R ELLIS1,2, M KATERELOS2,4, N COOK2, K PAIZIS2, S WALKER3, S CHOY2, G PELL3, P MOUNT1,2,4,5, D POWER1,2,4,5
1The University of Melbourne, Parkville, Australia, 2Austin Health, Heidelberg, Australia, 3Mercy Hospital for Women, Heidelberg, Australia, 4Institute for Breathing and Sleep Kidney Laboratory, Heidelberg, Australia, 5Equal senior authors
Aim: To detect and characterise the expression and phosphorylation of glycolytic regulatory proteins in urinary exosomes of normotensive non-pregnant (NC), normotensive pregnant (NP) and pre-eclamptic (PE) subjects.
Background: Some kidney diseases are characterised by a switch in energy metabolism from predominant fatty acid oxidation to aerobic glycolysis, where glycolysis without mitochondrial oxidation becomes the preferred energy source. The principal regulator of glycolysis in cells is 6-phosphofructokinase-2-kinase/fructose-2,6-bisphosphatase (PFK2). To determine whether there may be a switch to aerobic glycolysis in the kidney in women with pre-eclampsia, we studied expression and phosphorylation of PFK2 isozymes in urinary exosomes.
Methods: A cross sectional study of NC (n=19), NP (n=23) and PE (n=33) subjects was performed. Exosomes were isolated from urine samples using differential ultracentrifugation techniques. Exosomal content was analysed by Western blot and computer densitometry for expression and phosphorylation levels of three PFK2 isozymes (PFKFB2, PFKFB3 and PFKFB4).
Results: There was a 9.9-fold increase in PFKFB2 phosphorylated at Ser483 in PE compared to NP (p<0.0001). Expression of phosphorylated PFKFB2 at Ser466 was more common with PE, present in 72.72% (95% CI=55.6-85.1%) of PE and 8.70% (95% CI=1.3-28.0%) of NP samples (p<0.0001). PFKFB3 expression was more frequent with PE, detected in 93.9% (95% CI=79.4-99.3%) of PE and 8.70% (95% CI=1.3-28.0%) of NP samples (p<0.0001). PFKFB4 expression was increased 6.5-fold in PE compared to NP (p=0.0003). No significant differences between NP and NC groups were observed.
Conclusions: Renal expression and activating phosphorylation of PFK2 isoforms is increased in women with pre-eclampsia. Activity of these enzymes is predicted to increase glycolysis, suggesting that there may be a switch in metabolism in the kidneys of women with pre-eclampsia to aerobic glycolysis.
Rachael Ellis is a final year Doctor of Medicine student at The University of Melbourne, where she also obtained her Bachelor of Science in 2014. She has recently completed her project with the Nephrology Department at Austin Health and the Institute for Breathing and Sleep (Kidney Laboratory), looking into changes in renal metabolism that can be detected in urinary exosomes of patients with pre-eclampsia.