O2 affinity of cross-linked hemoglobins modifies O2 metabolism in proximal tubules

Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario M5G 1L5, Canada Submitted 3 March 2003 ; accepted in final form 22 April 2003 Previous experiments using cross-linked tetrameric hemoglobins (XLHb) to perfuse isolated rat kidneys showed that high-O 2 -affinity XLHb improved proximal tubule function more effectively than low-O 2 -affinity XLHb. To determine how function was improved, proximal tubule fragments were incubated with albumin, Hb 34 [half-saturation point (P 50 ) 34 Torr], or Hb 13 (P 50 13 Torr) with P O 2 values ranging from 22 to 147 Torr. ATP content reflected O 2 delivery to mitochondria. Both XLHb increased ATP, Hb 34 with P O 2 47 Torr and Hb 13 with P O 2 47 Torr. XLHb increased Na-K-ATPase activity ( 86 Rb uptake) in similar P O 2 -dependent patterns. O 2 consumption ( O 2 ) was measured in a closed, well-stirred chamber. Ouabain- and oligomycin-inhibited O 2 , reflecting Na-K-ATPase activity and oxidative phosphorylation, respectively, mirrored the P O 2 -dependent patterns of ATP and 86 Rb uptake. As P O 2 fell below the midpoint of XLHb desaturation, O 2 , uncoupled from oxidative phosphorylation, transiently increased. The increase was most pronounced with Hb 34 . Nitro- L -arginine methyl ester had no effect on O 2 . Inhibitors of NAD(P)H oxidases and diamine oxidase partially prevented the O 2 surge with Hb 34 . In conclusion, facilitated diffusion accounts for P O 2 -dependent XLHb effects on ATP content and Na-K-ATPase and for Hb 13 's effectiveness in hypoxic perfused kidneys. NO scavenging was not a factor. O 2 -binding characteristics influence XLHb effects on mitochondria and O 2 -sensitive enzymes such as oxidases. ATP; Na-K-ATPase; Rb uptake; NAD(P)H oxidases; diphenyleneiodonium; aminoguanidine; nitro- L -arginine methyl ester; deferroxamine; oxidative phosphorylation Address for reprint requests and other correspondence: A. D. Baines, Rm. 408 Banting Institute, 100 College St., Toronto, ON M5G 1L5, Canada (E-mail: andrew.baines{at}utoronto.ca ).