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Updated: Jan 28, 2026
A Modified Precipitation Method to Isolate Urinary Exosomes
Published on: January 16, 2015
Increased expression and phosphorylation of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase isoforms in urinary
R Ellis1,2, M Katerelos2,3, S W Choy2
1Faculty of Medicine, Dentistry and Health Sciences, University of Melbourne, Parkville, Australia.
Background:
Glycolysis is altered in various kidney diseases, but little is known about glycolysis in pre-eclampsia, a multi-system disorder with major pathological effects on the kidney. Urinary exosomes provide a non-invasive alternative for studying changes in kidney metabolism. This study aims to characterise the expression and phosphorylation of isozymes of the key glycolytic regulatory protein, 6-phosphofructokinase-2-kinase/fructose-2,6-bisphosphatase (PFK-2/FBPase-2), in urinary exosomes of subjects with pre-eclampsia (PE), compared to normotensive non-pregnant (NC) and normotensive pregnant (NP) controls.
Methods:
A cross-sectional study of NC (n = 19), NP (n = 23) and PE (n = 29) subjects was performed. Exosomes were isolated from urine samples by differential ultracentrifugation, and then analyzed by Western blot and densitometry for expression of PFK-2/FBPase-2 isozymes (PFKFB2, PFKFB3 and PFKFB4) and phosphorylation of PFKFB2 at residues Ser483 and Ser466 and PFKFB3 at Ser461.
Results:
PFKFB2 expression was increased 4.7-fold in PE compared to NP (p < 0.001). PFKFB2 phosphorylation at Ser483 was increased 2.6-fold in PE compared to NP (p = 0.002). Expression of phosphorylated PFKFB2/PFKFB3 at Ser466/Ser461 was increased in PE, being present in 77.4% (95% CI 59.9-88.9%) of PE and 8.3% (95% CI 1.2-27.0%) of NP samples (p < 0.001). PFKFB3 was more commonly expressed in PE, detected in 90.3% (95% CI 74.3-97.4%) of PE and 8.3% (95% CI 1.2-27.0%) of NP samples (p < 0.001). PFKFB4 had a 7.2-fold increase in expression in PE compared to NP (p < 0.001). No significant differences between NP and NC groups were observed.
Conclusion:
Regulatory proteins that increase glycolysis are increased in the urinary exosomes of subjects with pre-eclampsia, suggesting that renal glycolysis may be increased in this condition.
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