Ramnath, R. D. | Search bioRxiv

bioRxiv2026

The development of ToF-SIMS for in-situ glycosaminoglycan analysis

Glycosaminoglycans (GAGs) are linear polysaccharides with essential roles in a myriad of biological processes. Despite their biological importance, methods to determine both spatial and compositional information is limited. Time-of-flight secondary ion mass spectrometry (ToF-SIMS) provides spatially resolved compositional information of biological molecules without enzymatic digestion or label incorporation, enabling unbiased analysis independent of enzyme or label selectivity, overcoming many current limitations in GAG analysis. Here, we present the identification and validation of GAG discriminatory ions from biological samples by comparison of spectra from purified GAGs and cells with genetically modified GAG biosynthetic pathways. Ions discriminatory of specific GAG sub-families are identified and related to GAG structural components. The analysis is applied to human induced pluripotent stem cells engineered to lack heparan sulphate (HS), where compensatory changes in GAG display that link to function were observed. Furthermore, the broad applicability and spatial resolution of the technique is highlighted through detection of a disease-induced reduction in HS within the individual glomeruli of diabetic mice.

biochemistry↗

bioRxiv2025

Matrix Metalloproteinase (MMP) inhibition rescues endothelial glycocalyx damage and reduces neutrophil infiltration in sepsis-associated acute kidney injury

Endothelial glycocalyx (eGlx), a carbohydrate-rich endothelial coat, maintains vascular homeostasis, and its disruption contributes to several vascular diseases. We previously identified matrix metalloproteinase (MMP) 2 and 9-mediated eGlx shedding as a key mechanism in human glomerular endothelial cell (GEnC) damage and kidney dysfunction in diabetes. Here, we sought to determine whether this mechanism contributes to renal and systemic microvascular endothelial cell injury in sepsis-AKI. Sepsis-AKI was induced in mice by lipopolysaccharide (LPS) injection. Confocal microscopy demonstrated glomerular and peritubular eGlx damage, and increased circulating SDC4 demonstrated systemic eGlx shedding. These were associated with enhanced mRNA expression of eGlx components (syndecans 1 and 4) and endothelial inflammatory markers, Intercellular and Vascular Cell Adhesion Molecules (ICAM and VCAM) in the kidney. Additionally, sepsis-AKI markers, serum creatinine and urea, were increased. We found that renal and circulating MMP9, but not MMP2, levels were raised in sepsis-AKI. Moreover, MMP9 staining colocalises with neutrophil, endothelial and mesangial staining in glomeruli in sepsis-AKI. Treatment with MMP2 and 9 inhibitor 1, 1h before LPS injection, prevented glomerular, peritubular and systemic eGlx damage in sepsis-AKI. MMP inhibition also attenuated glomerular leucocyte numbers in sepsis-AKI. We confirmed increased MMP9 activity, shedding of eGlx syndecan 1 and elevated angiopoietin 2 levels, an endothelial damage marker, in sepsis-AKI in humans. Moreover, we used human glomerular endothelial cells (GEnC) in vitro to demonstrate that human sepsis-AKI serum directly damages GEnC eGlx. Our studies confirm that MMP-mediated eGlx damage is a key contributor to renal and systemic microvascular dysfunction in sepsis-AKI and represents a potential therapeutic target for protecting the microvasculature.

cell biology↗