Multiphoton Imaging Reveals Differences in Mitochondrial Function between Nephron Segments

Abstract

Mitochondrial dysfunction may play a role in the pathogenesis of several renal diseases. Although functional roles and metabolic demands differ among tubule segments, relatively little is known about the properties of mitochondria in different parts of the nephron. Clinically, the proximal tubule seems particularly vulnerable to mitochondrial toxicity. In this study, we used multiphoton imaging of live rat kidney slices to investigate differences in mitochondrial function along the nephron. The mitochondrial membrane potential was markedly higher in distal than proximal tubules. Inhibition of respiration rapidly collapsed the membrane potential in proximal tubules, but potential was better maintained in distal tubules. Inhibition of the F 1 F o -ATPase abolished this difference, suggesting that maintenance of potential via ATPase activity is more effective in distal than proximal tubules. Immunostaining revealed that the ratio of the expression of ATPase to IF1, an endogenous inhibitor of the mitochondrial ATPase, was lower in proximal tubules than in distal tubules. Production of reactive oxygen species was higher in proximal than distal cells, but inhibition of NADPH oxidase eliminated this difference. Glutathione levels were higher in proximal tubules. Overall, mitochondria in the proximal tubules were in a more oxidized state than those in the distal tubules. In summary, there are axial differences in mitochondrial function along the nephron, which may contribute to the pattern and pathophysiology of some forms of renal injury.

Illuminating Mitochondrial Function and Dysfunction using Multiphoton Technology

“…⌬⌿ m is the larger of the two components of proton motive force under both normal and pathologic conditions 9 ; 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 F 1 F O -ATPase reverses the flow of protons by hydrolyzing ATP to extrude protons and restore ⌬⌿ m .…”

“…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).…”

“…Parameters related to mitochondrial function including ⌬⌿ m , superoxide production, cellular GSH levels, and the redox state of NADH and FAD 2ϩ 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 TALϾproximal convoluted tubuleϾproximal 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.…”

“…In summary, the new multiphoton data provided by Hall et al 1 focus our understanding of mitochondrial function to individual cells with direct comparisons between adjacent PT and TAL cells. The data provide additional information as to why PT cells are more sensitive to ischemia.…”

“…1 The considerable clinical importance of Na ϩ and Cl Ϫ reabsorption by the DCT is illustrated by the therapeutic efficacy of thiazide diuretics and the clinical phenotype of Gitelman syndrome, 2 caused almost exclusively by loss-of-function mutations in the thiazide-sensitive Na ϩ -Cl Ϫ co-transporter (NCC). A gain-in-function of NCC occurs in pseudohypoaldosteronism type II (PHA-II; also known as Gordon syndrome or hereditary hypertension with hyperkalemia); however, this disorder is caused by mutations not in NCC but in two of the four WNK (with no K/lysine) kinases, so-named for the absence of a conserved catalytic lysine.…”