Targeting the periostin-integrin signaling pathway in PKD

Autosomal dominant polycystic kidney disease (ADPKD) is one of the most common inherited disorders affecting nearly 12 million people worldwide. ADPKD is characterized by relentless growth of numerous fluid-filled cysts causing kidney injury and fibrosis (scar tissue), leading to the progressive loss of kidney function. We discovered that kidneys of human ADPKD and autosomal recessive PKD (ARPKD), and PKD animal models have high expression of periostin, a molecule involved in tissue maintenance and repair and is highly expressed in fibrotic tissues. Periostin binds alpha(V)beta(3) [aVß3]-integrins (cell surface receptors that are linked to the cell’s cytoskeleton), leading to the activation of signaling pathways involved in repair, cell proliferation and survival. Periostin promotes the proliferation of human ADPKD cells, but not normal human kidney cells. This difference in the proliferative response is due to overexpression of aVß3-integrins by the cystic cells. Gene knockout of periostin in PKD mice significantly reduced renal cyst growth and fibrosis and extended the lifespan of the mice. Conversely, overexpression of periostin in the kidneys of PKD mice caused more rapid growth of cysts, fibrosis, and decline in kidney function. In preliminary studies, we found that an aVß3-integrin blocking antibody prevented periostin-induced proliferation of ADPKD cells. We propose that persistent overexpression of periostin and its receptor aVß3-integrin cause hyperactivation of aVß3-integrin signaling pathway causing misguided tissue repair that contributes to cyst growth and fibrosis, and that inhibition of the aVß3-integrin signaling pathway could be a therapeutic approach to slow the progression of ADPKD.

Darren Wallace is a Professor in the Department of Internal Medicine and a member of the Jared Grantham Kidney Institute at the University of Kansas Medical Center. Dr. Wallace earned his PhD from the Department of Molecular and Integrative Physiology and completed postdoctoral training in renal physiology and polycystic kidney disease at the University of Kansas Medical Center. His contributions to the PKD field include research on the molecular mechanisms involved in cAMP-dependent cell proliferation and Cl-dependent fluid secretion using primary cultures of human ADPKD cyst epithelial cells. Recently, his laboratory discovered that periostin, a matricellular protein, is highly overexpressed by cysts of ADPKD and ARPKD kidneys and it accumulates within the extracellular matrix adjacent to the cysts. Periostin binds to integrins on the cystic epithelial cells and stimulates repair mechanisms, including cell proliferation and matrix production, contributing to progressive cyst growth and fibrosis. Dr. Wallace is the Associate Director of the Kansas PKD Research and Translational Core Center and Director of PKD Biomarkers and Biomaterials Core, which is part of the national PKD Research Resource Consortium (PKD-RRC). He also served as a member of the PKD Foundation Scientific Advisory Committee from 2008 to 2019.

Intracranial aneurysms and vascular abnormalities in ARPKD

Unlike the dominant form of polycystic kidney disease (ADPKD), autosomal recessive polycystic kidney disease (ARPKD) has historically not been thought to cause an increased risk of intracranial aneurysms (ICA; outpouchings of arteries in the brain) or other problems with blood vessels (vascular abnormalities). However, there have now been six reported cases of ICA in children and young adults with ARPKD, and another two individuals reported with aneurysms in other parts of the body. In some cases, these aneurysms caused significant complications such as bleeding in the brain or even death. Despite the potentially devastating effects of ICAs and other blood vessel problems, we do not know how commonly they occur in individuals with ARPKD and whether they can be prevented, because to our knowledge there have been no prior systematic studies to investigate this issue. Our overall objective is to determine how common ICA and blood vessel abnormalities are in individuals with ARPKD, and to study potential risk factors for these problems, such as high blood pressure, abnormal function of the cells lining the blood vessels (endothelial dysfunction), and increased stiffness of the blood vessels. This study will yield important information to guide the care of individuals with ARPKD by helping to inform whether screening for ICA and other blood vessel problems is indicated, and by identifying potentially treatable risk factors to prevent vascular complications.

Erum Hartung, MD, MTR is a pediatric nephrologist at Children’s Hospital of Philadelphia and an Assistant Professor of Pediatrics at the University of Pennsylvania. Her clinical and research focus is in polycystic kidney disease, particularly autosomal recessive polycystic kidney disease (ARPKD). She co-directs the Combined Kidney/Liver Program at CHOP, which specializes in the care of children with ARPKD and other genetic kidney/liver diseases and ciliopathies. Her research aims to accelerate the development of new treatments for ARPKD through observational and database studies to better define the natural history and complications of ARPKD, and through imaging studies to develop new biomarkers of kidney and liver disease progression.

Caloric restriction in PKD: Mechanisms mediating efficacy and impact on immune cell function

The Autosomal Dominant Polycystic Kidney Disease (ADPKD) patient community is still in dire need of new treatment options despite the FDA approval of JYNARQUE (tolvaptan), which merely delays cyst growth, impacts quality of life, and only aids a select group of patients (rapid progressors). Changes in kidney metabolism are a characteristic of ADPKD and preclinical studies targeting these abnormalities have shown efficacy in alleviating disease severity. Correlatively, body mass index (BMI) has been shown to be an important predictor of ADPKD progression. Hence, there is rising interest in a variety of caloric intake regimes as a potential therapy for ADPKD. Preclinical studies in mouse models have shown a profound reduction in cyst growth with daily reduction in food intake, i.e. caloric restriction (CR). While daily CR may have health benefits, beyond impacting kidney cyst growth, in overweight/obese ADPKD patients, it is not a viable therapeutic option for ADPKD patients with normal BMI as it provokes health risks such as bone loss, decreased immune function, or anemia, to name a few. Besides, it is also very challenging to adhere to a life-long reduction in food intake. Alternate regimes such as intermittent fasting (IMF, alternate-day CR) or time restricted feeding (TRF, restricting the number of hours for food intake) are being considered for a variety of diseases including ADPKD.

We compared CR, IMF, and TRF using a clinically relevant ADPKD mouse model and found that neither IMF nor TRF were as efficacious in halting cyst growth as CR. Indeed, our results show a remarkable efficacy of CR in slowing PKD progression, even more so than tolvaptan. Hence, it is critical to understand the cellular and molecular mediators of the CR response in order to identify pharmaceutical-based alternatives to CR, which would eliminate the need for life-long food restriction and health risks associated with continued weight loss. In this study, we will use a clinically relevant mouse model to understand detailed mechanisms of the CR response in ADPKD kidneys in a cell type specific manner. We will use sophisticated methodologies such as single cell RNA sequencing and metabolomics as well as bioinformatic approaches of data integration to build a catalog of changes in cell composition, function, and communication that are modulated by CR. In additional, we will focus on the role of immune cells in the CR response. The reason for this focus is twofold, one, recent studies by others and us show that immune cells are key modulators of PKD severity, and two, in cancer, a disease with many parallels to PKD, CR improves disease and therapy outcome in part by modulating immune cell function. These studies will facilitate identification of novel drug targets for (pre-) clinical testing, which would not be feasible in humans due to limited kidney tissue availability. This study addresses the PKD Foundation special consideration areas of “lifestyle interventions” and “PKD drug discovery”.

Microbiota-Derived Bile Acids and Short Chain Fatty Acids as Markers of ADPKD

ADPKD is the most common hereditary kidney disease worldwide, characterized by bilateral kidney enlargement with numerous cysts and a variable rate of disease progression. Intrafamilial renal disease variability is a well-documented feature of ADPKD, making risk prediction at the level of the individual patient challenging, even among affected relatives. Modifying genes, epigenetic mechanisms and environmental factors, such as the here proposed gut microbiome, considerably influence the clinical course of the disease.

Despite tolvaptan’s approval, there is still critical need for identification of biomarkers and therapeutic targets for treatment of ADPKD. Liver is the most affected extrarenal organ in ADPKD and unfortunately tolvaptan has been shown to induce serious and potentially fatal liver injury. Gut microbiome and related gut-kidney-liver axis has been recognized as a main regulator of innate and adaptive immune systems. Despite the importance of gut microbiome in regulating inflammation and cellular injury, no studies have yet systematically evaluated microbiota-derived metabolites in ADPKD patients.

This proposal will be the first to validate bile acids (BAs) and short-chain fatty acids (SCFAs) as biomarkers of ADPKD severity and progression as measured by the change in total kidney volume and renal function. For this purpose, we will use samples already collected during HALT-PKD trials. BAs and SCFAs are chemically stable at low temperatures and HALT-PKD database contains information on considerable renal and cardiovascular clinical measures.
In the second part of this proposal, we will assess the potential of a simple resistant starch-based dietary intervention to slow down the progression of PKD in a PKD1RC/RC mouse model.

Our proposal will address the existing gap in knowledge about the role of microbiome metabolites in ADPKD. Collectively, our preliminary data suggest that ADPKD induces an imbalance in the hydrophobic BAs (accumulation) and SCFAs (depletion). In this scenario, intervention strategies such as the proposed dietary intervention with SCFA-producing resistant starch should be beneficial to re-establish the lost microbiota balance.

Jelena Klawitter, Ph.D. is an Associate Professor of Anesthesiology and Renal Medicine at the University of Colorado, Anschutz Medical Campus in Aurora. For the past 19 years, she has been investigating the mechanisms of cellular reprogramming first in malignant and later in cardiovascular diseases. In the past 9 years, since joining the PKD research group at UC Denver, Dr. Klawitter has participated in the group’s biomarker development efforts to understand the mechanisms underlying varying progression rates in patients with ADPKD. For Her laboratory utilizes a combination of lipidomic, metabolomic and proteomic approaches to explore the role of oxidative stress and inflammation, endothelial and mitochondrial dysfunction and recently that of the dysregulated microbiome in the development and progression of PKD.

Role of intracellular cholesterol in ADPKD

Autosomal dominant polycystic kidney disease is the most common monogenetic disorder in the United States and has no cure. Though significant progress has been made, we still do not have a complete understanding of the pathogenesis of this disorder. Understanding how kidney cysts develop and grow is necessary to identify new areas for drug development for the treatment of ADPKD. Recently, studies have highlighted the role of metabolism in modulating cyst growth. In particular, the effect of aberrant glycolysis and impaired fatty acid oxidation has been described in ADPKD. Whether other metabolic pathways play a role in cyst growth is not known. Cholesterol is an integral component of the cell membrane, serves as a signaling molecule and requires a significant amount of energy for synthesis. We have discovered that the synthesis of cholesterol may be reduced in the kidneys of mouse models of PKD. Moreover, our preliminary data suggests that inhibiting the synthesis of cholesterol in the kidney promotes cyst growth, whereas promoting cholesterol synthesis slows cyst growth. This leads us to hypothesize that the cholesterol biosynthesis pathway is downregulated in ADPKD and enhancing the synthesis of cholesterol in the kidney may slow cyst growth. To answer this question, we will first determine whether cholesterol biosynthesis is indeed reduced in the kidneys of mouse models of ADPKD. Then we will determine whether modulating cholesterol biosynthesis affects cyst growth in aggressive and long lived mouse models of ADPKD. By performing a comprehensive metabolic and phenotypic analysis, we will establish that cholesterol biosynthesis is a newly discovered modulator of cyst progression. These studies have the potential to add a dramatic new dimension to the understanding of metabolic rewiring in ADPKD and will pave the way for future studies to study the cholesterol biosynthesis pathway in detail to identify novel drug targets for the treatment of ADPKD.

Dr. Ronak Lakhia is an Assistant Professor of Medicine in the Division of Nephrology at the University of Texas Southwestern Medical Center in Dallas. She completed medical school at the University of Texas Southwestern Medical Center in Dallas and internal medicine residency at Baylor College of Medicine in Houston, TX. She then returned to UT Southwestern to pursue nephrology clinical and research fellowship at UT Southwestern and was appointed to the faculty at the completion of her training. Her research program focuses on understanding the role of metabolic and epigenetic aberrancies in the pathogenesis of ADPKD. In addition, Dr. Lakhia leads the PKD Clinic at UT Southwestern.

In vitro modelling of autosomal recessive polycystic kidney disease

Autosomal recessive polycystic kidney disease (ARPKD), caused by mutations in the PKHD1 gene, is a devastating kidney and liver disease affecting babies and children. Most babies are born unable to produce any urine which also affects lung development. These babies require prolonged intensive care, breathing support and dialysis from birth. They often remain in hospital for the first 4-6 months of life and remain on dialysis until kidney transplantation. Milder cases can present later but ultimately require dialysis and transplantation in childhood or young adulthood. Furthermore, 10-20% of patients will also require liver transplantation in childhood. Little is understood about how PKHD1 functions within the kidney and how PKHD1 mutation leads to cysts. Consequently, there are no established treatments for ARPKD.

Researchers often study genetic diseases in animals as a surrogate model for human disease. However, animals with PKHD1 mutations don’t develop kidney cysts like human ARPKD patients. PKHD1 therefore has unique functions in humans and needs to be studied using human kidney cells. Obtaining kidney cells from paediatric ARPKD patients by kidney biopsy is impractical and unethical.
Our laboratory is one of few in the world generating stem cells from patients with kidney disease and turning them into 3D mini-kidneys in a dish (called organoids). We have developed a new method to grow collecting duct (CD) cells, which are the cells that develop cysts in ARPKD. When we grow CD organoids from stem cells carrying PKHD1 mutations, they form large cysts. This represents an opportunity to study ARPKD in a human model without having to biopsy a human kidney.

In this proposal we will compare healthy and ARPKD-patient kidney organoids to better understand how defects in PKHD1 lead to cyst formation. In the short term, this will help us to understand the function of PKHD1 and possibly also allow the testing of treatments to reduce cyst growth. To move towards drug screening, we will miniaturise our cultures using a robotic cell handling and imaging platform. This will allow us to create almost 400 kidney models on one plate the size of a cell phone. Showing we can test drugs in this way will provide the foundation for future work screening potentially thousands of potential therapies to see which works best at reducing cyst growth in the ARPKD organoids. As such, this may lead to the development of the first treatments for ARPKD. The long term hope is to be able to grow an individual patient’s kidney cells within this system and find the best treatment for their particular PKHD1 mutation. This type of ‘personalised therapeutics’ would be a world first. In the long term this approach may also be applied to other diseases of the collecting duct, including ADPKD.

Urine extracellular vesicle miRNAs as prognostic biomarkers and to identify new therapeutic targets in ADPKD

The course of ADPKD can vary considerably between individuals even within the same family and there is a clinical need to identify those with more rapidly progressive disease who would benefit from early treatment (eg tolvaptan) or more intensive management. Extracellular vesicles are tiny microscopic particles that actively exported by most cells in the body including the kidney. They may be a common way through which cells communicate with each other since they contain both protein and genetic messengers including small ribonucleic acid (RNA) molecules. By isolating these particles in urine, we could potentially ‘sample’ the kidney without the need to do a needle biopsy (‘liquid biopsy’). In this study, we will examine whether the small RNA content of urine vesicles could be used to more accurately predict kidney lifespan, decide treatment options and provide clues for developing novel drugs. This is because tolvaptan is associated with rare cases of liver toxicity and is poorly tolerated in some patients. These features indicate that more effective, safer and better tolerated treatments are still needed.

Albert Ong is Professor of Renal Medicine, Head of Academic Nephrology and Deputy Director for Clinical Academic Training at the University of Sheffield, UK. He graduated in medicine from the University of Oxford and completed postgraduate training at University College London and Oxford. His interest in ADPKD began in the laboratory of Dr Peter Harris through a post-doctoral Kidney Research UK Senior Fellowship at the Weatherall Institute of Molecular Medicine, Oxford. Later, he successfully established a new research group and investigational program on ADPKD at Sheffield underpinned by a long-term Research Leave Award from the Wellcome Trust. His research goals are to define the molecular basis of cyst formation, initiate drug discovery and stimulate translational research in ADPKD. Dr Ong also directs the Sheffield Renal Genetics Service which serves to provide expert diagnosis and management for patients with ADPKD and other forms of inherited kidney disease. He is Chief Investigator of the CYSTic consortium and currently serves on the steering committees of the European ADPKD Forum, SONG-PKD initiative, the STAGED-PKD study and as External Faculty for the Mayo Clinic Translational PKD Centre (USA).

Targeting Trem2+ kidney resident macrophages to slow cyst growth

The current idea in the field is that macrophages promote cyst growth. However, the majority of data supporting this idea comes from rapidly progressing mouse models that do not accurately reflect the rate or timing of cyst growth observed in PKD patients, where cyst growth typically begins in teenage years and progresses over several decades. In this study, we address this concern by analyzing macrophages that are present in two slowly progressing models of cystic disease that mimic the timing and rate of cyst growth observed in patients. The macrophages from these models are compared to macrophages from commonly used, rapidly progressing cystic models and non-cystic control mice. Using this approach, we identify a single population of macrophages that are present only in the slowly progressing models of cystic disease but are absent in the rapidly progressing model of cyst growth and control mice, suggesting that these macrophages may restrict cyst growth. The goal of this study is to test the hypothesis that macrophages found only in slowly progressing models of cystic disease slow cyst growth and to test the idea that treatment with a therapeutic antibody, which is currently in clinical trials for other diseases, can be used to further activate these macrophages to further reduce cyst growth.