Center for Molecular Medicine Cologne

Thomas Benzing / Matthias Hackl - C 2

The role of slit diaphragm signalling of podocytes

Over the past decade we have pioneered the concept of slit diaphragm signalling and showed that podocyte viability and function is critically controlled through proteins that were initially thought to only serve as part of a filtration barrier. This project embarks on our previous work to develop innovative strategies and novel tools to study podocyte signaling in vivo by combining functional proteomics and multiphoton imaging technologies in genetically engineered animals.

Introduction

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. Over the past decade we showed that proteins residing eat the slit diaphragm form an evolutionarily conserved mechanosensitive multiprotein complex that controls podocyte viability and function. Studies in C. elegansrevealed a role for lipid-protein interactions at the slit diaphragm complex in mechanosensation and identified new components of the megadalton lipid-protein supercomplex at the filtration slit. These studies initiated new spurt of research worldwide that profoundly changed our view of glomerular filtration, the regulation of kidney function and our understanding of renal disease. However, most of these studies were based on cell culture experiments and the model organisms C. elegansand Drosophilaas podocyte signalling dynamics could not be studied in living mice. Over the past three years we have now generated new approaches to functionally characterize proteome and phosphoproteome changes as well as illuminate the signaling dynamics in living animals. Several essential questions can now be addressed. Here we focus on three unsolved questions in this new field of podocyte research:

  1. How does cGMP signaling affect intracellular calcium dynamics in podocytes? 
  2. How do intracellular cGMP and calcium levels change in podocytes stimulated with angiotensin II and purinergic activation? 
  3. Do alterations in cGMP and calcium affect glomerular circulation, permeation and convective flow and how does this change in podocyte disease models? 

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

In vivo imaging 

Podocyte research has been hampered by the lack of suitable in vitromodels 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.         

Perspectives

We are convinced that our experiments will help clarify the contribution of alterations of podocyte dynamics to CKD and enable us to attract additional new third-party funding as a result of the development of these novel tools for podocyte research.

Assady S., Benzing T., Kretzler M., Skoretzki K. (2019) Glomerular podocytes in kidney health and disease. Lancet 393, 856-858

Karaiskos N., Rahmatollahi M., Boltengagen A., Liu H., Hoehne M., Rinschen M.M., Schermer B., Benzing T., Rajewsky N., Kocks C., Kann M., Müller R.U. (2018) A single transcriptome atlas of the mouse glomerulus. J Am Soc Nephrol 29, 2060-2068

Schroeter C.B., Koehler S., Kann M., Schermer B., Benzing T., Brinkkoetter P.T., Rinschen M.M. (2018) Protein half-life determines expression of proteostatic networks in podocyte differentiation. FASEB J 32, 4696-4713

Höhne M., Frese C.K., Grahammer F., Dafinger C., Ciarimboli G., Butt L., Binz J., Hackl M.J., Rahmatollahi M., Kann M., Schneider S., Altintas M.M., Schermer B., Reinheckel T., Göbel H., Reiser J., Huber T.B., Kramann R., Seeger-Nukpezah T., Liebau M.C., Beck B.B., Benzing T., Beyer A., Rinschen M.M. (2018) Single nephron proteomes connect morphology and function in proteinuric kidney disease. Kidney Int 93, 1308-1319. 

Rinschen M.M., Hoppe A.K., Grahammer F., Kann M., Völker L.A., Schurek E.M., Binz J., Höhne M., Demir F., Malisic M., Huber T.B., Kurschat C., Kizhakkedathu J.N., Schermer B., Huesgen P.F., Benzing T. (2017) N-degradomic analysis reveals a proteolytic network processing the podocyte cytoskeleton. J Am Soc Nephrol 28, 2867-2878.

In revision:
Binz J., Jüngst C., Rinschen M.M., Koehler S., Zentis P., Schauss A., Schermer B., Benzing T., Hackl M.J. Injured podocytes are sensitized to Angiotensin II induced calcium signalling.

Butt L., Unnersjö-Jess D., Höhne M., Edwards A., Binz J., Reilly D., Hahnfeld R., Ziegler V., Fremter K., RinschenM.M., Helmstädter M., Eber L.K., Castorp H., Hackl M.J., Walz G., Brinkkötter P.T., Liebau M.C., Tory K., Brismar H., Blom H., Schermer B., Benzing T. Molecular Mechanism of Proteinuria.

 

Assady, S., Benzing, T., Kretzler, M., and Skorecki, K.L. (2019). Glomerular podocytes in kidney health and disease. Lancet 393, 856-8.

Braun, F., Blomberg, L., Brodesser, S., Liebau, M.C., Schermer, B., Benzing, T., and Kurschat, C.E. (2019). Enzyme Replacement Therapy Clears Gb3 Deposits from a Podocyte Cell Culture Model of Fabry Disease but Fails to Restore Altered Cellular Signaling. Cell Physiol Biochem 52, 1139-50.

Braun, F., Suarez, V., Dinter, J., Haneder, S., Quaas, A., Benzing, T., Nierhoff, D., and Muller, R.U. (2019). Successful use of TNFalpha blockade in a severe case of idiopathic non-granulomatous ulcerative jejunoileitis associated with thrombotic thrombocytopenic purpura. BMJ Open Gastroenterol 6, e000252.

Brinkkoetter, P.T., Bork, T., Salou, S., Liang, W., Mizi, A., Ozel, C., Koehler, S., Hagmann, H.H., Ising, C., Kuczkowski, A., Schnyder, S., Abed, A., Schermer, B., Benzing, T., Kretz, O., Puelles, V.G., Lagies, S., Schlimpert, M., Kammerer, B., Handschin, C., Schell, C., and Huber, T.B. (2019). Anaerobic Glycolysis Maintains the Glomerular Filtration Barrier Independent of Mitochondrial Metabolism and Dynamics. Cell Rep 27, 1551-66 e5.

Burgmaier, K., Kilian, S., Bammens, B., Benzing, T., Billing, H., Buscher, A., Galiano, M., Grundmann, F., Klaus, G., Mekahli, D., Michel-Calemard, L., Milosevski-Lomic, G., Ranchin, B., Sauerstein, K., Schaefer, S., Shroff, R.,

Ignarski, M., Rill, C., Kaiser, R.W.J., Kaldirim, M., Neuhaus, R., Esmaillie, R., Li, X., Klein, C., Bohl, K., Petersen, M., Frese, C.K., Hohne, M., Atanassov, I., Rinschen, M.M., Hopker, K., Schermer, B., Benzing, T., Dieterich, C., Fabretti, F., and Muller, R.U. (2019). The RNA-Protein Interactome of Differentiated Kidney Tubular Epithelial Cells. J Am Soc Nephrol 30, 564-76.

Jain, M., Kaiser, R.W.J., Bohl, K., Hoehne, M., Gobel, H., Bartram, M.P., Habbig, S., Muller, R.U., Fogo, A.B., Benzing, T., Schermer, B., Hopker, K., and Slaats, G.G. (2019). Inactivation of Apoptosis Antagonizing Transcription Factor in tubular epithelial cells induces accumulation of DNA damage and nephronophthisis. Kidney Int 95, 846-58.

Kaiser, R.W.J., Ignarski, M., Van Nostrand, E.L., Frese, C.K., Jain, M., Cukoski, S., Heinen, H., Schaechter, M., Seufert, L., Bunte, K., Frommolt, P., Keller, P., Helm, M., Bohl, K., Hohne, M., Schermer, B., Benzing, T., Hopker, K., Dieterich, C., Yeo, G.W., Muller, R.U., and Fabretti, F. (2019). A protein-RNA interaction atlas of the ribosome biogenesis factor AATF. Sci Rep 9, 11071.

Meyer, A.M., Becker, I., Siri, G., Brinkkotter, P.T., Benzing, T., Pilotto, A., and Polidori, M.C. (2019). The prognostic significance of geriatric syndromes and resources. Aging Clin Exp Res 10.1007/s40520-019-01168-9.

Meyer, A.M., Siri, G., Becker, I., Betz, T., Bodecker, A.W., Robertz, J.W., Krause, O., Benzing, T., Pilotto, A., and Polidori, M.C. (2019). The Multidimensional Prognostic Index in General Practice: one-year follow-up study. Int J Clin Pract 10.1111/ijcp.13403, e13403.

Phipps, E.A., Thadhani, R., Benzing, T., and Karumanchi, S.A. (2019). Author Correction: Pre-eclampsia: pathogenesis, novel diagnostics and therapies. Nat Rev Nephrol 15, 386.

Phipps, E.A., Thadhani, R., Benzing, T., and Karumanchi, S.A. (2019). Pre-eclampsia: pathogenesis, novel diagnostics and therapies. Nat Rev Nephrol 15, 275-89.

Schermer, B., and Benzing, T. (2019). Genome Editing with CRISPR/Cas9: First Steps Towards a new Era in Medicine? Deutsche Medizinische Wochenschrift 144, 276-81.

Spath, M.R., Bartram, M.P., Palacio-Escat, N., Hoyer, K.J.R., Debes, C., Demir, F., Schroeter, C.B., Mandel, A.M., Grundmann, F., Ciarimboli, G., Beyer, A., Kizhakkedathu, J.N., Brodesser, S., Gobel, H., Becker, J.U., Benzing, T., Schermer, B., Hohne, M., Burst, V., Saez-Rodriguez, J., Huesgen, P.F., Muller, R.U., and Rinschen, M.M. (2019). The proteome microenvironment determines the protective effect of preconditioning in cisplatin-induced acute kidney injury. Kidney Int 95, 333-49.

Volker, L.A., Burkert, K., Scholten, N., Grundmann, F., Kurschat, C., Benzing, T., Hampl, J., Becker, J.U., and Muller, R.U. (2019). A case report of recurrent membranoproliferative glomerulonephritis after kidney transplantation due to ventriculoatrial shunt infection. BMC Nephrol 20, 296.

Volker, L.A., Ehren, R., Grundmann, F., Benzing, T., Weber, L.T., and Brinkkotter, P.T. (2019). A newly established clinical registry of minimal change disease and focal and segmental glomerulosclerosis in Germany. Nephrol Dial Transplant 10.1093/ndt/gfz046.

 

 

Dafinger C, Rinschen MM, Borgal L, Ehrenberg C, Basten SG, Franke M, Hohne M, Rauh M, Gobel H, Bloch W, Wunderlich FT, Peters DJM, Tasche D, Mishra T, Habbig S, Dotsch J, Muller RU, Bruning JC, Persigehl T, Giles RH, Benzing T, Schermer B, and Liebau MC (2018). Targeted deletion of the AAA-ATPase Ruvbl1 in mice disrupts ciliary integrity and causes renal disease and hydrocephalus. Exp Mol Med 50, 75.

Grundmann F, Muller RU, Reppenhorst A, Hulswitt L, Spath MR, Kubacki T, Scherner M, Faust M, Becker I, Wahlers T, Schermer B, Benzing T, and Burst V (2018). 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.

Hagmann H, Mangold N, Rinschen MM, Koenig T, Kunzelmann K, Schermer B, Benzing T, and Brinkkoetter PT (2018). Proline-dependent and basophilic kinases phosphorylate human TRPC6 at serine 14 to control channel activity through increased membrane expression. FASEB J 32, 208-219.

Hohne M, Frese CK, Grahammer F, Dafinger C, Ciarimboli G, Butt L, Binz J, Hackl MJ, Rahmatollahi M, Kann M, Schneider S, Altintas MM, Schermer B, Reinheckel T, Gobel H, Reiser J, Huber TB, Kramann R, Seeger-Nukpezah T, Liebau MC, Beck BB, Benzing T, Beyer A, and Rinschen MM (2018). Single-nephron proteomes connect morphology and function in proteinuric kidney disease. Kidney Int 93, 1308-1319.

Karaiskos N, Rahmatollahi M, Boltengagen A, Liu H, Hoehne M, Rinschen M, Schermer B, Benzing T, Rajewsky N, Kocks C, Kann M, and Muller RU (2018). A Single-Cell Transcriptome Atlas of the Mouse Glomerulus. J Am Soc Nephrol 10.1681/ASN.2018030238.

Makitie, R.E., Hackl, M., Niinimaki, R., Kakko, S., Grillari, J., and Makitie, O. (2018). Altered MicroRNA Profile in Osteoporosis Caused by Impaired WNT Signaling. J Clin Endocrinol Metab 103, 1985-96.

Muller RU, and Benzing T (2018). Cystic Kidney Diseases From the Adult Nephrologist's Point of View. Front Pediatr 6, 65.

Rinschen MM, Godel M, Grahammer F, Zschiedrich S, Helmstadter M, Kretz O, Zarei M, Braun DA, Dittrich S, Pahmeyer C, Schroder P, Teetzen C, Gee H, Daouk G, Pohl M, Kuhn E, Schermer B, Kuttner V, Boerries M, Busch H, Schiffer M, Bergmann C, Kruger M, Hildebrandt F, Dengjel J, Benzing T, and Huber TB (2018). A Multi-layered Quantitative In Vivo Expression Atlas of the Podocyte Unravels Kidney Disease Candidate Genes. Cell Rep 23, 2495-2508.

Schroeter CB, Koehler S, Kann M, Schermer B, Benzing T, Brinkkoetter PT, and Rinschen MM (2018). Protein half-life determines expression of proteostatic networks in podocyte differentiation. FASEB J 10.1096/fj.201701307R, fj201701307R.

Siedek F, Persigehl T, Mueller RU, Burst V, Benzing T, Maintz D, and Haneder S (2018). Assessing renal changes after remote ischemic preconditioning (RIPC) of the upper extremity using BOLD imaging at 3T. MAGMA 31, 367-374.

Spath MR, Bartram MP, Palacio-Escat N, Hoyer KJR, Debes C, Demir F, Schroeter CB, Mandel AM, Grundmann F, Ciarimboli G, Beyer A, Kizhakkedathu JN, Brodesser S, Gobel H, Becker JU, Benzing T, Schermer B, Hohne M, Burst V, Saez-Rodriguez J, Huesgen PF, Muller RU, and Rinschen MM (2018). The proteome microenvironment determines the protective effect of preconditioning in cisplatin-induced acute kidney injury. Kidney Int 10.1016/j.kint.2018.08.037.

Volker LA, Maar BA, Pulido Guevara BA, Bilkei-Gorzo A, Zimmer A, Bronneke H, Dafinger C, Bertsch S, Wagener JR, Schweizer H, Schermer B, Benzing T, and Hoehne M (2018). Neph2/Kirrel3 regulates sensory input, motor coordination, and home-cage activity in rodents. Genes Brain Behav 10.1111/gbb.12516, e12516.

Welcker D, Jain M, Khurshid S, Jokic M, Hohne M, Schmitt A, Frommolt P, Niessen CM, Spiro J, Persigehl T, Wittersheim M, Buttner R, Fanciulli M, Schermer B, Reinhardt HC, Benzing T, and Hopker K (2018). AATF suppresses apoptosis, promotes proliferation and is critical for Kras-driven lung cancer. Oncogene 37, 1503-1518.

 

 

Benzing T (2017). Differential Diagnosis of Cystic Kidney. Nephrologe 12, 2-3.

Ebner K, Dafinger C, Ortiz-Bruechle N, Koerber F, Schermer B, Benzing T, Dotsch J, Zerres K, Weber LT, Beck BB, and Liebau MC (2017). Challenges in establishing genotype-phenotype correlations in ARPKD: case report on a toddler with two severe PKHD1 mutations. Pediatr Nephrol 32, 1269-73.

Franke M, Baessler B, Vechtel J, Dafinger C, Hohne M, Borgal L, Gobel H, Koerber F, Maintz D, Benzing T, Schermer B, and Persigehl T (2017). Magnetic resonance T2 mapping and diffusion-weighted imaging for early detection of cystogenesis and response to therapy in a mouse model of polycystic kidney disease. Kidney Int 92, 1544-54.

Grahammer F, Ramakrishnan SK, Rinschen MM, Larionov AA, Syed M, Khatib H, Roerden M, Sass JO, Helmstaedter M, Osenberg D, Kuhne L, Kretz O, Wanner N, Jouret F, Benzing T, Artunc F, Huber TB, and Theilig F (2017). mTOR Regulates Endocytosis and Nutrient Transport in Proximal Tubular Cells. J Am Soc Nephrol 28, 230-41.

Hagmann H, Mangold N, Rinschen MM, Koenig T, Kunzelmann K, Schermer B, Benzing T, and Brinkkoetter PT (2017). Proline-dependent and basophilic kinases phosphorylate human TRPC6 at serine 14 to control channel activity through increased membrane expression. FASEB J 10.1096/fj.201700309R.

Koehler S, Brahler S, Braun F, Hagmann H, Rinschen MM, Spath MR, Hohne M, Wunderlich FT, Schermer B, Benzing T, and Brinkkoetter PT (2017). Construction of a viral T2A-peptide based knock-in mouse model for enhanced Cre recombinase activity and fluorescent labeling of podocytes. Kidney Int 91, 1510-7.

Kohli P, Hohne M, Jungst C, Bertsch S, Ebert LK, Schauss AC, Benzing T, Rinschen MM, and Schermer B (2017). The ciliary membrane-associated proteome reveals actin-binding proteins as key components of cilia. EMBO Rep 18, 1521-35.

Rinschen MM, Grahammer F, Hoppe AK, Kohli P, Hagmann H, Kretz O, Bertsch S, Hohne M, Gobel H, Bartram MP, Gandhirajan RK, Kruger M, Brinkkoetter PT, Huber TB, Kann M, Wickstrom SA, Benzing T, and Schermer B (2017). YAP-mediated mechanotransduction determines the podocyte's response to damage. Sci Signal 10.

Siedek F, Persigehl T, Mueller RU, Burst V, Benzing T, Maintz D, and Haneder S (2017). Assessing renal changes after remote ischemic preconditioning (RIPC) of the upper extremity using BOLD imaging at 3T. MAGMA 10.1007/s10334-017-0658-4.

Voskarides K, Stefanou C, Pieri M, Demosthenous P, Felekkis K, Arsali M, Athanasiou Y, Xydakis D, Stylianou K, Daphnis E, Goulielmos G, Loizou P, Savige J, Hohne M, Volker LA, Benzing T, Maxwell PH, Gale DP, Gorski M, Boger C, Kollerits B, Kronenberg F, Paulweber B, Zavros M, Pierides A, and Deltas C (2017). A functional variant in NEPH3 gene confers high risk of renal failure in primary hematuric glomerulopathies. Evidence for predisposition to microalbuminuria in the general population. PLoS One 12, e0174274.

 

 

Prof. Dr. Thomas Benzing CMMC Cologne
Prof. Dr. Thomas Benzing

Chair - CMMC

Dept. II of Internal Medicine / RG location - CECAD Principal Investigator C 2

+49 221 478 89536

+49 221 478 4833

Chair - CMMC

Robert-Koch-Str. 21

50931 Cologne

https://cmmc.uni-koeln.de

CMMC Profile Page

Curriculum Vitae (CV)

Publications - Thomas Benzing

Link to PubMed

Dr. Matthias Hackl CMMC Cologne
Dr. Matthias Hackl

Dept. II of Internal Medicine / RG location - CECAD Building

Co-Principal Investigator C 2

Publications - Matthias Hackl

Link to PubMed

Group Members

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

Figure 1