The Contribution of Post-Ischemic Venous Obstruction to Tubular Injury in Rats

We recently reported that post-ischemic venous obstruction leads to red blood cell (RBC) trapping in the renal medulla and the extravasation of blood proteins into the cortical-medullary boundary zone. However, the extent to which plasma leakage and RBC trapping contribute to renal tubular injury and recovery remains unclear. This study aimed to characterize the relationship between plasma extravasation, RBC trapping, and tubular injury using a rat model of ischemia-reperfusion (IR) injury.

Experiments were conducted on 10–12-week-old male and female Sprague Dawley rats (n=4M, 4F per group), divided into sham and IR groups. Under isoflurane anesthesia (1–5%), flank incisions were made. In IR rats, both renal arteries were clamped for 45 minutes, with body temperature maintained throughout. After clamp removal, surgical wounds were closed, and rats were allowed to recover for 0, 1 or 3 hours before sacrifice. Sham rats underwent identical procedures without arterial clamping. To assess plasma extravasation following 45 minutes of ischemia, Evans blue dye (50 mg/kg) was injected via the tail vein prior to reperfusion.

Kidney volume increased rapidly upon reperfusion following 45 minutes of ischemia. Wet kidney weight per 100 g body weight rose from 0.32±0.01% in controls to 0.38±0.02% and 0.43±0.01% at 1 and 3 hours post-ischemia, respectively (p<0.0001). In Evans blue-injected IR rats, kidneys appeared dark blue, while control kidneys remained pale. Histological analysis showed unstained cortical glomeruli up to 3 hours post-ischemia, suggesting increased pre-glomerular vascular resistance. Despite the absence of filtration, Evans blue-stained blood proteins were present in proximal tubule lumens, especially in the boundary zone near the medulla. Control kidneys showed no extravasation, with dye confined to the vasculature.

Proximal tubular injury and the presence of absorption droplets were closely associated with Evans blue extravasation. Immunohistochemical staining confirmed albumin infiltration into the extravascular parenchyma. In control rats, albumin staining in proximal tubules was minimal. In IR rats with mild injury, albumin was observed in the paracellular space and brush borders of proximal tubules in the boundary zone. In severely injured rats, large cytosolic albumin droplets and widespread albumin staining were evident, including albumin casts in the tubular lumens and Bowman’s space of glomeruli.

RBC trapping was prominent in the outer stripe of the medulla and also strongly correlated with Evans blue extravasation. Tubular injury in this region consistent primarily of cast formation and tubular sloughing, with severity closely linked to the extent of RBC trapping.

Our findings demonstrate significant plasma protein extravasation upon kidney reperfusion. In the cortex, this leakage is most pronounced in the boundary zone, where blood proteins appear to infiltrate proximal tubules before being absorbed across the apical membrane. The strong correlation between plasma extravasation and proximal tubular injury—occurring early in reperfusion when filtration is absent—suggests that blood protein leakage is a primary driver of injury in this model. Injury to the outer-stripe of the medulla also correlated with plasma extravasation and was closely related to the degree of RBC trapping.