Illuminating mitochondrial function and dysfunction using multiphoton technology.

Studying differential cell function or dysfunction within the kidney is made difficult by cellular heterogeneity, alterations in regional blood flow, oxygen tension, and interstitial tonicity, resulting in extremely complex anatomic and physiologic arrangements. Creative investigators have developed techniques to reduce this heterogeneity but often with loss or alteration of three-dimensional cellular associations, anatomic and physiologic cell-to-cell interactions, and harsh isolation and experimental conditions. These limitations have made “absolutes” difficult to determine, especially in relation to pathologic processes such as cell type involvement in renal ischemia. In this issue of JASN, Hall et al.1 use multiphoton microscopy of kidney slices to compare mitochondrial parameters and differential cellular responses to stress. This approach allows them to quantify several key parameters of mitochondrial function and dysfunction in proximal tubular (PT) cells and directly compare different PT segments with each other and with distal tubular (DT) cells of the thick ascending limb (TAL). Because mitochondrial alterations play a central role in normal cell function and in response to injury, the article adds to our previous knowledge about an important area. To understand the significance and limitations of their contribution, one must first understand what is known about differences between PT and TAL cells. Abundant mitochondria in cortical and outer medullary renal epithelial cells are necessary to meet the ATP demands of sodium transport by high-capacity aerobic metabolism. They compose 33% of the volume of proximal convoluted tubular S1 cells, 39% of the cells in the S2 segment, and 22% of the volume of cells in the proximal straight S3 segment; in the medullary and cortical TAL cells, mitochondria account for 30 to 44% of cell volume.2 Differential tubular cell metabolism is also notable for the absence of aerobic and anaerobic glycolysis in the proximal convoluted tubule (S1),3,4 which removes an important mechanism for preserving cell ATP and viability during injury.5 In contrast, DT segments, including the TAL, have welldeveloped glycolytic pathways.3,4,6,7 Glycolysis occurs in the S3 segment of the proximal tubule, albeit to a lesser extent than in distal tubules, and contributes to maintaining ATP there when mitochondrial function is impaired.3,8 Mitochondrial ATP production depends on substratesupported, electron transport–mediated proton extrusion from the mitochondrial matrix that generates a proton gradient across the inner mitochondrial membrane. In turn, this is used to drive phosphorylation of ADP to ATP by proton movement down the gradient back into the matrix through the inner membrane F1FO-ATPase. Electron transport– driven proton extrusion is also responsible for the net potential across the inner membrane ( m), and m in combination with the pH gradient accounts for the net proton electrochemical gradient across the membrane, also called the proton motive force. m is the larger of the two components of proton motive force under both normal and pathologic conditions9; therefore, it serves as a valuable index of the state of the entire system, as shown in recent studies documenting a major role for nonesterified fatty acids in the persistent mitochondrial dysfunction seen in re-oxygenated proximal tubules.9,10 As discussed by Hall et al.,1 when m is low and ATP is available, the F1FO-ATPase reverses the flow of protons by hydrolyzing ATP to extrude protons and restore m. Fluorescence approaches, using both chemical probes and autofluorescence of components of the mitochondrial electron transport chain, provide powerful tools for dynamically following determinants of mitochondrial function and have been widely applied to studies of isolated mitochondria and whole cells. Parameters related to mitochondrial function including m, superoxide production, cellular GSH levels, and the redox state of NADH and FAD can be quantified in individual cells and m within individual mitochondria.11 Hall et al.1 show that mitochondrial uptake of two different lipophilic cationic probes (tetramethyl rhodamine methyl ester and rhodamine 123), as a measure of m, follows the pattern TAL proximal convoluted tubule proximal straight tubule. This pattern is not changed by inhibition of the multidrug resistance transporter with verapamil, suggesting it is due to differences in m between tubular segments rather than in transport of the probe across the plasma membrane of individual cells; however, during chemical anoxia, m is well maintained in TAL cells but not in PT cells. Inhibition of the F1FO-ATPase with oligomycin abolishes this difference, indicating that differences between PT and TAL cells are due to support of m by Published online ahead of print. Publication date available at www.jasn.org.