Red blood cell trapping in the kidney vasculature promotes mitochondrial dysfunction independent of ischemia time

Red blood cell (RBC) trapping presents as the expansion of- and obstruction, of the outer-medullary capillaries of the kidney with tightly packed RBCs. RBC trapping has been thought to promote hypoxic tubular injury by extending ischemia time to the outer-medulla. Recent evidence from our laboratory; however, indicates that RBC trapping may promote toxic tubular injury, secondary to the mitochondrial uptake of iron from hemoglobin released from damaged RBCs in nearby congested capillaries. In the current study, we tested the hypothesis that ‘RBC trapping promotes tubular mitochondrial dysfunction independent of ischemia’. We tested our hypothesis using 9–10-week-old male Wistar-Kyoto rats. In an initial study, rats underwent bilateral warm arterial ischemia for 45 minutes with 2 hours of reperfusion before harvesting the kidneys for analysis by transmission electron microscopy (n=3). In a second study, the left kidney from each of n=10 rats underwent either arterial, venous or sham control clamping for 45 minutes. An alternate procedure (either arterial, venous or sham clamping) was performed on the right kidney. To examine the effect of ischemia alone on mitochondrial function, renal arteries were clamped, and the kidney excised without releasing the clamp. To examine the effect of ischemia with RBC trapping on mitochondrial function, the renal vein was clamped. Renal venous clamping mimics the obstruction of the medullary venous vasculature that occurs during reperfusion from arterial clamping, resulting in both kidney ischemia and RBC trapping. In all animals body temperature was maintained using a servo-controlled heating table and heat lamp. At harvest the kidney medulla and cortex separated and mitochondria isolated from each region. Mitochondrial membrane potential (TMRM fluorescence) and hydrogen peroxide production (Amplex red) was then determined from n=6-7 kidneys for each intervention. Data were compared using ANOVA. Consistent with iron uptake into mitochondria, when viewed using transmission electron microscopy, following 2 hours of reperfusion from arterial clamping, tubular mitochondria near RBC congested vessels often appeared darkened. Both ischemia alone and ischemia with RBC trapping, resulted in a significant increase in mitochondrial H202 production compared to control (7063±562 and 7519±803 vs 5688±389 A.U per mg of protein, respectively, p<0.001). In contrast, ischemia alone resulted in a lower mitochondrial membrane potential (228±18 A.U) compared to control (253±18), whereas, mitochondrial membrane potential following ischemia with RBC trapping (308±26) was significantly greater than both ischemia alone and control (p<0.001). Our data supports the hypothesis that congestion of the kidney vasculature with RBCs directly promotes mitochondrial dysfunction independent of ischemia. This likely occurs secondary to mitochondrial uptake of iron from hemoglobin released from damaged RBCs. NIH NIDDK ISAC 21AU4231, NIH NHLBI P01 HL134604 and an American Heart Association Transformational Project Award 970585 to P.O and NIH NIDDK F31 DK127683 to S.R.M. NIH R01 HL148114 D.I. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.