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Abstract: SA-PO645

Cell-Surface Glycoprofiling of IgA1-Secreting Cells From Patients With IgA Nephropathy Reveals Subpopulations With Differential Cytokine Responses and Capacity to Produce Galactose-Deficient IgA1

Session Information

Category: Glomerular Diseases

  • 1302 Glomerular Diseases: Immunology and Inflammation

Authors

  • Reily, Colin, The University of Alabama at Birmingham College of Arts and Sciences, Birmingham, Alabama, United States
  • Nakazawa, Shigeaki, Osaka Daigaku Daigakuin Igakukei Kenkyuka Igakubu Igaku Senko, Suita, Osaka, Japan
  • Julian, Bruce A., The University of Alabama at Birmingham College of Arts and Sciences, Birmingham, Alabama, United States
  • Novak, Jan, The University of Alabama at Birmingham College of Arts and Sciences, Birmingham, Alabama, United States
Background

Galactose-deficient IgA1 (Gd-IgA1) is the main autoantigen in IgA nephropathy (IgAN) and its elevated circulatory levels predict poor kidney outcome. As Gd-IgA1 constitutes a small fraction of total circulatory IgA1, we hypothesize that only a few IgA1-secreting cells may be involved. Moreover, cytokine stimulation can increase Gd-IgA1 production in cultured IgA1-producing cells from IgAN patients. We report here that cell-surface glycoprofiling of immortalized IgA1+ cells with a GalNAc-Gal-specific lectin (peanut agglutinin, PNA) can identify subsets of cells with differential signaling and O-glycosylation of IgA1.

Methods

Immortalized IgA1-producing cells derived from peripheral blood mononuclear cells of healthy controls (HC) and IgAN patients were stimulated with mixture of cytokines (IL-4, IL-6, IL-21, CD40L) for 20 min, followed by cell-surface staining with PNA and IgA-specific antibody and FACS. Non-stimulated cells served as negative controls. The live-sorted cells with distinct PNA binding (low- and high-PNA-binding cells) were cultured and assessed for baseline and cytokine-mediated signaling and Gd-IgA1 production. Gd-IgA1 was determined by lectin ELISA, phosphoproteins by intracellular staining with antibodies and FACS.

Results

For both HC- and IgAN-derived IgA1+ cells without cytokine stimulation (i.e., baseline), high-PNA-binding cells exhibited more phosphorylated (P) ERK1/2, less P-65-NF-kB, and less P-STAT1 compared to low-PNA-binding cells (p<0.01, p=0.02, p<0.01, respectively). Moreover, there was less P-65-NF-kB in HC- vs. IgAN-derived cells, but only for the low-PNA-binding cells (p=0.01). For both HC- and IgAN-derived IgA1+ cells with cytokine stimulation, P-ERK1/2 was increased in PNA-high cells (p<0.01) and P-65-NF-kB was increased in PNA-low cells (p=0.01). In a pilot experiment, the high-PNA-binding cells from an IgAN patient produced more Gd-IgA1 compared to low-PNA-binding cells.

Conclusion

Glycoprofiling of IgA1+ cells with PNA lectin revealed cells with differential cell-surface glycosylation. These distinct cell types differed in baseline and cytokine-induced cell signaling and Gd-IgA1 production.

Funding

  • NIDDK Support