Isolation of Pure Mitochondria from Rat Kidneys and Western Blot of Mitochondrial Respiratory Chain Complexes.

Cardiac, neuronal and renal tubular epithelial cells are the most metabolically active cells in the body. Their fate depends largely on their mitochondria as the primary energy generating system which participates in the control of apoptosis, cell cycle and metabolism. Thus, mitochondrial dysfunction is a hallmark of many chronic diseases including diabetic nephropathy. A drop in mitochondrial bioenergetics efficiency is often associated with altered expression of respiratory chain complexes. Moreover, recent studies demonstrate that cellular proteins can shuttle to mitochondria and modify their function directly. Here we illustrate two mitochondria isolation protocols; one is recommended if the purity of the mitochondrial fraction is a priority such as if the mitochondrial localization of a protein has to be validated, the other if a high yield of intact functional mitochondria is required for functional studies and quantitative Western blotting. Next, we provide a detailed protocol for Western blotting of isolated mitochondria and renal cortex either to prove the purity of isolated fractions or to quantify complexes of the mitochondrial respiratory chain. We used this approach to identify classically cell membrane bound angiotensin II receptors in mitochondria and to study the effect of these receptors on mitochondrial function in early stages of diabetic nephropathy.

The angiotensin II type 2 receptors protect renal tubule mitochondria in early stages of diabetes mellitus.

Diabetic nephropathy correlates more closely to defective mitochondria and increased oxidative stress in the kidney than to hyperglycemia. A key driving factor of diabetic nephropathy is angiotensin II acting via the G-protein-coupled cell membrane type 1 receptor. The present study aimed to investigate the role of the angiotensin II type 2 receptor (AT2R) at the early stages of diabetic nephropathy. Using receptor binding studies and immunohistochemistry we found that the mitochondria in renal tubules contain high-affinity AT2Rs. Increased renal mitochondrial AT2R density by transgenic overexpression was associated with reduced superoxide production of isolated mitochondria from non-diabetic rats. Streptozotocin-induced diabetes (28 days) caused a drop in the ATP/oxygen ratio and an increase in the superoxide production of isolated renal mitochondria from wild-type diabetic rats. This correlated with changes in the renal expression profile and increased tubular epithelial cell proliferation. AT2R overexpression in tubular epithelial cells inhibited all diabetes-induced renal changes including a drop in mitochondrial bioenergetics efficiency, a rise in mitochondrial superoxide production, metabolic reprogramming, and increased proliferation. Thus, AT2Rs translocate to mitochondria and can contribute to reno-protective effects at early stages of diabetes. Hence, targeted AT2R overexpression in renal cells may open new avenues to develop novel types of drugs preventing diabetic nephropathy.