We will test whether STAT6 is activated in a scratch-wounding model. We will investigate the expression and localization/secretion of IL4/13 and IL4/13 receptor chains in response to STAT6 activation. Analysis of ADPKD tissue and polycystic mouse models will reveal whether IL4/13 receptors are up-regulated in cysts and whether they secrete IL4/13 into the lumen. Finally, we will test our hypothesis that the IL4/13/STAT6/PC1 pathway is activated in a mouse model of renal ischemia/reperfusion injury. In Aim 2, we will test whether crossing of STAT6 null mice with polycystic mouse models will result in suppression of renal cystic disease. Furthermore, we will investigate the amelioration of renal cystic disease by treatment with the STAT6 inhibitor and delineate the mechanism of action. In Aim 3, we will generate a transgenic mouse line over-expressing the soluble PC1 tail in a kidney-specific and doxycycline-inducible manner. Based on our results in MDCK cells and zebrafish, we anticipate that these animals will develop renal cystic disease and will mimic human ADPKD most closely mechanistically. Project Narrative ADPKD is the most common life-threatening genetic disease and affects over 600,000 patients in the US. Due to the need for kidney transplantation or life-long dialysis in most patients, the personal burden on patients and the burden on the health care system are enormous. Currently, there is no available treatment to prevent or slow the disease onset. Our work has led to the identification of two signaling mechanisms that are aberrantly regulated in ADPKD and involve the kinase mTOR and the transcription factor STAT6, respectively. We could validate both of these pathways as feasible drug targets. Based on animal experiments, two clinically approved drugs (the mTOR inhibitor rapamycin and the STAT6 inhibitor leflunomide) appear to be highly promising. In the case of mTOR inhibitors, this work has already led to at least three clinical trials that are in early stages. The present proposal is aimed at understanding the molecular mechanisms underlying one of the two pathways (STAT6) that we have discovered and to further test the feasibility of targeting this pathway with an existing, already FDA-approved drug. Therefore, this proposal is designed to yield new mechanistic insights and also to provide information that will be directly relevant for the guidance of future clinical trials. Leflunomide is currently used for long-term treatment of rheumatoid arthritis. Our results using animal models make us cautiously optimistic that leflunomide - or related compounds - have promise to become the first available treatment to slow the disease progression in ADPKD. Even a delay of the onset of end-stage renal disease by a number of years would be a tremendous advance and would dramatically improve the quality of life of a large number of patients. In addition to the immediate clinical relevance, our proposed work will also shed light on our unexpected discovery that renal tubular epithelial cells appear to possess a STAT6-dependent signaling pathway similar to the one previously described in immune cells. Compared to the wealth of knowledge in the immune system, little is known about the function of this pathway in renal tubular epithelial cells, or anywhere else outside of the immune system, despite the fact that STAT6 is widely expressed. We hypothesize that this signaling pathway regulates epithelial, proliferative repair responses after renal injury. If correct, then this would impact on most forms of acute and chronic renal damage.

DESCRIPTION (provided by applicant): SNARE proteins mediate membrane fusion events in virtually all cellular membrane trafficking pathways. We have discovered an unexpected, novel function of the SNARE protein syntaxin 3 (Stx3). Stx3 normally has a C-terminal trans-membrane anchor and is involved in trafficking to the apical plasma membrane domain of polarized epithelial cells. We found that Stx3 undergoes cleavage at an extremely conserved glutamine residue which removes its trans-membrane domain resulting in a soluble fragment, Stx3(1-225). Furthermore, a novel splice-isoform of Stx3 (Stx3E) lacks the trans-membrane anchor, and is expressed in human kidneys. Both, the cleavage fragment and Stx3E (collectively called soluble Stx3) bind to the nuclear import factor RanBP5, target to the nucleus and co-activate several transcription factors including ETV4. ETV4 is required for branching morphogenesis in kidney development, and associated with carcinogenesis and tumor metastasis. We found that kidneys from Autosomal Dominant Polycystic Kidney Disease (ADPKD) patients express a small Stx3 fragment - consistent with soluble Stx3 ! We hypothesize that cleavage and transcriptional regulation in the nucleus is a novel function that may be a common feature of syntaxin members of SNARE proteins. This may be a novel signaling mechanism that transduces information from cytoplasmic membrane trafficking events to the nucleus to affect changes in gene expression. If correct, this would introduce a new paradigm of SNARE function. More specifically, we hypothesize that soluble Stx3 plays a role in the regulation of renal epithelial morphogenesis, carcinogenesis and ADPKD. ! To test these hypotheses, we will pursue the following Specific Aims. In Aim 1, we will investigate the biological effects of soluble Stx3 in polarized epithelial cells. This will be done by expressing soluble Stx3 in epithelial cell lines - or knocking down Stx3E expression - and investigating possible effects on cellular parameters including morphology, proliferation, apoptosis and cell polarity. In Aim 2, we will identify the exact cleavage site of Stx3 in vitro and in vivo. We will investigate the expression of soluble Stx3 in epithelial cancers and ADPKD by using human tissue specimens and mouse models of ADPKD. In Aim 3, we will investigate the mechanism of transcriptional activity of soluble Stx3 and its regulation of ETV4. We will test whether soluble Stx3 interacts with the general transcription machinery and whether it regulates ETV4 stability or nuclear localization.

Project Summary/Abstract Autosomal-dominant polycystic kidney disease (ADPKD) is a very common, inherited disease affecting the world's population with a frequency of approximately 1:500. No approved treatment to slow or halt disease progression is currently available in the US. The disease progresses slowly to renal failure, typically in the 4-6th decades of life. However, for unknown reasons the rate of progression greatly varies from patient to patient even within the same family suggesting that environmental factors may affect disease progression. Recent results from animal studies suggest that renal insults are required - in addition to the gene mutation - for renal cysts to arise. However, rare forms of renal injury are unlikely to account for the constant pace of disease progression in humans. Our results suggest that a much more prevalent form of sub-clinical renal insult is the trigger of renal cyst formation that determines the rate of progression in ADPKD: microcrystals that are sporadically lodged in renal tubule lumens. We show that deposition of calcium oxalate (CaOx) crystals in renal tubules leads to rapid activation of the mTOR and Src/STAT3 signaling pathways, both of which are also strongly activated in ADPKD. In addition, CaOx crystal deposition leads to rapid tubule diameter widening that can be blocked by mTOR inhibition. Our results suggest that tubule dilation is a purposeful, and previously unrecognized, protective mechanism that facilitates crystal excretion. After crystal clearance, tubule diameters return to normal within a week. However, in mice lacking PC1 - the protein affected in ADPKD - CaOx challenge leads to persistent tubule dilation that ?overshoots? to cystic progression. This suggests that PC1 is required to re- establish normal tubule diameters after insults. We hypothesize (1) that tubule dilation is an innate renal protective mechanism against tubular crystals; and (2) that this mechanism inadvertently acts as a trigger for tubule dilation leading to cyst formation in ADPKD. If correct - these ?ndings immediately open a new and highly feasible avenue for therapeutic intervention because well-established treatments for recurring nephrolithiasis (dietary changes, increased water intake, citrate) should also be effective in slowing the progression of ADPKD. Using mouse and rat models of CaOx nephrolithiasis we will investigate tubule dilation and signaling pathway activation in response to crystal deposition and test whether citrate treatment prevents these effects (Aim 1). Using pharmacological inhibitors and genetic mouse models we will determine if tubule dilation is required for effective crystal clearance (Aim 2). Using conditional knockout mice for the ablation of cilia or PC1, respectively, we will determine if tubular crystal deposition acts as a trigger for cystogenesis (Aim 3). Using a mosaic PC1-KO mouse model and a rat model of PKD we will determine if crystal burden modulates disease severity in PKD.

Project Summary/Abstract We have previously shown that a mild reduction in food intake strongly inhibits progression of polycystic kidney disease (PKD) in an orthologous mouse model but we did not understand the mechanism. Now, we discovered that the metabolic state of ketosis is important, not caloric restriction per se. Dietary interventions leading to ketosis profoundly inhibit - and even reverse - PKD progression in orthologous and non-ortholgous mouse, rat and feline models of PKD. Remarkably, treatment with the ketone ?-hydroxybutyrate (BHB) alone is almost 100% effective in preventing PKD progression. Preliminary results suggest that BHB acts on PKD kidneys via its receptor GPR109a, a GPCR that suppresses cAMP signaling. Our results suggest that cyst cells in PKD are metabolically in?exible, depend on glucose and are unable to shift to utilizing fatty acids and ketone bodies. The main thrust of this proposal is to generate compelling results to justify clinical trials to investigate the ef?cacy of dietary interventions and/or BHB supplementation in ADPKD, and to inform the design of such trials. The main signi?cance of this proposal is the enormous potential for clinical translation. Dietary interventions to induce ketosis are well-established. Because dietary interventions frequently fail in clinical practice due to poor adherence, our ?nding that BHB (an FDA-classi?ed dietary supplement) has a dominant bene?cial effect could rapidly lead to a highly feasible therapy. To achieve our goals, we will treat rodent and feline models of PKD, with dietary interventions to induce ketosis (time-restricted feeding or ketogenic diets) or mimic ketosis by supplementation with BHB. Ef?cacy on parameters of PKD progression and effects on molecular mechanisms will be assessed. To determine whether BHB acts via GPR109a, we have crossed Gpr109a-/- mice with fast- and slowly-progressing Pkd1 mouse models. We will test whether Gpr109a knock-out affects disease progression and prevents the ef?cacy of ketogenic dietary intervention or BHB. Successful completion of the proposed work could lead to a disruptive change in ADPKD therapy by utilizing dietary interventions and/or dietary supplements without the need for pharmacological intervention.