Abstract 665: Cryofluorescence 3D Imaging Shows Mutation of p67phox Improves Metabolic Function and Reduces Oxidative Stress in the Renal Medulla of the Dahl Salt-sensitive Rat

Abstract

The Dahl salt-sensitive (SS) rat exhibits increased renal production of reactive oxygen species (ROS) especially in the renal outer medulla (OM) which is known to contribute importantly to the salt-induced hypertension. We have identified increased expression of the p67 phox cytosolic subunit of NAD(P)H oxidase and enhanced enzyme activity in the OM of Dahl salt-sensitive (SS). We found that ZFN knock down of the NADPH-oxidase cytosolic subunit p67 phox in SS rats (SS p67phox-/- ) resulted in a 40% reduction of hypertension and a substantial reduction of renal injury. To determine whether these protective effects were associated with alterations of regional metabolism and oxidative stress, a custom designed cryoimager was used to acquire multi-channel fluorescent images of sequential serial sections with a z-resolution of 30 μm yielding 400 slices per kidney. Computer reconstruction of the stacked sections provided a 3D image of regional changes of metabolic function and oxidative stress within the kidney. Kidneys obtained from SS and SS p67phox-/- rats fed a high salt diet (4% NaCl) for 21 days were flash frozen in liquid N 2 and fluorescent images of the mitochondrial electron transport chain carriers NADH and FAD were acquired. The naturally fluorescent NADH and FAD levels were acquired to provide a 3D representation of the metabolic state of the tissue and oxidative stress. The mean NADH redox ratio (NADH/FAD RR) was significantly higher in kidneys of SS p67phox-/- rats (1.46 ± 0.11 NADH/FAD RR; n=7) compared to SS kidneys (1.00 ± 0.07 NADH/FAD RR; n=4). This represents an average 46% increase in the electron transport chain metabolic activity and a reduction of oxidative stress in kidneys of SS p67phox-/- rats compared to the SS kidneys. Importantly, this was observed only in the region of the renal medulla as revealed by the 3D images of these kidneys. We conclude that p67(phox) is critically involved in cell energetics and ROS production in the renal medulla.