1. Research Background:

Acute kidney injury (AKI) is a very common, life-threatening condition and associated with an enormous burden, both in social as well as economic terms1. Patients affected by AKI are at a highly increased risk of developing not only dialysis-requiring endstage renal failure but also cardiovascular complications such as heart failure, heart attack and stroke2. Being an aging-associated disease the importance of AKI has been increasing in the last decades and will become a major challenge to  modernhealth-caresystems.Unfortunately,untilnowatargetedapproachforthe prevention and treatment of AKI has not been identified.

2. Research questions addresses by the group:

We approach this problem employing the power of the model organism C. elegans exploiting the fact that longevity in this model reflects increased stress resistance in an evolutionarily conserved manner3C. elegans is the perfect tool to elucidate the molecular mechanisms underlying this increased stress resistance. Understanding these mechanisms is the essential basis to developing novel strategies for the clinical setting, that are urgently required taking into account the quickly rising incidence of AKI due to demographic changes. Interestingly, pathways involved in lifespan regulation and stress resistance such as hypoxia-inducible factor signaling also play an important role in tumorigenesis4–6. This fact allows for the intriguing hypothesis of a tightly regulated balance between cellular stress tolerance on the one hand and tumor suppression on the other hand. Consequently, unraveling the molecular mechanisms behind this phenomenon holds the promise to allow for targeted strategies regarding not only the protection from organ damage7 but also anti-tumor therapy.
Regarding these questions, we are especially interested in the contribution of non- coding RNAs (ncRNAs)8–11 and RNA-binding proteins (RBPs)12,13. The regulatory network linking ncRNAs, RBPs and mRNAs has so far not been studied systematically regarding renal disease. However, linking RNA biology with the  power of model organisms such as C. elegans bears a huge potential to reveal novel approaches to fighting kidney disease as well as consecutive morbidity and mortality. Unraveling this potential and translating it to a clinical use is the declared aim of our group. In order to allow for a direct translation of the findings in the basic science lab all projects are accompanied by active clinical trial design that has already led to the completion of clinical trials on preconditioning-mediated organoprotection14.

3. Possible projects:

  • Project 1: A global view on RNA-binding proteins in stress resistance of the nematode

    Using RNAseq and proteomics in mouse models of acute injury15 and preconditioning as well as C. elegans samples  (long-lived mutants, worms after hypoxic preconditioning and exposure to heat stress) combined with the identification of novel RNA-binding proteins (RBPs) through RNA-interactome capture in the nematode,we have been able to generate a list of RBPs that are likely candidates to be involved with the mediation of increased organismal stress resistance. Now, we are going to couple this knowledge with a novel stress resistance screening approach using the lifespan machine16 that has recently been established as a novel tool in the lab. Doing so we will be able to confirm phenotypic consequences of loss-and gain-of-function of these RBPs in the response to heat-stress and longevity. A small number of key proteins identified in this screen will be further characterized regarding their molecular function focusing on the identification of RNA-interaction partners in mammalian cell culture as well as C. elegans using enhanced crosslinking and immunoprecipitation (eCLIP).

  • Project 2: Elucidating the role of lncRNAs in acute kidney disease

    Using RNAseq datasets of the kidney after cisplatin15 and ischemia-reperfusion induced acute kidney injury in a mouse model combined with datasets on mice pre- treated by caloric restriction, hypoxic preconditioning and prolyl hydroxylase inhibitors we were able to identify a number of known and novel lncRNAs to be significantly dysregulated as candidates to be involved in the protection from acute kidney injury. Based on this knowledge, we have designed the strategies for the CRISPR-Cas9 based generation of 5 lncRNA knockout mouse lines that were generated in the CECAD transgenesis facility. These mouse lines are now being analyzed focusing on renal phenotypes including susceptibility to AKI and preconditioning-mediated resistance. The phenotypic analyses will be coupled with molecular biology to unravel the entirely unknown molecular function of these lncRNAs employing state-of-the-art methods such as CHART-MS to identify protein, RNA and DNA interactors. Furthermore, the spatial resolution of their expression in the kidney will be taken to the cellular level using kidney scRNAseq17.

4. Applied Methods and model organisms:

  • model organisms: cell culture, C. elegans and mouse
  • all standard methods in molecular and cellular biology (such as molecular cloning, Western Blotting, Immunofluorescence etc.)
  • state-of-the-art methods regarding RNA-RNA and RNA-protein interactions, e.g. CLIP (crosslinking and immunoprecipitation), CHART-MS, RNA interactome capture, proteomics, RNA-sequencing (including single-cellRNAseq)
  • transgenesis and CRISPR-Cas9 mediated genome editing in mouse and nematode
  • basics in the bioinformatics analysis of the datasets obtained

5. Desirable skills and qualifications:

  • good training in and understanding of standard techniques in molecular and cellular biology
  • interest in tackling clinically relevant questions using these techniques in cell culture and model organisms
  • FELASA certificate would be desirable, but can also be obtained during the first months of the project
  • basic bioinformatics skills would be desirable, but can also be obtained during the first months of the project. However, an interest in the analysis of large-scale datasets under the supervision of a bioinformatician is required

6. References:

  1. Hsu RK, McCulloch CE, Dudley RA, Lo LJ, Hsu C: Temporal changes in incidence of dialysis-requiring AKI. J. Am. Soc. Nephrol. 24: 37–42, 2013
  2. Hsu C, Liu KD: Cardiovascular Events after AKI: A New Dimension. JASN 25: 425–427, 2014
  3. Müller R-U, Zank S, Fabretti F and Benzing T: Caenorhabditis elegans, a model organism for kidney research: from cilia to mechanosensation and longevity. Curr. Opin. Nephrol. Hypertens. 20: 400–408, 2011
  4. Bartram MP, Mishra T, Reintjes N, Fabretti F, Gharbi H, Adam AC, Göbel H, Franke M, Schermer B, Haneder S, Benzing T, Beck BB and Müller R-U: Characterization of a splice-site mutation in the tumor suppressor gene FLCN associated with renal cancer. BMC Med. Genet. 18: 53, 2017
  5. Gharbi H, Fabretti F, Bharill P, Rinschen MM, Brinkkötter S, Frommolt P, Burst V, Schermer B, Benzing T and Müller R-U: Loss of the Birt-Hogg-Dubé gene product folliculin induces longevity in a hypoxia-inducible factor-dependent manner. Aging Cell 2013
  6. Müller R-U, Fabretti F, Zank S, Burst V, Benzing T and Schermer B: The von Hippel Lindau tumor suppressor limits longevity. J. Am. Soc. Nephrol. 20: 2513–2517, 2009
  7. Siedek F, Persigehl T, Mueller R-U, Burst V, Benzing T, Maintz D ad Haneder S: Assessing renal changes after remote ischemic preconditioning (RIPC) of the upper extremity using BOLD imaging at 3T. MAGMA 2017
  8. Bartram MP, Höhne M, Dafinger C, Völker LA, Albersmeyer M, Heiss J, Göbel H, Brönneke H, Burst V, Liebau MC, Benzing T, Schermer B and Müller R-U: Conditional loss of kidney microRNAs results in congenital anomalies of the kidney and urinary tract (CAKUT). J. Mol. Med. 91: 739–748, 2013
  9. Bartram MP, Dafinger C, Habbig S, Benzing T, Schermer B and Müller R-U: Loss of Dgcr8-mediated microRNA expression in the kidney results in hydronephrosis and renal malformation. BMC Nephrol 16: 55, 2015
  10. Landgraf P, Rusu M, Sheridan R, Sewer A, Iovino N, Aravin A, Pfeffer S, Rice A, Kamphorst AO, Landthaler M, Lin C, Socci ND, Hermida L, Fulci V, Chiaretti S, Foà R, Schliwka J, Fuchs U, Novosel A, Müller R-U, Schermer B, Bissels U, Inman J, Phan Q, Chien M, Weir DB, Choksi R, De Vita G, Frezzetti D, Trompeter H-I, Hornung V, Teng G, Hartmann G, Palkovits M, Di Lauro R, Wernet P, Macino G, Rogler CE, Nagle JW, Ju J, Papavasiliou FN, Benzing T, Lichter P, Tam W, Brownstein MJ, Bosio A, Borkhardt A, Russo JJ, Sander C, Zavolan M, Tuschl T: A mammalian microRNA expression atlas based on small RNA library sequencing. Cell 129: 1401–1414, 2007
  11. Bartram MP, Amendola E, Benzing T, Schermer B, de Vita G and Müller R-U: Mice lacking microRNAs in Pax8-expressing cells develop hypothyroidism and end-stage renal failure. BMC Mol. Biol. 17: 11, 2016
  12. Kaiser R, Ignarski M, Nostrand ELV, Frese C, Jain M, Cukoski S, Heinen H, Schaechter M, Bunte K, Frommolt P, Keller P, Helm M, Bohl K, Höhne M, Schermer B, Benzing T, Höpker K, Dieterich C, Yeo GW, Müller R-U, Fabretti F: The RNA-binding protein AATF coordinates rRNA maturation. bioRxiv 446260,2018
  13. Ignarski M, Rill C, Kaiser RWJ, Kaldirim M, Neuhaus R, Esmaillie R, Li X, Klein C, Bohl K, Petersen M, Frese CK, Höhne M, Atanassov I, Rinschen MM, Höpker K, Schermer B, Benzing T, Dieterich C, Fabretti F, Müller R-U: The RNA-Protein Interactome of Differentiated Kidney Tubular Epithelial Cells. JASN ASN.2018090914, 2019
  14. Grundmann F, Müller R-U, Reppenhorst A, Hülswitt L, Späth MR, Kubacki T, Scherner M, Faust M, Becker I, Wahlers T, Schermer B, Benzing T and Burst V: Preoperative Short-Term Calorie Restriction for Prevention of Acute Kidney Injury After Cardiac Surgery: A Randomized, Controlled, Open-Label, Pilot Trial. J Am Heart Assoc 7: 2018
  15. Späth M, Bartram MP, Palacio-Escat N, Hoyer JR, Debes C, Demir F, Schroeter CB, Mandel AM, Grundmann F, Ciarimboli G, Beyer A, Kizhakkedathu JN, Brodesser S, Göbel H, Becker JU, Benzing T, Schermer B, Höhne M, Burst V, Saez-Rodriguez J, Huesgen PF, Müller R-U* and Rinschen MM*: The proteome microenvironment determines the protective effect of preconditioning in cisplatin-induced acute kidney injury. Kidney Int 2018 (accepted for publication)
    * shared corresponding authorship
  16. Stroustrup N, Ulmschneider BE, Nash ZM, López-Moyado IF, Apfeld J, Fontana W: The Caenorhabditis elegans Lifespan Machine. Nat Meth 10: 665–670, 2013
  17. Karaiskos N, Rahmatollahi M, Boltengagen A, Liu H, Hoehne M, Rinschen M, Schermer B, Benzing T, Rajewsky N, Kocks C, Kann M and Müller R-U: A Single-Cell Transcriptome Atlas of the Mouse Glomerulus. J. Am. Soc. Nephrol. 29: 2060–2068, 2018