Kidney Week

Abstract: FR-PO1067

Glomerular Endothelial Cells Lacking Neonatal Fc Receptor Have Decreased Major Histocompatibility Complex Type II Expression Due to Decreased Transcriptional Signaling

Session Information

• Glomerular Diseases: Immunology and Inflammation - II
  October 26, 2018 | Location: Exhibit Hall, San Diego Convention Center
  Abstract Time: 10:00 AM - 12:00 PM

Category: Glomerular Diseases

• 1202 Glomerular Diseases: Immunology and Inflammation

Authors

• Dylewski, James F., University of Colorado, Denver, Aurora, Colorado, United States

James F. Dylewski,

• Dobrinskikh, Evgenia, University of Colorado, Denver, Aurora, Colorado, United States

Evgenia Dobrinskikh,

• Lu, Sizhao, University of Colorado, Denver, Aurora, Colorado, United States

Sizhao Lu,

• Lewis, Linda, University of Colorado, Denver, Aurora, Colorado, United States

Linda Lewis,

• Blaine, Judith, University of Colorado Denver, Aurora, Colorado, United States

Judith Blaine,

Background

Glomerulonephritis (GN) requires intrinsic renal cells that express Major Histocompatibility complex type II (MHCII) to propagate the disease. Glomerular endothelial cells (GEnC) can express MHCII but it is unclear if they can present antigen. Neonatal Fc receptor (FcRn) is a trafficking protein that is required for antigen processing and presentation and is also expressed in GEnC. It has been demonstrated that when FcRn is globally knocked out, GN is prevented. The exact mechanism by which knock out of FcRn offers protection from GN is incompletely understood. An understanding of how FcRn is involved in MHCII expression by GEnC could lead to novel therapeutic options.

Methods

We isolated and purified GEnC from wild type (WT) & FcRn knockout (KO) mice by isolating glomeruli. We then used von Willebrand factor (vWF) coated dynabeads to isolate the endothelial cells. The cells were characterized using IF for vWF and a podocyte specific marker (WT1). Once characterized & purified, we used a temperature sensitive SV40 virus to conditionally immortalize both the cell lines. The cells were grown under permissive conditions at 33^oC and differentiated at 37^oC. Quantitative PCR (qPCR) was used to demonstrate appropriate expression of endothelial cell markers and verify the expression of FcRn in WT and absence in KO. We then treated our GEnCs with INFγ and TNFα and used flow cytometry to evaluate for the expression of CD146 (an endothelial cell marker) and MHCII. We also used qPCR to evaluate mRNA expression of MHCII, MHCII chaperone protein (CD74 also known as invariant chain), and transcript regulator protein (CIITA) to evaluate for differences in level of expression of MHCII and its key transcriptional components.

Results

We demonstrated by flow cytometry that while IFNγ significantly upregulated expression of MHCII in both WT and KO, KO GEnC had significantly less expression of MHCII compared to WT. We also found significantly less MHCII, CD74, and CIITA mRNA in KO compared to WT GEnC.

Conclusion

These findings suggest that the absence of FcRn alters the signaling needed for MHCII transcription. This finding may provide new insight and offer potential new targets for therapies GN.

Funding

• NIDDK Support