Mutations in Complement Factor H Impair Alternative Pathway Regulation on Mouse Glomerular Endothelial Cells in Vitro

Area of Science:

• Immunology
• Molecular Biology
• Renal Medicine

Background:

• Complement factor H (FH) regulates the complement system, preventing host tissue damage.
• FH interacts with glomerular endothelium, crucial for kidney health.
• FH dysfunction is implicated in renal diseases like atypical hemolytic uremic syndrome (aHUS).

Purpose of the Study:

• To identify glycosaminoglycans (GAGs) mediating FH binding to mouse glomerular endothelial cells (mGEnCs).
• To assess the functional impact of aHUS-associated FH mutations on complement regulation.
• To develop a novel assay for screening FH mutant pathogenicity.

Main Methods:

• ELISA to measure FH and FH19-20 binding to mGEnCs.
• Flow cytometry to quantify C3b deposition on mGEnCs.
• GAG modification (desulfation, digestion) and competition assays.

Main Results:

• FH and FH19-20 bind dose-dependently to mGEnCs, with binding enhanced by TNF-α.
• Heparan sulfate (HS) is the primary GAG mediating FH binding; other GAGs are not involved.
• aHUS-associated FH mutants exhibit reduced ability to inhibit complement activation on mGEnCs.

Conclusions:

• FH binding to glomerular endothelial cells is specifically mediated by HS.
• HS plays a critical role in FH's protective function in the kidney.
• A new assay allows for patient serum-independent pathogenicity screening of FH mutants.