Linking polarity signaling to mechanotransduction in glomerular podocytes
Final Report Abstract
Podocytes are highly specialized epithelial cells of the kidney filtration barrier. Due to their position and function, they are exposed to constant biomechanical forces such as shear stress and hydrostatic pressure. These forces increase during disease like diabetes and hypertension, resulting in podocyte injury and finally loss. To counterbalance losses to the podocyte population, remaining cells exhibit adaptive mechanisms that include hypertrophic growth; this is important as podocytes are post-mitotic and cannot be replenished through cell division. However, the mechanisms by which biomechanical forces are sensed and transduced into an adaptive and protective molecular response remain largely unknown. Previous studies revealed an increase of the mechanosensor Filamin B in different in vitro and in vivo injury models as well as human FSGS patient tissue. But, whether this increase in protein level has a protective or pathological role remains unknown. Hence within this project I explored the hypothesis that the mechanosensor Cheerio, the Filamin homologe in the fly, is central to the ‘mechano-protective’ mechanism during injury using the Drosophila nephrocyte model. Cheerio is expressed in nephrocytes and co-localizes with Duf, the NEPH homologe, suggesting Cheerio and Filamin B are part of the slit diaphragm multi-protein complexe. To mimic the increased Filamin B levels observed during injury, I expressed an over-active mechanosensor region variant of Cheerio, which resulted in a significant hypertrophy phenotype and caused a rescue of filtration function in injured nephrocytes. Interestingly, the expression of a closed variant, which needs higher biomechanical forces to be activated, also presented with a partial rescue of filtration function. Additional analysis revealed, that Cheerio is activated during injury and localizes to the cell periphery as previously published. This translocation was also observed during injury in cells expressing the closed Cheerio variant, suggesting an activation during nephrocyte injury. To investigate whether this protective role is conserved throughout evolution, I generated flies expressing human Filamin B. The presence of human Filamin B in injured nephrocytes also resulted in a partial rescue of the filtration function, confirming the mechano-protective role. To delineate the mechano-protective pathway acting downstream to Cheerio I studied the candidates: TOR, WNT and YAP, as all of them were previously linked to cell growth. Activation of these pathways resulted in nephrocyte hypertrophy phenotype. Interestingly, TOR and YAP repression reversed the Cheerio-mediated hypertrophy, suggesting TOR and YAP to be novel downstream targets of Cheerio and to be responsible for the hypertrophy phenotype. The repression of WNT however, resulted in the absence of nephrocyte confirming the essential role of WNT in nephrocyte and podocyte differentiation. Additional experiments revealed a decrease in cell size when repressing TOR and YAP exclusively. Based on these findings I cannot exclude that TOR and YAP act independtly from Cheerio. As the ecessive expression and activation of other mechanosensors such as TRPC6 causes a podocyte phenotype and glomerular disease, I analysed the effect of further increase of Cheerio and Filamin B protein levels on nephrocyte biology. These data showed that although Cheerio and Filamin B mediate a mechano-protective role in the face of injury, their excessive expression resulted in a severe morphological and functional phenotype, emphasizing the need for tight control of activity levels. Taken together, my data shows that the expression and activation of Cheerio and human Filamin B induces hypertrophic growth as a mechano-protective response to nephrocyte injury. This hypertrophy phenotype might be mediated via TOR and YAP signalling. To exhibit this mechano-protective role the protein levels of Cheerio and human Filamin B need to be tightly controlled. Future studies should exploit these findings and use Cheerio and human Filamin B as a therapeutic target for glomerular disease.
Publications
- Drosophila Filamin exhibits a mechano-protective role during nephrocyte injury via induction of hypertrophic growth
Koehler, S and Denholm, B.
(See online at https://doi.org/10.1101/2021.08.20.457058) - The mechano-sensitive ion channel Piezo mediates Rho activation and actin stress fibre formation in Drosophila nephrocytes
Koehler, S and Denholm, B
(See online at https://doi.org/10.1101/2021.10.23.465463)
