Mitochondrial proteomic analysis reveals deficiencies in oxygen utilization in medullary thick ascending limb of Henle in the Dahl salt-sensitive rat.
The renal medullary thick ascending limb (mTAL) of the Dahl salt-sensitive (SS) rat is the site of enhanced NaCl reabsorption and excess superoxide production. In the present studies we isolated mitochondria from mTAL of SS and salt-resistant control strain SS.13(BN) rats on 0.4 and 8% salt diet for 7 days and performed a proteomic analysis. Purity of mTAL and mitochondria isolations exceeded 93.6 and 55%, respectively. Using LC/MS spectral analysis techniques we identified 96 mitochondrial proteins in four biological mTAL mitochondria samples, run in duplicate, as defined by proteins with a false discovery rate <5% and scan count ≥2. Seven of these 96 proteins, including IDH2, ACADM, SCOT, Hsp60, ATPA, EFTu, and VDAC2 were differentially expressed between the two rat strains. Oxygen consumption and high-resolution respirometry analyses showed that mTAL cells and the mitochondria in the outer medulla of SS rats fed high-salt diet exhibited lower rates of oxygen utilization compared with those from SS.13(BN) rats. These studies advance the conventional proteomic paradigm of focusing exclusively upon whole tissue homogenates to a focus upon a single cell type and specific subcellular organelle. The results reveal the importance of a largely unexplored role for deficiencies of mTAL mitochondrial metabolism and oxygen utilization in salt-induced hypertension and renal medullary oxidative stress.
Department of Physiology, Medical College of Wisconsin, Milwaukee, Wisconsin 53226, USA.
Nadezhda N
NN
Zheleznova
Chun
C
Yang
Robert P
RP
Ryan
Brian D
BD
Halligan
Mingyu
M
Liang
Andrew S
AS
Greene
Allen W
AW
Cowley
D000818:Animals; D015153:Blotting, Western; D002853:Chromatography, Liquid; D007521:Isocitrate Dehydrogenase / Q000378:metabolism; D008138:Loop of Henle / Q000378:metabolism* / Q000502:physiology; D013058:Mass Spectrometry; D008856:Microscopy, Fluorescence; D024101:Mitochondrial Proteins / Q000235:genetics / Q000378:metabolism*; D010101:Oxygen Consumption / Q000502:physiology*; D040901:Proteomics / Q000379:methods*; D051381:Rats; D020303:Rats, Inbred Dahl / Q000235:genetics / Q000378:metabolism* / Q000502:physiology
D024101:Mitochondrial Proteins; D007521:Isocitrate Dehydrogenase