Kidney Week

Abstract: TH-OR062

High Glucose and High Osmolarity Modulate Function of FRMD3/Protein 4.1O: A Candidate Gene of Diabetic Nephropathy

Session Information

• Diabetic Kidney Disease: Discovery of Molecular Mechanisms
  November 07, 2019 | Location: 207, Walter E. Washington Convention Center
  Abstract Time: 05:18 PM - 05:30 PM

Category: Diabetic Kidney Disease

• 601 Diabetic Kidney Disease: Basic

Authors

• Koenigshausen, Eva, Medical Faculty Heinrich-Heine-University Düsseldorf, Düsseldorf, Germany

Eva Koenigshausen, MD

• Rieckmann, Sonja, Medical Faculty Heinrich-Heine-University Düsseldorf, Düsseldorf, Germany

Sonja Rieckmann,

• Bajraktarevic, Aida, Medical Faculty Heinrich-Heine-University Düsseldorf, Düsseldorf, Germany

Aida Bajraktarevic,

• Ohlsson, Sinja, Medical Faculty Heinrich-Heine-University Düsseldorf, Düsseldorf, Germany

Sinja Ohlsson,

• Schönberger, Marie, Medical Faculty Heinrich-Heine-University Düsseldorf, Düsseldorf, Germany

Marie Schönberger,

• Wiech, Thorsten, Department of Pathology, University Hospital Hamburg Eppendorf, Hamburg, Germany

Thorsten Wiech,

• Quack, Ivo, Klinikum Konstanz, Konstanz, Germany

Ivo Quack,

• Haller, Hermann G., Medical Faculty, Hannover, Germany

Hermann G. Haller,

• Rump, Lars C., Medical Faculty Heinrich-Heine-University Düsseldorf, Düsseldorf, Germany

Lars C. Rump,

• Schiffer, Mario, University Hospital Erlangen, Erlangen, Germany

Mario Schiffer,

• Sellin, Lorenz, Medical Faculty Heinrich-Heine-University Düsseldorf, Düsseldorf, Germany

Lorenz Sellin,

Background

FRMD3 has been proposed as a candidate gene for susceptibility of diabetic nephropathy (DN) in type 1 diabetes. FRMD3 encodes for protein 4.1O, which is a member of the 4.1 protein family. The molecular function of FRMD3/protein 4.1O is unknown so far.
Albuminuria is the earliest sign of DN and results from a defect in the glomerular filtration barrier. Linkage of the slit diaphragm protein nephrin to the actin cytoskeleton via adapter proteins are essential for the integrity of the glomerular slit diaphragm.

Methods

RNA was isolated from human podocytes and qPCR for FRMD3 was performed. Nephrin and protein 4.1O were stained in mouse glomeruli. In Cos7 cells, protein 4.1O and actin were visualized via immunofluorescence. Zebrafish larvae were treated with morpholinos against the orthologue of FRMD3 in zebrafish. Injection of fluorescently labeled FITC-dextran was monitored via eye fluorescence. A reduction of fluorescence was an indirect sign of glomerular tracer loss. Cells expressing protein 4.1O, its truncations and nephrin were subjected to cell lysis. Co-immunoprecipitation and Western blot analysis were performed. Kidney samples from patients with T1DN or T2DN were stained for protein 4.1O. Cells were treated with different glucose concentrations and mannitol for osmolarity control.

Results

Protein 4.1O is expressed in human podocytes. Protein 4.1O interacts with nephrin and actinin vitro andin vivo. Injection of frmd3 paralogue morpholinos in zebrafish larvae leads to zebrafish yolk sac edema, slit diaphragm disruption and increase in glomerular permeability. The increase in glomerular permeability can be rescued by reconstitution of protein 4.1O AA 506-553, the nephrin binding domain. Protein 4.1O expression is increased in human T1 and T2DN. High glucose levels increase protein 4.1O expression while high osmolarity increases nephrin protein 4.1O interaction and protein 4.1O threonine phosphorylation.

Conclusion

Protein 4.1O is a novel linker of nephrin to the actin cytoskeleton and essential for the glomerular filtration barrier. High glucose increases expression of protein 4.1O while high osmolarity leads to posttranslational modifications on protein 4.1O mediating an increased interaction with nephrin.