Center for Molecular Medicine Cologne

Benzing, Thomas | Hackl, Matthias - C 02

Cellular crosstalk of endothelial cells and podocytes in glomerular injury and repair

Introduction

Over the past decade we showed that slit diaphragm signalling in podocytes is essential for podocyte viability and function and the integrity of the kidney filtration barrier.

Here we will now extend these studies on interactions with the underlying glomerular basement membrane and particularly glomerular endothelial cells in closest vicinity of podocytes. This project embarks on our previous work where we developed innovative strategies to monitor signalling in vivo in genetically engineered mice.

We are convinced that the proposed experiments will help clarify the contribution of alterations of podocyte dynamics to CKD and enable us to to examine the therapeutic potential of our findings.

Chronic kidney disease (CKD) is becoming an increasingly prevalent condition affecting almost 10% of the population in the Western societies.

The majority of kidney diseases that progress to CKD start in the glomerulus, the renal filtration unit, as a consequence of a limited capacity of glomeruli for regeneration and the limited ability of terminally differentiated glomerular podocytes for self-renewal. Podocytes enwrap the glomerular capillaries and elaborate primary and interdigitating secondary extensions that are connected by a membrane like cell junction, called the slit diaphragm.

Together with the underlying glomerular basement membrane and specialized fenestrated glomerular endothelial cells podocytes constitute the kidney filter. Over the past decade, we showed that proteins residing at the slit diaphragm, a structure that connects adjacent podocyte processes, form an evolutionarily conserved mechanosensitive multiprotein complex controls podocyte viability and function. Studies in C. elegans revealed a role for lipid-protein interactions at the slit diaphragm complex in mechanosensation and identified new components of the lipid-protein supercomplex at the filtration slit. Although podocyte function is more and more understood the complex crosstalk of all layers at the filtration barrier is much less understood.

The major problem in studying crosstalk is that it cannot be studied in cell culture systems or in the model organisms C. elegans and Drosophila melanogaster. Therefore, we developed new in vivo approaches to illuminate the signalling dynamics in living animals using multiphoton microscopy. Using these techniques we will now focus on three unsolved questions in field of glomerular biology.

We believe that this project will pave the way into a new era of glomerular research and ultimately help develop therapeutic interventions in a very important group of human disorders.

Our Aims

We will study :

1) how endothelial-cell derived NO modulates podocyte homeostasis

2) how podocytes respond to signals from endothelial cells induced by increased shear stress

3) how mechanosensation in podocytes affects these endothelial cell dependent cGMP and calcium responses in podocytes

Previous Work

In vivo imaging

Podocyte research has been hampered by the lack of suitable in vitro models recapitulating the glomerular microenvironment. Especially as we learned in the recent years that the cross-talk between podocytes and endothelial cells is crucial for glomerular function.

Using multiphoton microscopy enables us, by using 2 or 3 photons of a longer wavelength, to penetrate deep into the kidney tissue reaching glomeruli in the intact mouse kidney. Using this technique is the only possibility to study podocytes in their native environment, close the endothelial, mesangial and parietal cells and exposed to blood flow, filtration pressure and urine flow.

The high temporal resolution allows to study glomerular dynamics in the time range from seconds to hours and repetitive imaging allows to follow a single glomerulus over several days. The use of transgenic mice with genetically encoded indicators enables us to visualize intracellular signaling molecules like calcium or cGMP cell specific and without the need for loading target cells with a dye.

Proteomics

In recent years our group made significant advances in proteomic analysis of podocytes, including deep mapping of the podocyte proteome with nearly 10.000 proteins, cross-species analysis in humans, cows and mice, analysis of isolated glomeruli after the induction of disease and single-cell proteomics in health and disease.

This technique allows us to compare podocyte protein expression at baseline and after induction of disease. The data sets containing several thousand proteins are then compared and significantly up- or downregulated proteins are identified. These proteins are then grouped into cellular pathways, which provide a comprehensive picture of the podocyte response to this specific disease condition. 

Genetic engineering

New developments in genetic engineering, sparked by the discovery of the CRISPR/Cas9 system, allow us to quickly introduce point mutations in proteins of the slit diaphragm and their signalling molecules. By recapitulating human mutations we gain valuable insight into protein interactions and protein function and its disturbance in diseases.

Due to the short generation times, even the combination of compound heterozygous mutations is feasible, allowing to mimic patient cases, in which not a homozygous mutation in a single gene is causing a disease, but the combination of heterozygous mutations in the same or in different proteins are suspected to influence disease progression. This is much more frequent in patients than homozygous mutations.       

  • Affeldt P, Di Cristanziano V, Grundmann F, Wirtz M, Kaiser R, Benzing T, Stippel D, Kann M & Kurschat C (2021) Monitoring of hepatitis E virus RNA during treatment for chronic hepatitis E virus infection after renal transplantation. Immun Inflamm Dis 9, 513–520.
  • Butt L, Unnersjo-Jess D, Hohne M, Hahnfeldt R, Reilly D, Rinschen MM, Plagmann I, Diefenhardt P, Brahler S, Brinkkotter PT, Brismar H, Blom H, Schermer B, and Benzing T (2022). Super-Resolution Imaging of the Filtration Barrier Suggests a Role for Podocin R229Q in Genetic Predisposition to Glomerular Disease. J Am Soc Nephrol33, 138-154. doi:10.1681/ASN.2020060858.
  • Butt L, Unnersjo-Jess D, Hohne M, Schermer B, Edwards A, and Benzing T (2021). A mathematical estimation of the physical forces driving podocyte detachment. Kidney Int100, 1054-1062. doi:10.1016/j.kint.2021.06.040.
  • Dafinger C, Benzing T, Dotsch J, Schermer B, and Liebau MC (2021). Targeted deletion of Ruvbl1 results in severe defects of epidermal development and perinatal mortality. Mol Cell Pediatr8, 1. doi:10.1186/s40348-021-00111-1.
  • He B, Chen P, Zambrano S, Dabaghie D, Hu Y, Moller-Hackbarth K, Unnersjo-Jess D, Korkut GG, Charrin E, Jeansson M, Bintanel-Morcillo M, Witasp A, Wennberg L, Wernerson A, Schermer B,Benzing T, Ernfors P, Betsholtz C, Lal M, Sandberg R, and Patrakka J (2021). Single-cell RNA sequencing reveals the mesangial identity and species diversity of glomerular cell transcriptomes. Nat Commun12, 2141. doi:10.1038/s41467-021-22331-9.
  • Hoyer-Allo KJR, Spath MR, Hanssen R, Johnsen M, Brodesser S, Kaufmann K, Kiefer K, Koehler FC, Gobel H, Kubacki T, Grundmann F, Schermer B,Bruning J, Benzing T, Burst V, and Muller RU (2021). Modulation of Endocannabinoids by Caloric Restriction Is Conserved in Mice but Is Not Required for Protection from Acute Kidney Injury. Int J Mol Sci22. doi:10.3390/ijms22115485.
  • Kann M & Benzing T (2021) [Update 2021: COVID-19 from the perspective of nephrology]. Dtsch Med Wochenschr 146, 915–917.
  • Koehler S, Odenthal J, Ludwig V, Jess DU, Hohne M, Jungst C, Grawe F, Helmstadter M, Janku JL, Bergmann C, Hoyer PF, Hagmann HHH, Walz G, Bloch W, Niessen C, Schermer B, Wodarz A, Denholm B, Benzing T, Iden S, and Brinkkoetter PT (2021). Scaffold polarity proteins Par3A and Par3B share redundant functions while Par3B acts independent of atypical protein kinase C/Par6 in podocytes to maintain the kidney filtration barrier. Kidney Int. doi:10.1016/j.kint.2021.11.030.
  • Lagolio E, Demurtas J, Buzzetti R, Cortassa G, Bottone S, Spadafora L, Cocino C, Smith L, Benzing T, and Polidori MC (2021). A rapid and feasible tool for clinical decision making in community-dwelling patients with COVID-19 and those admitted to emergency departments: the Braden-LDH-HorowITZ Assessment-BLITZ. Intern Emerg Med. doi:10.1007/s11739-021-02805-w.
                                            • Mangold N, Pippin J, Unnersjoe-Jess D, Koehler S, Shankland S, Brahler S, Schermer B, Benzing T, Brinkkoetter PT, and Hagmann H (2021). The Atypical Cyclin-Dependent Kinase 5 (Cdk5) Guards Podocytes from Apoptosis in Glomerular Disease While Being Dispensable for Podocyte Development. Cells10. doi:10.3390/cells10092464.
                                            • Müller FM, Meyer AM, Pickert L, Heeß A, Becker I, Benzing T & Polidori MC (2021) An interdisciplinary intervention is associated with overall improvement of older inpatients in a non-geriatric setting: A retrospective analysis of an observational, longitudinal study with one-year follow up. Geriatric Care 7.
                                            • Noetzel N, Meyer AM, Siri G, Pickert L, Heeß A, Verleysdonk J, Benzing T, Pilotto A, Barbe AG & Polidori MC (2021) The impact of oral health on prognosis of older multimorbid inpatients: the 6-month follow up MPI oral health study (MPIOH). Eur Geriatr Med 12, 263–273.
                                            • Pickert L, Meyer AM, Becker I, Heeß A, Noetzel N, Brinkkötter P, Pilotto A, Benzing T & Polidori MC (2021) Role of a multidimensional prognosis in-hospital monitoring for older patients with prolonged stay. Int J Clin Pract 75, e13989.
                                            • Rarek MP, Meyer AM, Pickert L, Pilotto A, Benzing T, Burst V & Polidori MC (2021) The prognostic signature of health-related quality of life in older patients admitted to the emergency department: a 6-month follow-up study. Aging Clin Exp Res 33, 2203–2211.
                                            • Reusch B, Bartram MP, Dafinger C, Palacio-Escat N, Wenzel A, Fenton RA, Saez-Rodriguez J, Schermer B, Benzing T, Altmuller J, Beck BB, and Rinschen MM (2022). MAGED2 controls vasopressin-induced aquaporin-2 expression in collecting duct cells. J Proteomics252, 104424. doi:10.1016/j.jprot.2021.104424.
                                                                                                                  • Talyan S, Filipow S, Ignarski M, Smieszek M, Chen H, Kuhne L, Butt L, Gobel H, Hoyer-Allo KJR, Koehler FC, Altmuller J, Brinkkotter P, Schermer B, Benzing T, Kann M, Muller RU, and Dieterich C (2021). CALINCA-A Novel Pipeline for the Identification of lncRNAs in Podocyte Disease. Cells10. doi:10.3390/cells10030692.
                                                                                                                  • Benzing T, and Salant D (2021). Insights into Glomerular Filtration and Albuminuria. N Engl J Med384, 1437-1446. doi:10.1056/NEJMra1808786.
                                                                                                                  • Hohfeld J, Benzing T, Bloch W, Furst DO, Gehlert S, Hesse M, Hoffmann B, Hoppe T, Huesgen PF, Kohn M, Kolanus W, Merkel R, Niessen CM, Pokrzywa W, Rinschen MM, Wachten D, and Warscheid B (2021). Maintaining proteostasis under mechanical stress. EMBO Rep22, e52507. doi:10.15252/embr.202152507.
                                                                                                                  • Polidori MC, Sies H, Ferrucci L, and Benzing T (2021). COVID-19 mortality as a fingerprint of biological age. Ageing Res Rev67, 101308. doi:10.1016/j.arr.2021.101308.
                                                                                                                  • Seufert L, Benzing T, Ignarski M, and Muller RU(2021). RNA-binding proteins and their role in kidney disease. Nat Rev Nephrol. doi:10.1038/s41581-021-00497-1.
                                                                                                                  • Binz-Lotter J, Jungst C, Rinschen MM, Koehler S, Zentis P, Schauss A, Schermer B, Benzing T, and Hackl MJ (2020). Injured Podocytes Are Sensitized to Angiotensin II-Induced Calcium Signaling. J Am Soc Nephrol 31, 532-42.
                                                                                                                     
                                                                                                                  • Braun F, Rinschen M, Buchner D, Bohl K, Plagmann I, Bachurski D, Richard Spath M, Antczak P, Gobel H, Klein C, Lackmann JW, Kretz O, Puelles VG, Wahba R, Hallek M, Schermer B, Benzing T, Huber TB, Beyer A, Stippel D, Kurschat CE, and Muller RU (2020). The proteomic landscape of small urinary extracellular vesicles during kidney transplantation. Journal of extracellular vesicles 10, e12026.
                                                                                                                     
                                                                                                                  • Butt L, Unnersjo-Jess D, Hohne M, Edwards A, Binz-Lotter J, Reilly D, Hahnfeldt R, Ziegler V, Fremter K, Rinschen MM, Helmstadter M, Ebert LK, Castrop H, Hackl MJ, Walz G, Brinkkoetter PT, Liebau MC, Tory K, Hoyer PF, Beck BB, Brismar H, Blom H, Schermer B, and Benzing T (2020). A molecular mechanism explaining albuminuria in kidney disease. Nat Metab2, 461-474. doi:10.1038/s42255-020-0204-y.
                                                                                                                  • Dafinger C, Mandel AM, Braun A, Goebel H, Burgmaier K, Massella L, Mastrangelo A, Doetsch J, Benzing T, Weimbs T, Schermer B & Liebau MC (2020) The carboxy-terminus of the human ARPKD protein fibrocystin can control STAT3 signalling by regulating SRC-activation. J. Cell. Mol. Med. 24, 14633–14638.
                                                                                                                  • Grundmann F, Muller RU, Hoyer-Allo KJR, Spath MR, Passmann E, Becker I, Pfister R, Baldus S, Benzing T, and Burst V (2020). Dietary restriction for prevention of contrast-induced acute kidney injury in patients undergoing percutaneous coronary angiography: a randomized controlled trial. Sci Rep 10, 5202.
                                                                                                                     
                                                                                                                  • Johnsen M, Kubacki T, Yeroslaviz A, Spath MR, Morsdorf J, Gobel H, Bohl K, Ignarski M, Meharg C, Habermann B, Altmuller J, Beyer A, Benzing T, Schermer B, Burst V, and Muller RU (2020). The Integrated RNA Landscape of Renal Preconditioning against Ischemia-Reperfusion Injury. J Am Soc Nephrol 31, 716-30.
                                                                                                                     
                                                                                                                  • Kann M, and Benzing T (2020). COVID-19 from the Perspective of Nephrologist. Deut Med Wochenschr 145, 1068-73.
                                                                                                                     
                                                                                                                  • Koehler S, Kuczkowski A, Kuehne L, Jungst C, Hoehne M, Grahammer F, Eddy S, Kretzler M, Beck BB, Hohfeld J, Schermer B, Benzing T, Brinkkoetter PT, and Rinschen MM (2020). Proteome Analysis of Isolated Podocytes Reveals Stress Responses in Glomerular Sclerosis. J Am Soc Nephrol 31, 544-59.
                                                                                                                     
                                                                                                                  • Matin M, Morgelin M, Stetefeld J, Schermer B, Brinkkoetter PT, Benzing T, Koch M, and Hagmann H (2020). Affinity-Enhanced Multimeric VEGF (Vascular Endothelial Growth Factor) and PlGF (Placental Growth Factor) Variants for Specific Adsorption of sFlt-1 to Restore Angiogenic Balance in Preeclampsia. Hypertension 76, 1176-84.
                                                                                                                     
                                                                                                                  • Meyer AM, Becker I, Siri G, Brinkkotter PT, Benzing T, Pilotto A, and Polidori MC (2020). The prognostic significance of geriatric syndromes and resources. Aging Clin Exp Res 32, 115-24.
                                                                                                                     
                                                                                                                  • Schermer B, Fabretti F, Damagnez M, Di Cristanziano V, Heger E, Arjune S, Tanner NA, Imhof T, Koch M, Ladha A, Joung J, Gootenberg JS, Abudayyeh OO, Burst V, Zhang F, Klein F, Benzing T, and Muller RU (2020). Rapid SARS-CoV-2 testing in primary material based on a novel multiplex RT-LAMP assay. PloS one 15, e0238612.
                                                                                                                     
                                                                                                                  • Unnersjo-Jess D, Butt L, Hohne M, Witasp A, Kuhne L, Hoyer PF, Patrakka J, Brinkkotter PT, Wernerson A, Schermer B, Benzing T, Scott L, Brismar H, and Blom H (2020). A Fast and Simple Clearing and Swelling Protocol for 3D In-Situ Imaging of the Kidney across Scales. Kidney Int 10.1016/j.kint.2020.10.039.
                                                                                                                     
                                                                                                                  • Suarez V, Norello D, Sen E, Todorova P, Hackl MJ, Huser C, Grundmann F, Kubacki T, Becker I, Peri A, and Burst V (2020). Impairment of Neurocognitive Functioning, Motor Performance, and Mood Stability in Hospitalized Patients With Euvolemic Moderate and Profound Hyponatremia. Am J Med 10.1016/j.amjmed.2019.12.056.
                                                                                                                  Prof. Dr. Thomas Benzing CMMC Cologne
                                                                                                                  Prof. Dr. Thomas Benzing

                                                                                                                  Clinic II of Internal Medicine

                                                                                                                  CMMC - Chair

                                                                                                                  CMMC - PI - C 02

                                                                                                                  +49 221 478 89536

                                                                                                                  +49 221 478 4833

                                                                                                                  Clinic II of Internal Medicine

                                                                                                                  Robert-Koch-Str. 21

                                                                                                                  50931 Cologne

                                                                                                                  https://cmmc.uni-koeln.de

                                                                                                                  CMMC Profile Page

                                                                                                                  Curriculum Vitae (CV)

                                                                                                                  Publications - Thomas Benzing

                                                                                                                  Link to PubMed

                                                                                                                  Publications - Matthias Hackl

                                                                                                                  Link to PubMed

                                                                                                                  Figure 1
                                                                                                                  Group Members

                                                                                                                  Linus Butt (Postdoc)
                                                                                                                  Julie Binz (Graduate Student)
                                                                                                                  Nelli Rutkowski (Graduate Student)