Abstract: PUB201
Optimizing Lipoprotein Isolation Methods to Assess Lipoprotein Composition in Glomerular Diseases
Session Information
Category: Glomerular Diseases
- 1401 Glomerular Diseases: Mechanisms, including Podocyte Biology
Authors
- Gye, Haley, University of Miami Miller School of Medicine, Miami, Florida, United States
- Fontanella, Antonio Miguel, University of Miami Miller School of Medicine, Miami, Florida, United States
- Njeim, Rachel, University of Miami Miller School of Medicine, Miami, Florida, United States
- Molina David, Judith T., University of Miami Miller School of Medicine, Miami, Florida, United States
- Mendez, Armando, University of Miami Miller School of Medicine, Miami, Florida, United States
- Pennathur, Subramaniam, University of Michigan Medical School, Ann Arbor, Michigan, United States
- Merscher, Sandra, University of Miami Miller School of Medicine, Miami, Florida, United States
- Fornoni, Alessia, University of Miami Miller School of Medicine, Miami, Florida, United States
Background
High-density lipoprotein (HDL) plays a crucial role in renal health, with alterations in its composition linked to glomerular diseases (GDs), such as Alport syndrome (AS). Apolipoprotein M (APOM), primarily associated with HDL, is involved in lipid transport and endothelial protection. We hypothesize that a decrease in APOM bound to HDL contributes to renal injury in GDs.
Methods
To assess HDL composition, particularly APOM association, we compared three lipoprotein isolation methods using normal human serum as well as plasma isolated from Col4a3+/+ and Col4a3-/- mice, a model of AS: (1) 2-step density gradient ultracentrifugation separating HDL from APOB-containing fractions; (2) 2-step density gradient ultracentrifugation followed by fast protein liquid chromatography (FPLC) for further purification; and (3) 5-step density gradient ultracentrifugation isolating VLDL, IDL, LDL, HDL2, and HDL3. We evaluated each method for HDL yield using BCA assay, and assessed APOM detectability and contaminant presence using SYPRO Orange staining and western blotting.
Results
Our findings demonstrate that both the 2-step and 5-step ultracentrifugation methods preserved the association of APOM with HDL. Post-isolation analysis of the remaining serum infranatant by western blot showed no detectable APOM signal, suggesting that ultracentrifugation did not dissociate APOM from HDL particles. While FPLC effectively reduced albumin levels as observed by SYPRO Orange staining, it also decreased HDL yield, limiting its utility. Importantly, western blot analysis revealed persistent albumin presence in HDL across all methods in both normal human serum and plasma from Col4a3+/+ and Col4a3-/- mice. These findings suggest that albumin may be an intrinsic component of HDL particles rather than a contaminant.
Conclusion
Taken together, our findings reveal that both the 2-step and 5-step ultracentrifugation methods are effective for isolating HDL without detectable loss of APOM. The 5-step method offers the advantage of separating VLDL, IDL, LDL, HDL2, and HDL3, enabling future investigations into APOM distribution and composition across lipoprotein subfractions. Given its versatility, we will continue to employ the 5-step protocol for downstream lipoprotein profiling and functional studies.
Funding
- NIDDK Support