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.

We will study :

• how endothelial-cell derived NO modulates podocyte homeostasis
• how podocytes respond to signals from endothelial cells induced by increased shear stress
• how mechanosensation in podocytes affects these endothelial cell dependent cGMP and calcium responses in podocytes

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.