Effect of Lipocalin‐2 expression on mitochondrial function in renal tubular cells in chronic kidney disease

Kidney cells metabolic status requires high abundance of mitochondria. It has been reported in chronic kidney disease (CKD) a loss of mitochondrial mass and function leading to reactive oxygen species (ROS) production and subsequent tubular cell death. Hence, mitochondrial dysfunction participates to CKD onset. Nevertheless, the cellular mechanisms inducing mitochondrial dysfunction in kidney cells are poorly understood. We have recently shown that in CKD, Lipocalin-2 (Lcn2) expression is strongly induced in kidney cells leading to ROS production and apoptosis. However, the mechanisms by which Lcn2 triggers these phenomena are unknown. Considering that mitochondria can induce oxidative stress and apoptosis, we hypothesized that Lcn2 participates to CKD onset via an effect on mitochondrial dependent mechanisms. Using renal tubular cell lines overexpressing (OE) or knock-down (KD) for Lcn2, we aimed at studying the effect of Lcn2 protein expression on mitochondrial function. First, we demonstrated that Lcn2 regulates mitochondrial dynamics, mass and function. Indeed, we observed by immunofluorescence and electron microscopy that kidney cells expressing Lcn2 present smaller and rounded mitochondria while Lcn2 KD induces mitochondrial elongation. Moreover, Lcn2 expression increases mitochondrial fission marker abundance and decreases fusion marker expression. Mitotracker staining and mtDNA content measurement experiments demonstrated that Lcn2 expression decreased mitochondrial mass while Lcn2 KD increased it. Furthermore, we observed that Lcn2 expression decrease mitochondrial membrane potential and accordingly increased Lactate production compared to cell KD for Lcn2. Using iron chelator or Lcn2 mutant unable to transport iron, we demonstrated that Lcn2 expression induces mitochondrial dysfunction independently of its iron transport effect. In addition, we observed that extracellular Lcn2 did not rescue Lcn2 effects on mitochondria while intracellular Lcn2 did. Finally, using a subtotal nephrectomy mice model, that recapitulates different features of human CKD, we demonstrated that Lcn2 invalidation by knock-out in mice decreased the mitochondrial fission and the subsequent mitochondrial fragmentation and mass loss in kidney tubular cells normally observed during CKD onset in wild-type mice. Altogether, these results demonstrated that Lcn2 expression regulates mitochondrial dynamics, mass and function in kidney tubular cells. This suggests that Lcn2 driven mitochondrial dysfunction might by responsible at least in part of CKD onset.