Kidney Week

Abstract: FR-PO944

Studying the Role of Fibronectin in Mechanically Stressed Podocytes

Session Information

• Glomerular Diseases: Podocyte Biology - II
  November 08, 2019 | Location: Exhibit Hall, Walter E. Washington Convention Center
  Abstract Time: 10:00 AM - 12:00 PM

Category: Glomerular Diseases

• 1204 Podocyte Biology

Authors

• Kliewe, Felix, University Medicine Greifswald, Greifswald, Germany

Felix Kliewe,

• Kaling, Sören, University Medicine Greifswald, Greifswald, Germany

Sören Kaling,

• Lötzsch, Henriette, University Medicine Greifswald, Greifswald, Germany

Henriette Lötzsch,

• Artelt, Nadine, University Medicine Greifswald, Greifswald, Germany

Nadine Artelt,

• Schindler, Maximilian, University Medicine Greifswald, Greifswald, Germany

Maximilian Schindler,

• Rogge, Henrik, University Medicine Greifswald, Greifswald, Germany

Henrik Rogge,

• Schröder, Sindy, University Medicine Greifswald, Greifswald, Germany

Sindy Schröder,

• Scharf, Christian, University Medicine Greifswald, Greifswald, Germany

Christian Scharf,

• Amann, Kerstin U., University Hospital Erlangen, Erlangen, Germany

Kerstin U. Amann,

• Daniel, Christoph, University Hospital Erlangen, Erlangen, Germany

Christoph Daniel,

• Lindenmeyer, Maja, University Medical Center Hamburg-Eppendorf, Hamburg, Germany

Maja Lindenmeyer,

• Cohen, Clemens D., Klinikum Munchen, Munchen, Germany

Clemens D. Cohen,

• Endlich, Karlhans, University Medicine Greifswald, Greifswald, Germany

Karlhans Endlich,

• Endlich, Nicole, University Medicine Greifswald, Greifswald, Germany

Nicole Endlich,

Background

Glomerular hypertension induces mechanical load to podocytes in situ, often resulting in podocyte detachment and the development of glomerulosclerosis. Although it is well known that podocytes are mechanosensitive, the mechanosensor and mechanotransducer, respectively, are still unknown. Extracellular matrix proteins could function as such a mechanosensor.

The objective was to clarify the potential significance of the extracellular matrix protein fibronectin which became up-regulated in cultured podocytes 2-fold after the exposure to mechanical strain. Furthermore, biopsies of patients suffering from diabetic nephropathy were used to study the expression of fibronectin.

Methods

Mouse podocytes were cultured on silicone membranes that were connected to the stretch apparatus for three days (0.5 Hz and 5% extension). To study the role of fibronectin in cultured podocytes under mechanical stretch, fibronectin was knocked down (Fn1 KD) by specific siRNAs. Additionally, we established a fibronectin knockout podocyte cell line (Fn1 KO) by CRISPR/Cas9. LC-MS as well as qRT-PCR were performed from mechanically stretched podocytes.

Results

Here, we demonstrate that the extracellular matrix protein fibronectin is essential for the attachment of podocytes during mechanical stress. By qRT-PCR as well as by LC-MS, we found a significant up-regulation of fibronectin in cultured podocytes after three days of mechanical stretch. Additionally, we observed a significant loss of Fn1 KD as well as Fn1 KO podocytes (> 80%) compared to controls in the presence of mechanical strain. Beside this, a significant down-regulation of the focal adhesion proteins talin, vinculin and paxillin and a reduced cell spreading was observed in Fn1 KO podocytes indicating an important role of fibronectin for the adhesion of cultured podocytes. Analyzing kidney biopsies of patients suffering from diabetic nephropathy, we found a significant up-regulation of fibronectin especially in podocytes in contrast to control biopsies.

Conclusion

Fibronectin plays an important role in the adaptation and adhesion of cultured podocytes in the presence of mechanical stretch and could serve as a mechanosensor in podocytes.