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 signalling 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. elegans revealed 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. elegans and Drosophila as 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 signalling 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 signalling 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 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.        

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.

Selected publications

Koehler, S., Brähler, S., Kuczkowski, A., Binz, J., Hackl, M.J., Hagmann, H., Höhne, M., Vogt, M.C., Wunderlich, C.M., Wunderlich, F.T., Schweda, F., Schermer, B., Benzing, T., Brinkkoetter, P.T. (2016) Single and Transient Ca2+ Peaks in Podocytes do not induce Changes in Glomerular Filtration and Perfusion, Sci Rep. 6, 35400

Rinschen, M.M., Pahmeyer, C., Pisitkun, T., Schnell, N., Wu, X., Maaß, M., Bartram, M.P., Lamkemeyer, T., Schermer, B., Benzing, T., Brinkkoetter, P.T. (2015) Comparative phosphoproteomic analysis of mammalian glomeruli reveals conserved podocin C-terminal phosphorylation as a determinant of slit diaphragm complex architecture. Proteomics. 15, 1326-31

Rinschen, M.M., Bharill, P., Wu, X., Kohli, P., Reinert, M.J., Kretz, O., Saez, I., Schermer, B., Höhne, M., Bartram, M.P., Aravamudhan, S., Brooks, B.R., Vilchez, D., Huber, T.B., Müller, R.U., Krüger, M., Benzing, T. (2016). The ubiquitin ligase Ubr4 controls stability of podocin/MEC-2 supercomplexes. Hum Mol Genet 25, 1328-44

Rinschen, M.M., Wu, X., König, T., Pisitkun, T., Hagmann, H., Pahmeyer, C., Lamkemeyer, T., Kohli, P., Schnell, N., Schermer, B., Dryer, S., Brooks, B.R., Beltrao, P., Krueger, M., Brinkkoetter, P.T., Benzing, T. (2014). Phosphoproteomic analysis reveals regulatory mechanisms at the kidney filtration barrier. J Am Soc Nephrol. 25, 1509-22 


Prof. Dr. Thomas Benzing

Chair CMMC

Prof. Dr. Thomas Benzing

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

cmmc-office@uni-koeln.de

Work +49 221 478 89536

Fax (Work) +49 221 478 4833

CMMC Research Building
Robert-Koch-Str. 21
50931 Cologne

https://cmmc.uni-koeln.de

Publications - Thomas Benzing

Link to PubMed


Dr. Matthias Hackl

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

Dr. Matthias Hackl

Co-Principal Investigator C 2

matthias.hackl@uk-koeln.de

Publications - Matthias Hackl

Link to PubMed

Group Members

Julia Binz (doctoral student)
Linus Butt (PostDoc)

Figure 1

Calcium wave travelling over the glomerulus after laser injury of a single podocyte. Images from a time lapse movie of in vivo imaging of a glomerulus of a mouse expressing the fluorescent calcium indicator GCaMP3 specifically in podocytes. Increases in intracellular calcium levels are visualized by increased fluorescence emission of the calcium indicator. (A) before laser injury, (B) directly after laser injury, (C) calcium wave travelling from the site of injury to the other end of the glomerulus.