Proximal tubular epithelial cells (PTC) are highly energy demanding kidney cells. Their energy need is covered mostly from mitochondrial fatty acid oxidation. If the high energy demand is not met with sufficient ATP production, tubular epithelial cells undergo apoptosis and atrophy. Derailments in mitochondrial fatty acid metabolism are therefore the main underlying candidate mechanisms in tubular cell death. It has recently been discovered in type 2 diabetic mice and humans that the diabetic kidney exhibits increased fatty acid metabolism and oxidation, but this is not matched with ATP production. The kidney cortex accumulates incompletely oxidized metabolic products which is typical of mitochondrial overload. Our central hypothesis is that this incomplete fatty acid oxidation causes proximal tubule apoptosis through pathways critical to mitochondrial function. Our compelling set of preliminary data show that mitochondrial overload in cells causes energy deficit and oxidant production. In our new mouse model, incomplete fatty acid oxidation and mitochondrial overload causes kidney disease. This was achieved by PTC-specific deletion of carnitine-acetyltransferase (CrAT). The enzyme shuttles excess fatty acid products out of the mitochondria; therefore, lack of CrAT models mitochondrial overload. Our goal is to define the mechanisms that are forerunners of tubular apoptosis due to mitochondrial overload. Three interconnected but independent aims will test our hypothesis using state-of-the- art approaches of both molecular and redox biology.
In Aim 1, we will test the prediction that incomplete fatty acid oxidation causes mitochondrial energy deficit, which is then detrimental to tubular cells.
Aim 2 will test the hypothesis that incomplete fatty acid oxidation leads to excess mitchondrial ROS production and apoptosis. The first two aims will use loss-of-function approaches (PTC-specific CrAT knockout mice alone or in combination with obesity and type 2 diabetes), mechanistic studies from primary PTCs isolated from these models and advanced biophysical measurements (extracellular flux analyzer, electron spin resonance spectroscopy).
In Aim 3, we will test whether alleviating mitochondrial overload by enhancing the efflux of incompletely oxidized products offers prevention. We will use in vitro and in vivo gain-of-function experiments (CrAT overexpression and re-expression) as rescue experiments. The experimental strategy is designed to establish the role of incomplete mitochondrial fatty acid oxidation in tubular injury, decipher the underlying biochemical mechanisms and address whether such pathways can offer the basis for tubular cell preservation well before the appearance of apoptosis, in the context of obesity and type 2 diabetes. Provided that these mechanisms are forerunners of tubular cell apoptosis, targeting mitochondrial fatty acid overload can be a prominent new area to prevent, rather than treat tubular atrophy and chronic kidney disease.
Kidney disease is a major complication of obesity and type 2 diabetes. Outcome predictions of progression to end-stage disease correlate best with the loss of specific kidney cells ? proximal tubular cells. Our projects focus on discovering new mechanisms leading to tubular cell loss, specifically in the context of lipid metabolism alterations and redox imbalance. This proposal addresses the role of fatty acid metabolism derailments in kidney proximal tubular epithelial cell loss. Our hope is that by understanding these mechanisms, future interventions can be designed not only to treat, but to prevent tubular cell injury and kidney failure.
