MSACL 2025 Abstract

Identification of New Urine Glycan Biomarkers for Lupus Nephritis Using Spatial N-Glycomic Profiling of Biopsy Tissues Linked With Patient Matched Biofluids

Richard R. Drake, Aaron Angerstein, Caroline Kittrell, Jim Oates, Tamara Nowling
Medical University of South Carolina, Charleston, SC

Richard Drake, PhD (Presenter) Medical University of South Carolina

Presenter Bio: Dr. Drake earned his PhD in Biochemistry and Molecular Biology from the University of Kentucky in 1990. Over the course of his career, first as a faculty member at the University of Arkansas for Medical Sciences, then the Eastern Virginia Medical School, and finally for the past seven years at the Medical University of South Carolina (MUSC), Dr. Drake has published over 150 manuscripts in peer-reviewed journals, developed 4 patents, and edited 2 books. He has been Director of the MUSC Proteomics Center since 2011, and in close collaboration with Dr. Ball will serve as Director of the DDRCC Proteomics Core. The Core will provide highly specialized expertise and advanced instrumentation for the application of imaging mass spectrometry (IMS) technologies to gastrointestinal and liver research questions (includes both experimental and clinical projects).

Since 2002, Dr. Drake has developed multiple MS-based approaches for profiling clinical biofluids and tissues, and specializes in the analysis of glycans and glycoprotein biomarkers. In the past five years, his laboratory has developed a robust and highly novel IMS approach for the analysis of N-linked glycans in tissues. This method is of great utility as it can be used with any FFPE (formalin-fixed paraffin-embedded) or frozen tissue, ranging from clinical samples to tissues harvested from genetically engineered animal models. His glycan IMS approach continues to generate high-content N-glycome maps which provide detailed information on potential marker functions and localization in tissues. This in turn is facilitating further IMS method development for other types of glycan targets, such as O-glycans and heparin/chondroitin sulfate glycosaminoglycans, as well as glycoprotein post-translational modifications (PTMs).

Relevant Financial Disclosures (within past 24 months, reported on Jul 17, 2025) No relevant financial relationship(s) to disclose. Conflict mitigation NOT REQUIRED.

INTRODUCTION:
Systemic Lupus Erythematosus (SLE) is a chronic autoimmune disease primarily affecting women of childbearing age. Approximately 50% of SLE patients develop Lupus Nephritis (LN), a severe kidney complication linked to increased morbidity and mortality. Current urine biomarkers, like the urine protein-to-creatinine ratio (UPr:Ucr), lack specificity for glomerular damage, often requiring repeated biopsies to monitor disease progression, highlighting the need for sensitive, non-invasive alternatives. Our previous studies in urine identified elevated glycosphingolipids and an altered N-glycome as potential LN markers.

OBJECTIVES:
We hypothesize that glycosylation changes precede irreversible kidney damage detectable by current urine markers. Using N-glycomic profiling of LN patient matched tissues, urine and serum by matrix-assisted laser desorption ionization mass spectrometry imaging (MALDI-MSI), the goal was to identify LN-specific urinary glycan biomarkers, providing insights into renal pathology and potentially be predictive of treatment response.

METHODS:
We analyzed patient-matched urine, serum, and FFPE kidney biopsy samples from fourteen LN patients to characterize N-glycan composition using established MALDI-MSI workflows1-4. Analysis of healthy control samples for renal biopsies, urine and serum were available for reference. FFPE kidney biopsies were dewaxed, antigen-retrieved (citraconic buffer [pH 3.0], 95°C, 30 min), and prepared for analysis. Urine (1 µL) and serum (1µL) were suspended in 2µL sodium bicarbonate (pH 8.0), and 1 µL was spotted in duplicate onto amine-reactive slides. Samples were treated with a molecular coating of PNGaseF PRIME (0.1 µg/µL) via an HTX M5 Sprayer, followed by α-cyano-4-hydroxycinnamic acid matrix coating using an HTX Sublimate apparatus. N-glycan imaging was performed using timsTOF fleX MALDI-QTOF at 10 micron spatial resolution for tissue, with spectra processed in SCiLS Lab and annotated using an in-house N-glycan database based on accurate mass determinations.

RESULTS:
A key for obtaining high resolution MALDI-MSI spatial data for the LN kidney biopsies was use of matrix sublimation via an HTX Sublimate, which minimized N-glycan delocalization. This approach enabled the identification of distinct glycosylation signatures associated with key renal structures, including the glomerulus and distal/proximal convoluted tubules. In healthy control tissues, glomeruli have distinct tri- and tetra-antennary N-glycans that are also multiply sialylated. Proximal and distal convoluted tubules have abundant bisecting bi-,tri- and tetra-antennary structures with 1-4 fucoses, and the antennae number progressively increases along the length of the tubule. Analysis of the fourteen patient-matched urine, serum, and biopsy samples resulted in a peak list of 78 N-glycans present in each sample. Overall, there was a strong correlation between the urinary glycomic signatures and those associated with glomeruli histology. Thirteen N-glycans were elevated in both biopsy and urine samples and minimally detected in serum, This suggests that the associated glomerular damage for LN can be detected in the patient urines as reflective of glomerular specific N-glycans.

DISCUSSION:
Overall, having determined the uniqueness of the spatial N-glycome in normal healthy kidney glomeruli and distal/proximal tubules allows direct assessment of the LN-associated glycome and underlying histopathology changes that could provide unique biomarker targets. A urine-based glycomic assay in the LN setting could provide a companion biomarker assay for monitoring disease progression and treatment responses.

REFERENCES:
1. McDowell CT, et al. (2021) Applications and continued evolution of glycan imaging mass spectrometry. Mass Spectrom Rev. 42(2):674-705.
2. Drake RR, et al. (2018) In Situ Imaging of N-Glycans by MALDI Imaging Mass Spectrometry of Fresh or Formalin-Fixed Paraffin-Embedded Tissue. Curr Protoc Protein Sci. 94(1):e68.
3. Blaschke CRK, et al. (2020) Rapid N-Glycan Profiling of Serum and Plasma by a Novel Slide-Based Imaging Mass Spectrometry Workflow. J Am Soc Mass Spectrom. 31(12):2511-2520.
4. Blaschke CRK, et al. (2021) Direct N-Glycosylation Profiling of Urine and Prostatic Fluid Glycoproteins and Extracellular Vesicles. Front Chem. 9:734280.