Enhanced protection of the renal vascular endothelium improves early outcome in kidney transplantation: Preclinical investigations in pig and mouse

Ischemia reperfusion injury is one of the major complications responsible for delayed graft function in kidney transplantation. Applications to reduce reperfusion injury are essential due to the widespread use of kidneys from deceased organ donors where the risk for delayed graft function is especially prominent. We have recently shown that coating of inflamed or damaged endothelial cells with a unique heparin conjugate reduces thrombosis and leukocyte recruitment. In this study we evaluated the binding capacity of the heparin conjugate to cultured human endothelial cells, to kidneys from brain-dead porcine donors, and to murine kidneys during static cold storage. The heparin conjugate was able to stably bind cultured endothelial cells with high avidity, and to the renal vasculature of explanted kidneys from pigs and mice. Treatment of murine kidneys prior to transplantation reduced platelet deposition and leukocyte infiltration 24 hours post-transplantation, and significantly improved graft function. The present study thus shows the benefits of enhanced protection of the renal vasculature during cold storage, whereby increasing the antithrombotic and anti-adhesive properties of the vascular endothelium yields improved renal function early after transplantation.


Human dermal microvascular endothelial cells (HDMEC #C-12215; PromoCell
GmbH, Heidelberg, Germany) were cultured in complete endothelial cell growth medium microvascular (ECGM MV #C-22020; PromoCell) and used until the 6 th passage. For quantification of the uptake of CHC on HDMEC, 50,000 cells were seeded in 12-well plates. Triplicate wells were treated with increasing concentrations of CHC-FITC diluted in University of Wisconsin preservation solution (UW; ViaSpan; Bristol-Myers Squibb, New York City, NY, USA) for 4 h at 4 °C. After treatment, the cells were washed twice with phosphate buffered saline (PBS) and subsequently lysed with radio-immunoprecipitation assay (RIPA) buffer (Thermo Scientific, Rockford, IL, USA). Lysates were transferred to a 96-well plate for quantification of the FITC content with a Victor 2 fluorescent reader (PerkinElmer, Waltham, MA, USA). The background, consisting of cells treated with UW alone, was subtracted from all samples, which were then compared to a standard curve of known CHC-FITC concentrations. The experiment was repeated either three or four times for each CHC concentration, and results were imported into GraphPad Prism (GraphPad Software, La Jolla, CA, USA) and fitted with a function of the specific binding by nonlinear regression, assuming only one binding site on the endothelial cells, as presented in equation 1: where L is unbound CHC, R is a binding site on the endothelial cells, LR is the complex formed between CHC and the binding site, k a is the association rate constant, and k d is the dissociation rate constant.

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In total, 300,000 HDMEC in 4 mL ECGM MV were seeded in a 10-cm cell culture dish placed in a tilted position to allow the cells to adhere only to a section of the dish. pressures in addition to the mean arterial pressure were continuously monitored via the subclavian artery.
The intracranial pressure was increased in a stepwise manner by inserting a

SPECT/CT imaging
Single-photon emission computed tomography (SPECT)/computed tomography (CT) imaging was performed using the Triumph Trimodality system (TriFoil Imaging Inc., Northridge, CA, USA), an integrated small-animal positron emission tomography (PET)/SPECT/CT scanner. The settings for the CT scan were 50 kV, 2 frames, and 512 projections. SPECT acquisition was performed using 5-pinhole 75A10 collimators with a dual-head, solid-state, cadmium zinc telluride detector. The acquisition parameters for SPECT were 64 projections × 15 s. SPECT raw data were reconstructed with FLEX SPECT software (TriFoil Imaging), which uses an orderedsubset expectation maximization iterative reconstruction algorithm. CT raw files were 6 reconstructed by filtered backprojection. SPECT and CT data were fused and analyzed in PMOD 3.5 (PMOD Technologies Ltd., Zurich, Switzerland).

Ex vivo autoradiography
Biopsies were obtained from the kidneys imaged with SPECT/CT. The biopsies were immediately covered with CO 2 powder and stored at -80 °C until use. For ex vivo autoradiography, 20-µm thick sections were generated with a microtome, placed onto glass slides, dried, and exposed for 7 days (2.5 half-lives of 111 In) to storage phosphor screens (Super Resolution; Perkin Elmer, Downers Grove, IL, USA). The plates were scanned in a Cyclone Plus imager (Perkin Elmer) with a resolution of 600 dpi.

Ex vivo perfusion of murine kidneys
Donor kidneys were carefully perfused with 500 μL cold saline with or without 500

Imaging and image analysis
Brightfield images were captured with a Leica Biosystems APERIO AT2 scanner (Leica, Bromma, Sweden). Confocal image scans were captured with a Zeiss LSM710 (Carl Zeiss, Jena, Germany). Scans consisted of 3 × 4 tiles with z-stacks obtained with a 20X objective (NA=0.8). Images were analyzed with ImageJ (National Institutes of Health, Bethesda, MD, USA). Briefly, the whole section area was determined by the size of the DAPI stained area. The CD41 + area and the TUNEL + area were quantified after thresholding, and then divided by the corresponding DAPI + area. CD11b was quantified as the number of positive particles after thresholding, and normalized according to the corresponding size of the DAPI staining.

QPCR gene expression analysis
First-strand complementary DNA was synthesized using in a two-step reaction. 1.0 μg total RNA was incubated with 0.5 μg Oligo (dT) and 1 μg random primers (Life Victoria, Australia). The relative expression of each gene was calculated as follows: Cycle threshold (Ct) was corrected against the geometric mean of the reference genes glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and ribosomal 18 s (DCt).
Relative expression is expressed as 2 −Δ Ct .

Multiplex cytokine analysis
The levels of cytokines and chemokines in mouse serum samples collected 24 hours post-transplantation were measured using Meso Scale Discovery (MSD, Rockville, Maryland, US) V-PLEX mouse cytokine 19-Plex kit (MSD, #K15255D-1). The samples were analyzed according to the manufactures instructions. In short, the serum samples were pre-diluted 1:4 or 1:10 in Eppendorf low-bind tubes. After activation of the plates, the samples were added to the plates and incubated overnight at 4°C on a shaking plate set to 750 rpm. After washing the secondary antibodies were added and incubated for two hours at RT and then washed to be ready for the reading buffer. The plates were read and analyzed on MSD Sector Imager 2400 within 5 minutes after the addition of the reading buffer. Cytokines and chemokines undetectable above the lowest detection range in the standard curve were excluded in the analysis (IFN-g, IL-2, IL-4, IL12p70, IL-27 and IL-9). The data was the further analyzed using GraphPad Prism.