Expanding the Horizons of Pre-Transplant Renal Vascular Assessment Using Ex Vivo Perfusion

Abstract

Recently, immense efforts have focused on improving the preservation of (sub)optimal donor organs by means of ex vivo perfusion, which enables the opportunity for organ reconditioning and viability assessment. However, there is still no biomarker that correlates with renal viability. Therefore, it is essential to explore new techniques for pre-transplant assessment of organ quality to guarantee successful long-term transplantation outcomes. The renal vascular compartment has received little attention in machine perfusion studies. In vivo, proper renal vascular and endothelial function is essential for maintaining homeostasis and long-term graft survival. In an ex vivo setting, little is known about vascular viability and its implications for an organ's suitability for transplant. Seeing that endothelial damage is the first step in a cascade of disruptions and maintaining homeostasis is crucial for positive post-transplant outcomes, further research is key to clarifying the (patho)physiology of the renal vasculature during machine perfusion. In this review, we aim to summarize key aspects of renal vascular physiology, describe the role of the renal vasculature in pathophysiological settings, and explain how ex vivo perfusion plays a role in either unveiling or targeting such processes. Additionally, we discuss potentially new vascular assessment tools during ex vivo renal perfusion.

Keywords: endothelial cells; ex vivo perfusion; homeostasis; kidney; transplantation; vasculature; viability assessment.

Figure 1

Schematic image of the renal (vascular) structure. Black labels indicate the nephron structures, red labels indicate the vascular structures, and blue labels indicate the filtration processes. Blood flow is directed to an area of glomeruli via interlobular arteries (ILA). Glomerular capillary flow—which is surrounded by Bowman’s capsule—is flanked by two resistance vessels: inflow via afferent arteries and outflow via efferent arteries. In this structure, the ultrafiltrate (UF) is formed. The distal end of the efferent artery can follow one of two paths depending on the location of the glomerulus: if the glomerulus resides in the outer cortex (CG), the effluent blood will run alongside the proximal (PCT) and distal convoluted tubules (DCT) into the peritubular capillaries (PTC), and if the glomerulus resides in the juxtamedullary region (JMG), the effluent blood will run parallel to the loops of Henle (LH) into the vasa recta (VR). The ultrafiltrate formed in the glomeruli flows past the PCT to re-absorb amino acids, ions, glucose, and water, and as it runs through the descending (DL) and ascending limb (AL) of the Loop of Henle, it continues to re-absorb water and sodium chloride into the circulation to control the urine osmolarity. Finally, the urine is fine-tuned in the DCT by exchanging water and ions, and flows into the collecting duct (CD), then to the renal pelvis, and down to the bladder via the ureter, where it is stored until urination. Image created with BioRender.

Figure 2

Schematic image of vascular contractility pathways in the endothelial cells (ECs). Purple labels indicate the nitric oxide (NO) signaling cascade, blue labels indicate the prostacyclin (PGI₂) cascade, red labels indicate the thromboxane (TXA₂) cascade and green labels indicate the endothelin-1 (ET-1) cascade. The main pathway of NO production starts with the activation of the endothelial enzyme nitric oxide synthase (eNOS) by an intracellular influx of calcium (Ca⁺) and/or an agonist signal to release Ca²⁺ from the endoplasmic reticulum (ER). Ca²⁺ is then modified by calmodulin (CaM) so that it can bind to eNOS, which then results in the production of NO. Once Ca²⁺ levels are generally depleted and more long-term vasodilation is needed, an alternative pathway is activated to stimulate NO production directly. Namely, the phosphorylation of eNOS via protein kinases (PK) in response to sheer stress on the cell surface. Both pathways culminate in sending NO to the smooth muscle cells (SMCs), which stimulates a decrease in Ca²⁺ levels and promotes relaxation; The PGI₂ pathway, a vasodilatory pathway that compensates for low NO levels, is mediated and catalyzed by cyclooxygenase enzyme 2 (COX-2), which is activated by sheer stress and ECs are damaged and exposed to inflammatory cytokines. The conversion of arachidonic acid (AA) into prostaglandin H₂ (PGH₂) by COX-2 is the initial step to synthesize PGI₂. PGI₂ then activates platelets and SMCs by binding to a prostacyclin receptor (IP). In response, SMCs produce adenosine monophosphate (cAMP) and activate protein kinase (PKA), which, in turn, relaxes the SMCs in the same way as NO. The binding of circulating PGI₂ to their EC IP receptor can also promote vasodilation by stimulating eNOS; In contrast, the production of TXA₂ induces vasoconstriction and platelet aggregation. Cyclooxygenase enzyme 1 (COX-1) converts AA into PGH₂, which stimulates the isomerization of TXA₂. In sequence, TXA₂ binds to thromboxane-prostanoid receptors (TP) located in SMCs and platelets, therefore inducing platelet aggregation. Vasoconstriction by TXA₂ can also occur by stimulating CA²⁺ release by the endoplasmic reticulum in SMCs. Lastly, the ET-1 pathway is regulated by inflammatory cells (interleukins and TNF-α) stimulation and reduction in NO and PGI₂ levels. ET-1 receptors are present both in ECs (ET_B1) and SMCs (ET_A and ET_B2). When SMC ET-1 receptors are activated, there is an influx of calcium due to the opening of the Ca²⁺ channels, causing vasoconstriction in a similar way as TXA₂. In contrast, activation of ET_B1 receptors in ECs causes vasodilation by stimulating the release of PGI₂ and NO. When endothelial dysfunction occurs, EC ET-1 receptors are downregulated, and SMC ET-1 receptors are upregulated, therefore enhancing a vasoconstrictive environment. Image created with BioRender.

Figure 3

Schematic image of possible applications of vascular viability assessment and targeted treatment from other fields of research in ex vivo perfusion research. Real-time analysis during perfusion could be achieved by administering boluses of vasoactive agents during perfusion, and assessment could be performed by observing flow/vascular resistance variations via ultrasound or laser speckle contrast imaging to observe changes in perfusion profiles; Iontophoresis assesses the nitric oxide availability in the microvasculature by placing two watertight chambers on the skin that conduct an electrical current to transfer positive or negatively charged vasoactive agents through resistance vessels locally. To measure vascular changes, Laser Doppler Flowmetry (LDF) or Laser Doppler Imaging (LDI) can be used; Venous Occlusion Plethysmography entails stopping venous return while there is arterial inflow. This flow occlusion causes the forearm to increase volume over time in proportion to the incoming arterial flow. The increase in forearm volume is measured as the increase in length of the strain-gauge causes a change in electrical resistance; arterial stiffness is measured as branching points in the arteries cause the heartbeat pulsatile pressure waves to be reflected, meaning that the stiffer an artery is, the faster these waves travel back. The most common techniques to assess arterial stiffness are Pulse Wave Analysis (PWA) and Pulse Wave Velocity (PWV); Functional MRI (fMRI) imaging during renal NMP has recently been applied to investigate the impact of relevant events in the transplant process (such as warm/cold ischemia times) on grafts, and it can add an extra set of variables to organ quality prediction models by acquiring information on general vessel architecture and regional blood flow distribution. Sample analysis of vascular viability could be performed by flow cytometry. This technique could be applied to monitor the shedding of endothelial/epithelial cells as a marker of cellular damage; Microcomputed tomography is a relatively new technology that can be applied to image samples inside and out in a non-destructive way and acquire high-resolution images with fast 3D cross-section reconstruction. Voxel sizes can be adjusted to image structures as small as afferent/efferent arterioles; Lightsheet fluorescence microscopy is an ex vivo tissue microscopy technique that enables optical sectioning of the sample while still in a three-dimensional architecture, therefore enabling whole organ imaging. Last but not least, ex vivo perfusion can be used as a platform for targeted treatment, which can be achieved by administering medications (such as drugs, micro RNAs, small interfering RNAs, or even nanoparticles) targeting fibrinolysis inflammation, antibody-mediated rejection (HLA), fibrosis (TGF-β), ischemia-reperfusion injury, hypoxia (HIF), angiogenesis (VEGF). Image created with BioRender.