Keratin containing vascular casts obstruct venous drainage of the post-ischemic kidney.

Acute kidney injury (AKI) remains a major clinical problem; however, the pathogenesis of ischemic acute kidney injury remains unclear. Red blood cell (RBC) trapping presents as the accumulation of many, tightly packed, RBC in the outer-medullary plexus, and is a hallmark of ischemic AKI in both humans and rodents. We have recently reported that extravasation of blood proteins from the RBC congested medullary capillaries is a significant cause of tissue injury. The current study was designed to identify the location of the obstruction responsible for RBC trapping in the rat kidney following ischemia/reperfusion (IR) and to characterize this obstruction

All studies and kidney procurements were approved by the Augusta University IRB and IACUC. Studies were performed in 10–12-week-old male and female Sprague Dawley rats (n=10) and human kidneys from patients with and without AKI that were rejected for transplant (n=10). Rats were anesthetized with isoflurane and renal ischemia caused by placing clamps on the left renal artery for 45 minutes followed by a 1-hour reperfusion period. The right kidney was used as a non-ischemic control. To identify the location of the vascular obstruction in the kidney, prior to the end of the ischemic period, 50mg/kg of the plasma protein marker Evans blue was injected into the tail vein. As Evans blue labels both RBCs and the surrounding vascular wall, this enabled us to identify which vessels were being perfused upon reperfusion of the kidney, as well as allowing us to distinguish between older unstained RBCs and new RBCs entering the kidney post-ischemia. For mass spectrometry analysis, a laser dissection microscope was utilized to collect tubular and venous casts free of surrounding tissue and shotgun proteomics analysis performed. Tissue samples without casts and membrane-only sections without tissue were run as controls. 

Following 1 hour of reperfusion from IR, congested, Evans blue stained RBCs were observed in both the vasa recta bundles and surrounding outer-medullary plexus capillaries. In 6 of 10 congested, Evans blue stained RBC could be observed within the arcuate veins and in 4 of 10 samples these were observed in segmental veins. Congested RBCs in these large veins appeared to be surrounded by a homogeneous material. Scanning electron microscopy imaging of this material revealed it to be a solid that conformed to the shape of the enclosed RBC. Mass spectrometry analysis of the large venous casts identified common blood proteins as well as keratins 1b, 2, 5, 6a, 7, 10 and 14 as the major proteins enriched in the casts.  Similar appearing material was identified in the lumens of damaged tubules and vasa recta of human and rat kidneys with AKI. Immunohistochemical staining confirmed that both vascular and tubular casts stained positive for keratins. 

Our data indicates that, in the post-ischemic rat kidney, a solid material obstructs both vascular and tubular lumens early in reperfusion. This material appears to be responsible for obstruction of venous drainage of the medulla. Our data indicates that this material consists largely of insoluble keratins. Importantly, we demonstrate that tubular and vascular casts containing the same keratin isoforms are prominent in human kidneys with AKI. Keratin casts appear to be responsible for vascular obstruction in the kidney post-ischemia and likely contribute to the pathogenesis of AKI.