Student

Prinicipal Investigator

Scientific interest within the context of the graduate college:

In our project, we focus on maladaptive immune responses to prevent multi-morbidity in patients with chronic kidney disease (CKD). Based on our longstanding interest in microbiome-immune interactions in cardiovascular and renal diseases, the planned project involves first proof-of-concept clinical studies side by side with experimental in vitro assays. Our translational project addresses a molecular mechanism that is crucial to maintain health and opens up areas for preventative strategies in line with the Re-Thinking Health program.

Project description:

Introduction: Patients with chronic kidney disease (CKD) have a 500-1000-fold increase in cardiovascular mortality, irrespective of age (Jankowski et al. Circulation 2021). This heightened risk is in part due to inflammatory processes that result from abnormal immune system status (Ridker et al. European Heart Journal 2022). We recently demonstrated that even very young patients with CKD have specific alterations to their gut microbiome and immune system (Holle et al. JASN 2022). The main disease-associated changes we observed were a reduction of gut microbial production of the short-chain fatty acid (SCFA) propionate and a reduction of circulating regulatory T cells (Treg). Furthermore, we have shown with animal model studies and in vitro assays a potent and Treg-dependent positive effect of propionate in cardiovascular disease (Bartolomaeus et al. Circulation 2019). We offer a project based on well-established previous work by us and others which aims to investigate the effects of propionate on Treg abundance and function in a translational study in healthy participants and CKD patients.

Aim 1: Analyzing the effects of propionate on Treg induction and function in vitro.

Aim 2: Demonstrating the effects of oral propionate treatment on Treg abundance and function in healthy individuals.

Aim 3: Investigating the effects of propionate treatment in a double-blind placebo-controlled study in adolescent patients with CKD.

The clinical studies are approved by the local ethics board. To focus on truly CKD-related effects, children with CKD will be enrolled in a multicenter-fashion, as they do not suffer from additional comorbidities, which might affect our results.

References

Student

Prinicipal Investigator

Scientific interest within the context of the graduate college:

The gastrointestinal epithelium is organized into clonal crypts that represent sophisticated anatomical and functional tissue units. The epithelium is intimately associated with the mesenchymal stroma network, and various mesenchymal cell types are essential constituents of the stem cell niche that regulates epithelial homeostasis. The gastrointestinal stem cells give rise to differentiated cells. This process is important to maintain the nutritive absorptive functions of the epithelium as well as to build a barrier against pathogens and toxins from the environment. Recently, it has become increasingly evident that interactions between the epithelium and stroma are vital in regulating the barrier function, allowing tissue adaptations to environmental perturbations^1,2. Our research aims at understanding the interplay between the epithelium, stroma and the microbiota. We would like to understand how tissues respond to microbiota alterations or exposure to pathogenic bacteria as well as their toxins. To address this, we are also developing new organoid and assembloid models to recapitulate the cellular networks observed in vivo.

Project description:

Introduction: The cellular organization of gastrointestinal crypts is regulated by various cells in the surrounding mesenchymal niche, which guide stem cell self-renewal and turnover. Environmental factors such as bacterial virulence, chemicals, and radiation can alter the mesenchymal niche. The interaction between the epithelium and its microenvironment allows the mucosal barrier to react quickly to harmful factors and maintain homeostasis. Exploring how the epithelial and mesenchymal compartments communicate and self-organize could provide new insights into disease prevention and therapy development.

Currently, 3D gastrointestinal organoids are the standard for studying epithelial cell behavior, but the absence of mesenchymal cells in this system limits the analysis of the interplay between the epithelium and connective tissue⁴. Therefore, we aim to establish a novel co-culture system, called assembloids, by combining human colonic epithelial and mesenchymal cells in a single structure, to dissect epithelial-mesenchymal interactions in a tractable manner. Using the assembloid system, we can investigate whether stromal cells promote the formation of true colonic crypts that resemble the in vivo anatomy and cellular organization. Next, we plan to establish a vessel network and incorporate immune cells in assembloids to mimic a functional mucosal barrier. We will also study whether the assembloid model can resemble the mucosal response to environmental factors, helping us understand how health is maintained in the human gastrointestinal tract.

Aim 1: Generate human colon assembloids, characterize the various stromal cells, investigate how the stromal compartment in assembloids guides epithelial crypt maturation.

Aim 2: Optimize the culture conditions required for the formation of vessel networks and incorporation of macrophages in assembloids. Explore the interplay between macrophages and stromal cells.

Aim 3: Expose assembloids to bacteria and their virulence factors, to explore mechanisms that guide the response of mucosa to environmental factors.

References

1. Sigal M, Logan CY, Kapalcynska M, […], Nusse R, Amieva MR, Meyer TF. Stromal R-spondin orchestrates gastric epithelial stem cells and gland homeostasis. Nature. 2017; 548:451-455. doi: 10.1038/nature23642.
2. Kapalczynska M, Lin M, Maertzdorf J, […], Tacke F, Meyer TF, Sigal M. BMP feed-forward loop promotes terminal differentiation in gastric glands and is interrupted by H. pylori-driven inflammation. Nat Commun. 2022; 13:1577. doi: 10.1038/s41467-022-29176-w.
3. Heuberger J, Trimpert J, Vladimirova D, […], Tacke F, Osterrieder N, Sigal M. Epithelial response to IFN-gamma promotes SARS-CoV-2 infection. EMBO Mol Med. 2021; 13:e13191. doi: 10.15252/emmm.202013191.
4. Sato T, Vries RG, Snippert HJ, […], Kujala P, Peters PJ, Clevers H. Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche. Nature. 2009; 459:262-265. doi: 10.1038/nature07935.

Student

Prinicipal Investigator

Scientific interest within the context of the graduate college:

Diabetics have a higher risk of various infectious diseases including pneumonia.^1-3 Current estimates suggest that 450 million people worldwide have diabetes, and this number will increase to approximately 700 million by 2045.⁴ The increase in diabetes prevalence is thus likely to cause an increase in pneumonia-related morbidity and mortality. A better understanding of the mechanisms underlying diabetes-related dysregulation of the antibacterial immune response may allow to develop more targeted prophylactic strategies to prevent pneumonia in diabetic individuals.

Project description:

Lower respiratory tract infections represent the fifth leading cause of death worldwide. In the coming years, pneumonia-associated morbidity and mortality may continue to rise as the number of individuals with risk factors for pneumonia, such as diabetes mellitus (among others) growths in many parts of the world.⁴ However, the mechanism underlying the enhanced susceptibility of diabetic patients to pneumonia is incompletely understood. Preliminary results indicate that diabetic animals exhibit decreased IFNg production and influx of monocyte-derived macrophages into the lung and are higher susceptibility to Legionella pneumophila infection. Moreover, IFNs are crucial for innate defense against L. pneumophila infection.^5,6 The aim of the proposed project is to further characterize the impact of diabetes on early IFN-dependent antibacterial immune defense. A better understanding of these mechanisms has the potential to enable more targeted prophylactic strategies to prevent pneumonia in individuals with diabetes mellitus.

Aim 1: To characterize the effect of diabetes mellitus on susceptibility towards L. pneumophila:

• diabetic animals (db/db, TallyHo) and controls
• use of established mouse model of L. pneumophila-induced pneumonia^5,7

Aim 2: To characterize the effect of diabetes mellitus on IFN-dependent defense:

• Which cells produce IFNg?
• How are upstream regulators of IFNg such IL-12 and IL-18 influenced by diabetes?
• What is the consequence of impaired IFNg production on macrophage-intrinsic antibacterial defense?

Aim 3: To explore prophylactic intervention strategies to rescue impaired antibacterial immunity in diabetic animals – treatment with e.g. rIFNg, rIL-12 or rIL-18 in diabetic animals and controls to rescue antibacterial defense

References

1. Muller LMAJ, Groter KJ, Hak E, […], Schellevis FG, Hoepelman AIM, Rutten GEHM. Increased risk of common infections in patients with type 1 and type 2 diabetes mellitus. Clin Infect Dis. 2005; 41: 281-288. doi: 10.1086/431587.
2. Lepper PM, Ott S, Nüesch E, […], Jüni P, Bals L, Rohde G; German Community Acquired Pneumonia Competence Network. Serum glucose levels for predicting death in patients admitted to hospital for community acquired pneumonia: prospective cohort study. BMJ. 2012; 344:e3397. doi: 10.1136/bmj.e3397.
3. Yende S, von der Poll T, Lee M, […], Bauer D, Satterfield S, Angus DC; GenIMS and Health ABC study. The influence of pre-existing diabetes mellitus on the host immune response and outcome of pneumonia: analysis of two multicentre cohort studies. Thorax. 2010; 65:870-877. doi: 10.1136/thx.2010.136317.
4. Cho NH, Shaw JE, Karuranga S, […], da Rocha Fernandes JD, Ohlrogge AW, Malanda B. IDF Diabetes Atlas: Global estimates of diabetes prevalence for 2017 and projections for 2045. Diabetes Res Clin Pract. 2018; 138:271-281. doi: 10.1016/j.diabres.2018.02.023.
5. Naujoks J, Tabeling C, Dill BD, […], Hilbi H, Trost M, Opitz B. IFNs Modify the Proteome of Legionella-Containing Vacuoles and Restrict Infection Via IRG1-Derived Itaconic Acid. PLoS Pathog. 2016; 12:e1005408. doi: 10.1371/journal.ppat.1005408.
6. Opitz B, van Laak V, Eitel J, Suttorp N. Innate immune recognition in infectious and noninfectious diseases of the lung. Am J Respir Crit Care Med. 2010; 181:1294-1309. doi: 10.1164/rccm.200909-1427SO.
7. Ruiz-Moreno JS, Hamann L, Shah JA, […], Schumann RR, JinL, Hawn TR, Opitz B; CAPNETZ Study Group. The common HAQ STING variant impairs cGAS-dependent antibacterial responses and is associated with susceptibility to Legionnaires’ disease in humans. PLoS Pathog. 2018; 14:e1006829. doi: 10.1371/journal.ppat.1006829.

Student

Prinicipal Investigator

Scientific interest within the context of the graduate college:

The airway mucosa represents the first line of defense of the respiratory system against pathogens, pollutants, and irritants that are constantly inhaled during tidal breathing. Elimination of these potentially harmful stimuli by mucociliary clearance is an important innate defense mechanism of the lung, which operates through the coordinated function of (i) the motile cilia, (ii) the airway surface liquid layer, and (iii) the mucus layer. Abnormalities in mucociliary clearance contribute to the pathogenesis of a spectrum of chronic lung diseases, such as cystic fibrosis, where the underlying ion and fluid transport defect results in viscous, thick mucus that can not be cleared properly from the lungs. Recent evidence suggests that the SLC26A9 chloride transporter plays important roles in coordinated epithelial ion and fluid transport and is crucial for the maintenance of airway mucus clearance during inflammation.¹ Furthermore, genetic association studies demonstrated a link between SLC26A9 and lung function in health, as well as in chronic lung diseases, suggesting that SLC26A9 is an attractive therapeutic target to improve mucociliary clearance.² However, the cellular and molecular mechanisms that maintain proper mucociliary clearance upon pro-inflammatory stimuli, and the role of SLC26A9 upregulation in this process, are poorly understood.

Project description:

In our lab, we use patient-derived, highly differentiated airway epithelial cultures to model key aspects of airway physiology and mucosal defense.³ The aim of this translational research project is to utilize this model system to investigate the coordinated regulation of SLC26A9-mediated chloride transport and mucociliary clearance in health and during inflammation, to identify physiologic changes that adapt mucociliary clearance to environmental challenges, as well as changes that promote disease development. For this purpose, airway epithelial cultures from healthy donors and cystic fibrosis patients will be challenged with pro-inflammatory cytokines and proteases, and will be studied in vitro under near physiological air-liquid-interface conditions. This will include studies on (i) epithelial ion transport, (ii) airway mucus characteristics, and (iii) mucociliary transport. Further, to identify molecules and signaling pathways that may serve as therapeutic targets to enhance mucociliary clearance, we will conduct RNA sequencing and proteome analysis. This experimental MD thesis will apply complex primary cell culture methods, basic molecular biology techniques, electrophysiology assays and state of the art live cell microscopy imaging to obtain mechanistic insights into the pathobiology of chronic inflammatory airway diseases.

WP1: Effect of inflammatory mediators and proteases on ion transport properties of the airway epithelium

WP2: Effect of inflammatory mediators and proteases on mucus properties of the airway epithelium

WP3: Effect of inflammatory mediators and proteases on mucociliary transport of the airway epithelium

WP4: Molecular signatures and therapeutic targeting of airway epithelial inflammation

References

1. Balázs A, Mall MA. Role of the SLC26A9 Chloride Channel as Disease Modifier and Potential Therapeutic Target in Cystic Fibrosis. Front Pharmacol. 2018; 9:1112. doi: 10.3389/fphar.2018.01112.
2. Gong J, He G, Wang C, […], Ratjen F, Rommens JM, Strug LJ. Genetic evidence supports the development of SLC26A9 targeting therapies for the treatment of lung disease. NPJ Genom Med. 2022; 7:28. doi: 10.1038/s41525-022-00299-9.
3. Balázs A, Millar-Büchner P, Mülleder M, […], Röhmel J, Ralser M, Mall MA. Age-Related Differences in Structure and Function of Nasal Epithelial Cultures From Healthy Children and Elderly People. Front Immunol. 2022; 13:822437. doi: 10.3389/fimmu.2022.822437.

Student

Prinicipal Investigator

Scientific interest within the context of the graduate college:

Acute respiratory distress syndrome (ARDS) is a serious complication of infectious or sterile lung inflammation, typically as a consequence of pneumonia or sepsis, with high morbidity and mortality (30%-40%) and presently no pharmacological or mechanistic treatment strategies. This critical knowledge and treatment gap became strikingly evident in the recent COVID-19 pandemic, with ARDS as the main cause of death.^1,2 ARDS is characterized by a breakdown of the lung vascular barrier and the leak of fluid from the blood into the airspaces, preventing normal lung mechanics and gas exchange. Traditionally, mechanisms of ARDS are studied in animal models, which have, however, translated poorly into the clinical scenario. Here, we will assess mechanisms of lung vascular barrier integrity and regeneration in a novel microphysiological Microvasculature-on-Chip (MOC) model that allows to track vascular morphology and leak as well as the dynamics of individual cell types and their interaction in an unprecedented temporal and spatial context. Here, we will employ this model for the first time to study lung vascular barrier integrity, and to devise strategies for its maintenance in experimental settings mimicking ARDS.

Project description:

Vascular barrier integrity is critical for normal lung function, as it preserves the compartmentalization between blood and airspaces of the lung, and prevents systemic entry and dissemination of inhaled pathogens and environmental pollutants. In the past, we and others have extensively studied mechanisms of lung vascular barrier integrity in animal as well as cell culture models,^3-6 which, however, proved challenging to translate into the clinical scenario, and/or provide only limited spatial and temporal information on critical structural and functional changes in a realistic, multicellular 3D arrangement.

Pericytes are important regulators of endothelial homeostasis and vascular barrier integrity in the systemic circulation. As perivascular cells, they are encased within the basement membrane and surround vessels; their attachment to and interaction with endothelial cells stabilizes microvascular networks. Pericytes may be expected to serve similar vessel- and barrier-stabilizing functions in the lung, and ARDS may potentially drive barrier failure by causing pericyte loss or detachment. Counterintuitively, however, ablation of pericytes was recently reported to increase lung injury in a mouse model of ARDS.⁷ In the present proposal, we want to utilize a recently developed MOC model^8,9 that we have – in collaboration with colleagues from the University of Berne and Stanford University – successfully adapted to study formation and integrity of lung microvascular networks formed by primary human lung endothelial cells and pericytes. Specifically, we aim to address the following research questions:

Question 1: What is the role of pericytes in lung vascular barrier integrity? To this end, we will generate self-assembling lung microvascular networks from primary human endothelial cells and pericytes, and probe for pericyte-endothelial interaction (e.g. via gap junctions, integrins, or paracrine mediators by use of high resolution structural and functional imaging, single cell RNA sequencing, and mass-spectrometry based proteomics, metabolomics and glycomics) and the functional role of pericytes and these interactions for barrier integrity (e.g. by targeted pericyte ablation using diphtheria toxin receptor expressing cells).

Question 2: How does ARDS affect pericyte-endothelial interaction, and how does this contribute to barrier failure in lung microvascular networks? In established lung microvascular networks composed of endothelial cells and pericytes, we will assess the effect of ARDS-characteristic stimuli (e.g. proinflammatory cytokines, bacterial exotoxins, infectious pathogens) on pericyte-endothelial interaction and its functional consequences on vascular integrity.

Question 3: Can we target pericyte-endothelial interaction to stabilize and/or restore vascular barrier integrity in ARDS-like settings in vitro? Based on results from questions 1 & 2, we will devise targeted strategies to preserve or restore homeostatic pericyte-endothelial interaction and as such, barrier integrity. Priority will be given to measures that may be implemented into the clinical scenario, e.g. by drug repurposing.
The results from this work are expected to provide novel insights into the intrinsic mechanism that regulate and preserve lung vascular barrier integrity and their exploitation as therapeutic strategy for the treatment of ARDS.

References

1. Voelkel NF, Bogaard HJ, Kuebler WM. ARDS in the time of corona: context and perspective. Am J Physiol Lung Cell Mol Physiol. 2022; 323:L431-L437. doi: 10.1152/ajplung.00432.2021.
2. Ahmad S, Matalon S, Kuebler WM. Understanding COVID-19 susceptibility and presentation based on its underlying physiology. Physiol Rev. 2022; 102:1579-1585. doi: 10.1152/physrev.00008.2022.
3. Erfinanda L, Zou L, Gutbier B, […], Mall MA, Witzenrath M, Kuebler WM. Loss of endothelial CFTR drives barrier failure and edema formation in lung infection and can be targeted by CFTR potentiation. Sci Transl Med. 2022; 14:eabg8577. doi: 10.1126/scitranslmed.abg8577.
4. Jiang T, Samapati R, Klassen S, […], Nüsing R, Uhlig S, Kuebler WM. Stimulation of the EP3 receptor causes lung oedema by activation of TRPC6 in pulmonary endothelial cells. Eur Respir J. 2022; 60:2102635. doi: 10.1183/13993003.02635-2021.
5. McVey MJ, Maishan M, Foley A, […], Goldenberg NM, Khursigara CM, Kuebler WM. Pseudomonas aeruginosa membrane vesicles cause endothelial barrier failure and lung injury. Eur Respir J. 2022; 59:2101500. doi: 10.1183/13993003.01500-2021.
6. Michalick L, Weidenfeld S, Grimmer B, […], Witzenrath M, Hippenstiel S, Kuebler WM. Plasma mediators in patients with severe COVID-19 cause lung endothelial barrier failure. Eur Respir J. 2021; 57:2002384. doi: 10.1183/13993003.02384-2020.
7. Hung CF, Wilson CL, Chow YH, […], Gharib SA, Altemeier WA, Schnapp LM. Effect of lung pericyte-like cell ablation on the bleomycin model of injury and repair. Am J Physiol Lung Cell Mol Physiol. 2022; 322:L607-L616. doi: 10.1152/ajplung.00392.2021.
8. Bichsel CA, Wang L, Froment L, […], Schmid RA, Guenat OT, Hall SRR. Increased PD-L1 expression and IL-6 secretion characterize human lung tumor-derived perivascular-like cells that promote vascular leakage in a perfusable microvasculature model. Sci Rep. 2017; 7:10636. doi: 10.1038/s41598-017-09928-1.
9. Bichsel CA, Hall SR, Schmid RA, Guenat OT, Geiser T. Primary human lung pericytes support and stabilize in vitro perfusable microvessels. Tissue Eng Part A. 2015; 21:2166-76. doi: 10.1089/ten.TEA.2014.0545.

Student

Prinicipal Investigator

Scientific interest within the context of the graduate college:

Sulfate is an ion that is indispensable for human health. It is necessary for the formation of connective tissues, including bone and cartilage.¹ The kidney plays a central role in body ion homeostasis by reabsorbing electrolytes from the tubular fluid. Specifically, the proximal tubule is a major site for fluid, protein, and nutrient retrieval.^2,3 Our working groups recently described a patient who presented with unexplained chronic chest pain and a kidney stone. By combining clinical and genetic analyses with functional expression assays, our groups demonstrated that the mutation in the sulfate transporter SLC26A1 we identified in this patient impaired the function of this transporter, thereby causing sulfate deficiency due to sulfate loss into the urine.⁴ To extend these findings to a population level, we used genetic data of >5,000 individuals and identified 43 variants in the SCL26A1 gene. Of note, variants affecting transporter function were significantly associated with lower plasma sulfate concentrations. In view of recent evidence by others linking sulfate homeostasis to bone disorders, we concluded that the kidney may play an important role in musculoskeletal health by retaining sulfate in the body. The current research proposal seeks to examine the role of the sulfate transporter SLC26A1 in musculoskeletal health in more depth.

Project description:

During the last three months, we identified a second patient with a novel, homozygous SLC26A1 mutation. The patient has been suffering from aortic root dilatation, early cataract formation, and increased arm length. Based on his clinical symptoms the diagnosis of Marfan syndrome was made. However, testing for known genes involved in Marfan syndrome has not yielded any results. We identified a novel mutation of the sulfate transporter gene SLC26A1 in the affected patient. The doctoral thesis will examine the hypothesis that a defect in SLC26A1-mediated sulfate transport may explain the patient’s clinical symptoms. In a first step, the doctoral student will be examining whether the novel mutation in fact leads to a sulfate transport defect by comparing radioactive sulfate fluxes in  Xenopus laevis oocytes expressing WT and mutant transporter. In a second step, the doctoral student will examine kidney tubuloids derived from the patient. To this end, pluripotent stem cells (iPSC) will be programmed to kidney tubuloids and examined for sulfate transport. Lastly, we would like to examine cartilage formation in the patient and in SLC26A1-deficient mice.

Aim 1: Functional analyses of the detected SLC26A1 mutation in Xenopus laevis oocytes.

Aim 2: Characterization of sulfate transport in kidney tubuloids from patient-derived pluripotent stem cells.

Aim 3: Assess bone health and cartilage formation in the patient and SLC26A1-deficient mice.

References

1. Langford R, Hurrion E, Dawson PA. Genetics and pathophysiology of mammalian sulfate biology. J Genet Genomics. 2017; 44:7-20. doi: 10.1016/j.jgg.2016.08.001.
2. Novarino G, Weinert S, Rickheit G, Jentsch TJ. Endosomal chloride-proton exchange rather than chloride conductance is crucial for renal endocytosis. Science. 2010; 328:1398-1401. doi: 10.1126/science.1188070.
3. Knauf F, Yang CL, Thomson RB, Mentone SA, Giebisch G, Aronson PS. Identification of a chloride-formate exchanger expressed on the brush border membrane of renal proximal tubule cells. Proc Natl Acad Sci U S A. 2001; 98:9425-9430. doi: 10.1073/pnas.141241098.
4. Pfau A, Lopez-Cayuqueo KI, Scherer N, […], Kottgen A, Jentsch TJ, Knauf F. SLC26A1 is a major determinant of sulfate homeostasis in humans. J Clin Invest. 2023; 133:e161849. doi: 10.1172/JCI161849.

Student

Prinicipal Investigator

Scientific interest within the context of the graduate college:

Not Everything Is “Genetic”, but Genes Are Involved in Everything (adapted from Kenneth M. Weiss).

Our group is interested in the identification and investigation of genetic, clinical, and environmental factors determining the onset of chronic kidney disease (CKD) and kidney survival. We make use of next-generation sequencing techniques and deep-phenotyping to identify genetic variants that are predictive for disease progression or convey protection from organ failure. We functionally evaluate identified germline variants in vitro in order to understand underlying molecular mechanisms leading to CKD on the one hand or protecting from kidney failure on the other. By doing so, we aim at defining and targeting molecular switches responsible for health maintenance and disease alleviation.

Project description:

Introduction: One of six patients undergoing renal transplantation has autosomal-dominant polycystic kidney disease (ADPKD) caused by heterozygous germline mutations in one of two main disease genes, namely PKD1 or PKD2. ADPKD is the commonest genetic disorder leading to CKD including kidney failure (KF), cystic liver disease, and CNS-involvement in terms of intracranial aneurysms.^1-3 KF from ADPKD commonly occurs between ages 30-80 years. While PKD1-associated disease is generally more severe than PKD2-disease, exemplified by a 20-year difference in mean age at KF (55 versus 75 years), current genotype-phenotype correlations only explain part of the observed disease variability.^2,3 For example, some patients with even identical PKD2-mutations vary dramatically in their progression. We demonstrated that additional non-diagnostic hypomorphic PKD1/2-germline variants as well as variants in PKD-interactors mechanistically add to the mutational effect.^3,4 Recently, collaborators of ours identified a single PKD2-mutation that collectively accounts for about 18% of all PKD2-cases in Taiwan (n=200): c.2407C>T, p.Arg803*.⁵ By a first genetic screening, we also found this variant in several families in Germany and France (n=30). This situation is unique in the field and strongly facilitates studying disease variability, as joint cohorts with the same diagnostic PKD-variant allow for completely new approaches to explain why some individuals experience kidney failure in mid-life and others seem to be protected. We aim to learn from families harboring this distinct PKD2 variant in Asia and Europe for transethnic comparison, proposing the following two specific aims:

Aim 1 / WP1: Identification and characterization of additional European cases with PKD2-Arg803*

While we have access to genetic and clinical data from the Taiwan cohort, we aim to further extend the European counterpart for transethnic comparison. Therefore, we will use platforms of the European Rare Kidney Disease Network (ERKNet)⁶ and the European Renal Association (ERA)⁷ to run online surveys across centers in whole Europe. We estimate to identify at least another 30-50 individuals with the PKD2-Arg803* variant for complete clinical characterization of kidney-, liver-, and CNS-involvement. Additionally, we also aim to capture environmental factors by sending out patient questionnaires. Lastly, we will run exome sequencing in all individuals available (n=60-80) for identification of additional germline variants likely accounting for disease progression versus disease protection.

Aim 2 / WP2: Functional studies with PKD2-Arg803* and comparison with other PKD-disease variants Characterization of urinary renal epithelial cells from individuals with PKD2-p.Arg803*. Overexpression of PKD2-Arg803* in established cell-culture models and consecutive comparison to established PKD2 gene variants. Consecutive use of established cellular read-outs on RNA and protein level. Planned analyses include qRT-PCR, Western Blot, and immunofluorescence imaging.

References

1. Lanktree MB, Haghighi A, Guiard E, […], Harris PC, Paterson AD, Pei Y. Prevalence Estimates of Polycystic Kidney and Liver Disease by Population Sequencing. J Am Soc Nephrol. 2018; 29:2593-2600. doi: 10.1681/ASN.2018050493.
2. Cornec-Le Gall E, Alam A, Perrone RD. Autosomal dominant polycystic kidney disease. Lancet. 2019; 393:919-935. doi: 10.1016/S0140-6736(18)32782-X.
3. Schönauer R, Baatz S, Nemitz-Kliemchen M, […], Neuber S, Bergmann C, Halbritter J. Matching clinical and genetic diagnoses in autosomal dominant polycystic kidney disease reveals novel phenocopies and potential candidate genes. Genet Med. 2020; 22:1374-1383. doi: 10.1038/s41436-020-0816-3.
4. Durkie, M. et al. Biallelic inheritance of hypomorphic PKD1 variants is highly prevalent in very early onset polycystic kidney disease. Genet Med. 2021; 23:689-697. doi: 10.1038/s41436-020-01026-4.
5. Yu CC, Lee AF, Kohl S, […], Hildebrandt F; Taiwan PKD Consortium; Hwang DY. PKD2 founder mutation is the most common mutation of polycystic kidney disease in Taiwan. NPJ Genom Med. 2022; 7:40. doi: 10.1038/s41525-022-00309-w.
6. https://www.erknet.org
7. https://www.era-online.org/about-us/working-groups/g-k-working-group/