Introduction: Ischemia-reperfusion injury (IRI) results in cellular damage, production of free oxygen radicals, and subsequent apoptosis. During partial nephrectomy, the renal hilum is temporarily clamped to optimize resection of renal masses in a bloodless field. The trade-off for this maneuver is IRI. It is not known definitively how long a period of renal artery clamping will result in irreversible cellular death, nor in which region of the kidney the injury primarily occurs. An established marker of inflammation and ischemic injury is P-selectin, a cell adhesion molecule expressed on endothelial cells and activated platelets. The goal of this project was to use targeted microbubbles outfitted with anti-P-selectin antibodies to quantitate for the first time the microvascular reperfusion injury and regional blood flow in the kidney during IRI. Materials and Methods: Using the protocol approved by the Institutional Animal Care and Use Committee (IACUC), 20 renal units obtained from C57/BL6J female mice were studied. The left renal artery and vein were ligated for 30 minutes. Microbubbles coated with anti-P-selectin antibodies were injected after hilar unclamping, and both kidneys were scanned. As a control, perfusion and re-perfusion of the left kidney was performed using pulse-wave Doppler mode. Subsequently, a Vevo 770 micro-ultrasound system with a resolution of 40μm was used to noninvasively measure the microvascular flow and quantitate targeted microbubbles bound to P-selectin. Negative controls consisted of sham animals and microbubbles coated with isotype serum. Customized software produced digital subtraction video intensity units (VIU), which allowed comparison of the different regions of the kidney. Results: Regional blood flow was measured in three areas: cortex, medulla, and corticomedullary junction (CMJ). In the sham left kidney, the CMJ had the highest blood volume and flow (141.1) compared with renal medulla (43.7) and cortex (100.6) VIU (p< 0.01). After hilar unclamping, blood flow rate to the left kidney decreased from 554mm/s to 182mm/s, despite improvement in the color of the kidney from cyanotic to pink. After 30 minutes of ischemia, P-selectin expression increased by 41%, 25%, and 14% in the CMJ, cortex, and the medulla, respectively, compared to controls. P-selectin expression, and therefore the greatest area of ischemic injury, was highest in the CMJ region (432.1) compared with the cortex (369.4) and medulla (86.5) (p< 0.01). Conclusion: This pilot study quantitates for the first time in an in-vivo model of IRI that the CMJ region sustains the highest degree of nephron and microvascular damage. This region is the most susceptible to ischemic injury as indicated by 41% increase in expression of P-selectin immediately postunclamping.
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