Glycolysis, but not Mitochondria, responsible for intracellular ATP distribution in cortical area of podocytes

Abstract

BACKGROUND:

Podocyte dysfunction is a common feature of chronic kidney disease. Understanding how these cells maintain their physiological function may be the first step in preventing podocyte injury. Nevertheless, substantial level of ATP is required to maintain podocyte structure and function, but the precise energy metabolism in these cells remains unknown. This study evaluated the roles of two ATP production systems, mitochondrial oxidative phosphorylation (OXPHOS) and glycolysis in podocytes.

METHODS:

To examine the role of glycolysis in production of ATP, glycolysis was blocked by 2-deoxyglucose (2-DG) in cultured podocytes while basic mitochondrial function was preserved with pyruvate administration. In turn, the role of OXPHOS in podocytes was evaluated by Antimycin. In addition, adenoviruses carrying shRNAs for phosphofructokinase (PFK) -M or PFK-L were also used as an alternative way to examine the effects of blocking glycolysis without inhibitors. We sought a condition in which total cellular ATP under blocking glycolysis was identical to that under blocking OXPHOS. Lamellipodia length was quantified by tracing perimeters of whole-cell and the lamellipodia. Cellular migration was assessed with scratch assay. Intracellular ATP was monitored using fluorescence resonance energy transfer (FRET) - based ATP indicator (ATeam1.03). Mitochondria were visualized with Mito Tracker. In vivo study, transmission electron microscopy was used to examine mitochondrial structures and foot process morphology in mouse podocytes. The expressions of nephrin, a marker of podocyte and PFK-L, a marker of glycolysis, were examined by immunofluorescence.

RESULTS:

While mitochondria occupied the central part of cell body, blocking mitochondria showed only minor effects on lamellipodia and migratory ability. In contrast, blocking glycolysis significantly reduced the formation of lamellipodia and decreased the cell migratory ability. Consistently, the local ATP production in lamellipodia was predominantly regulated by glycolysis. Finally, mitochondria were predominantly located at the center of cytosol in adult mouse podocytes while no mitochondria was detected in foot processes. In contrast, phosphofructokinase was expressed in foot processes, suggesting that ATP production in foot process is predominantly regulated by glycolysis, but not mitochondria.

CONCLUSIONS:

This study suggests that glycolysis could be a major regulator of ATP production in cortical area of podocytes, including podocyte foot process.