The role of Crumbs2 in podocyte function and disease.

• Applicant: Professor Dr. Thomas Weide
• Subject Area: Nephrology
• Term: since 2021
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 469263956

Kidney diseases affect more than 100 million people worldwide and are a major cause of morbidity and mortality. In many cases proteinuria precedes renal inflammation and renal failure. Thus, postmitotic podocytes of the renal filtration barrier are particularly interesting. They form highly branched cell protrusions (foot processes, FPs) to build the slit diaphragm (SD), a unique cell contacts of the glomerular filtration barrier (GFB) that prevents the loss of serum proteins into the urine. This indicates that podocyte depletion and/or SD injury causes most if not all forms of severe proteinuria. Especially studies of monogenetic forms of proteinuric renal diseases uncovered details about SD composition and resulted in a better understanding of pathogenic mechanisms explaining proteinuria on a molecular level. In a preceding project we therefore focused on Crumbs2 (Crb2) as some mutations in this gene are linked to inherited forms of steroid-resistant nephrotic syndrome (SRNS). Using mice lacking Crb2 exclusively in podocytes and cell lines expressing GFP-tagged versions of Crb2 wildtype and mutants, we identified Crb2 as a central SD component, organized in clusters and able to interact with the SD protein Nephrin and the intracellular adapter proteins Pals1. Moreover, our in vitro data showed that SRNS-mutants accumulate in the endoplasmic reticulum (ER) and trigger ER stress. Therefore, we hypothesize, that Crb2 is of similar relevance as Nephrin but with complementary or additional functions. To address this, we will investigate the Crb2 interactome of podocytes. In addition, we assume that the majority of Crb2-associated renal diseases are caused by a dysfunctional export of Crb2 to the cell surface or - in vivo - to the slit diaphragm. In other words: Crb2 mutants or variants that are improperly folded or factors like ER stress that indirectly inhibit Crb2 processing, will ultimately result in SD injury and proteinuria. Thus, we think, that ER stress is a major and perhaps underestimated co-factor in Crb2-associated renal diseases. These aspects will be address by cell biological approaches.The outcomes of this project will give us an in-depth understanding of the Crb2 biology in podocytes at the SD. This will be the basis for the development of a cell-based drug-screening systems, suitable for testing of the pathological potential of novel uncharacterized Crb2 protein variants as well as for evaluating the therapeutic (or toxic) potential of agents influencing Crb2 folding and export to the cell surface (or SD). Finally, an understanding of Crb2’s role at the SD may also be a starting point for gene therapeutic approaches for rare inherited forms of SRNS-podocytopathies (e.g. by using recombinant viruses for replacing non-functional Crb2 variants).

• DFG Programme: Research Grants