(I-331) Unraveling the Role of Formins in Podocyte Dynamics and Function

Chronic kidney disease could progress to end-stage kidney disease which is lethal. Podocytes, with foot processes attaching to the glomerular basement membrane, play a crucial role in kidney filtration. Injured podocytes were recently found to develop sarcomeres-like structures (SLS) in response to injury, with formins likely responsible for elongation and rearrangements. INF2, one of the formins, is widely expressed in kidney podocytes and tubules, with most mutations occurring there. It is suggested that formin proteins like INF2 are involved in maintaining the dynamic structure and stability of podocyte foot processes and the filtration slits. Maintaining the healthiness of podocytes and the integrity of the glomerular filtration barrier is crucial for overall kidney function. However, limited data exists on renal formins' role in podocyte dysfunction due to the hardships of studying podocytes outside of their native microenvironment, we therefore used a novel in vitro culture system of podocytes to study the functions of formins, with the use of differentiated podocyte cell lines and the help of siRNA inhibition to determine their functionality in the cytoskeleton organization and maintenance of podocytes.

Podocyte cell line culture and differentiation; To visualize the behavior of sarcomere like structures, podocyte cell lines were maintained at 33°C, and then incubated at 37°C for 7 days for differentiation. Last, the cells were trypsinized and seeded on glass bottom dishes for live imaging.

Fluorescent staining and imaging; Frozen mice unfixed kidney tissue was sectioned 7 micrometers thick using Leica cryostats, then were mounted on glass slides for immunostaining. The immunofluorescence staining consisted of using 2% BSA blocking solution to reduce nonspecific binding, then incubated overnight with primary antibodies, secondary antibodies were used to recognize the first antibody and its fluorescent dye for observation. Last, the samples were mounted and observed under fluorescent, confocal microscope.

Live imaging: Cells for live imaging were maintained in CO2-independent medium to preserve morphology and then observed under the confocal microscope.

Transfection of plasmid: 4mm cuvettes were used for the electroporation of INF2, RFP-actin and GFP-Myosin plasmids into differentiated podocyte cell lines to analyze the physiology and location of the proteins within the cells.

SiRNA inhibition of Formins: Chemical transfection of siRNA for INF2, FHOD3 and MDIA1 formins was employed on the differentiated podocyte cells lines using Lipofectamine 3000 Transfection Reagent to observe how silencing the formins affect the behavior of SLS’s.

Frozen mice unfixed kidney tissue was sectioned 7 micrometers thick using Leica cryostats, then were mounted on glass slides for immunostaining. Samples were analyzed under the confocal microscope, where it was observed that the formin INF2 (blue) colocalizes with the foot processes of podocytes (green).

Chemical transfection of siRNA was employed of the INF2 formin on differentiated podocyte cell lines. Then, the samples were fixed for fluorescence staining. It was observed that there is a colocalization between cortactin and the INF2 formin as the cell spreads in the surface. A higher signal of INF2 was perceived whenever cortactin, a marker for lamellipodia was present. At the same time, synaptopodin positive stress fibers were noticed to be avoiding the lamellipodia area, suggesting a potential role of INF2 for the early-stage formation of the SLSs.

Chemical transfection of siRNA of INF2, MDIA1 and FHOD3 formins was employed in differentiated podocyte cell lines. The cells were fixed for fluorescence staining of INF2, synaptopodin, and cortactin, where it was observed, that the arrangements of sarcomere-like structures are independent of formins, meaning, that there is presence of actin-myosin arrangements even when formins are silenced. This suggest that the presence of formins in cells are not strictly required for SLSs development.

However, after using the bright-field to compare the cell number, it is suggested that there is a potential role of formins in attachment as it heavily decreased in silenced cells.

The potential of cortactin and INF2 for cell regeneration in a mechanobiological approach:

·      Gene Therapy: Modulating the expression of cortactin and INF2 genes could be used to enhance or direct cell migration, proliferation, and tissue remodeling during regeneration.

·      Biomaterials Design: Developing biomaterials that can release or interact with cortactin and INF2 could provide localized cues to promote cell attachment, migration, and differentiation at the site of tissue injury.

·      Tissue Engineering: Incorporating cortactin and INF2 into tissue-engineered constructs may improve the functionality and integration of engineered tissues with the host tissue during regeneration.