Alpha-1 Acid Glycoprotein in Serum, Urine, and Neutrophils of Patients with Lupus Nephritis

Background: Alpha-1 acid glycoprotein (AGP) is a highly glycosylated protein that has been associated with lupus nephritis (LN). We and others have reported elevated AGP concentrations in LN patient urine, suggesting that it is a promising biomarker for LN morbidity and activity. While AGP is predominately synthesized by the liver, it is also a component of neutrophil granule proteins. LN patient kidneys contain increased neutrophil abundance, suggesting that they may serve as the source of AGP found in LN patient urine. The goal of this study is to identify the cellular origin of AGP in LN patients to further our understanding of the immune system’s contribution to glomerular injury associated with LN. Methods: Serum was isolated from whole blood samples and stored at -80°C. Urine was collected, centrifuged to remove debris, and stored at -80°C. LN patient-matched serum and urine were retrieved from our biorepository during states of active (n=10) and inactive (n=10) disease, as well as from healthy controls (n=10). Neutrophils were isolated from whole blood collected from LN patients during active and inactive disease and from healthy controls, and they were stimulated with f-MLF to promote AGP exocytosis by degranulation. Two-step column chromatography was used to purify AGP from each sample. The presence of AGP in the resulting elution fractions was confirmed by immunoblot. A commercially available AGP isolated from healthy human plasma was used as a control. Results: Both active and inactive LN sera contain AGP of various molecular weights (~45, 65, 150, and 200 kDa), while healthy control serum only contains a 45 kDa version that matches a commercially available AGP standard. Urine from active LN patients contained a single AGP band around 45 kDa, while AGP was not detected in HC urine. Conclusion: These results indicate that there are multiple circulating forms of AGP in serum, while the urine contains less diversity. Future studies will characterize AGP glycosylation by mass spectrometry to elucidate if neutrophils are the cellular origin of the AGP found in LN patient serum and urine. These results will contribute to improving our understanding of the physiological mechanisms of LN as well as the future of precision medicine. Funding: NIDDK Support