Integrative omics reveals subtle molecular perturbations following ischemic conditioning in a porcine kidney transplant model.

BACKGROUND: Remote Ischemic Conditioning (RIC) has been proposed as a therapeutic intervention to circumvent the ischemia/reperfusion injury (IRI) that is inherent to organ transplantation. Using a porcine kidney transplant model, we aimed to decipher the subclinical molecular effects of a RIC regime, compared to non-RIC controls. METHODS: Kidney pairs (n = 8 + 8) were extracted from brain dead donor pigs and transplanted in juvenile recipient pigs following a period of cold ischemia. One of the two kidney recipients in each pair was subjected to RIC prior to kidney graft reperfusion, while the other served as non-RIC control. We designed an integrative Omics strategy combining transcriptomics, proteomics, and phosphoproteomics to deduce molecular signatures in kidney tissue that could be attributed to RIC. RESULTS: In kidney grafts taken out 10 h after transplantation we detected minimal molecular perturbations following RIC compared to non-RIC at the transcriptome level, which was mirrored at the proteome level. In particular, we noted that RIC resulted in suppression of tissue inflammatory profiles. Furthermore, an accumulation of muscle extracellular matrix assembly proteins in kidney tissues was detected at the protein level, which may be in response to muscle tissue damage and/or fibrosis. However, the majority of these protein changes did not reach significance (p < 0.05). CONCLUSIONS: Our data identifies subtle molecular phenotypes in porcine kidneys following RIC, and this knowledge could potentially aid optimization of remote ischemic conditioning protocols in renal transplantation.

Dynamics of circulating dendritic cells and cytokines after kidney transplantation-No effect of remote ischaemic conditioning.

Inflammation resulting from ischaemia/reperfusion injury can cause kidney graft dysfunction, increase the risk of delayed graft function and possibly reduce long-term graft survival. Remote ischaemic conditioning may protect against ischaemia/reperfusion injury and mitigate the immunological response to the graft. We investigated the immunological effects of remote ischaemic conditioning on kidney transplantation from deceased donors in the randomized CONTEXT study. Three circulating dendritic cell (DC) subtypes identified in peripheral blood from kidney transplant recipients [myeloid DCs, plasmacytoid DCs and immunoglobulin-like transcript (ILT)3+ DCs] were measured at baseline, days 1, 3 and 5 and 1 and 3 months after transplantation. We also quantified 21 cytokines at baseline, days 1 and 5 and 3 months after transplantation. Neither DC counts nor cytokine levels differed between patients receiving remote ischaemic conditioning and controls; however, several parameters exhibited dynamic and parallel alterations in the two groups over time, reflecting the immunological response to the kidney transplantation and immunosuppression.

Local Pressure Drives Low-Density Lipoprotein Accumulation and Coronary Atherosclerosis in Hypertensive Minipigs.

BACKGROUND: The mechanisms by which hypertension accelerates coronary artery disease are poorly understood. Patients with hypertension often have confounding humoral changes, and to date, no experimental models have allowed analysis of the isolated effect of pressure on atherosclerosis in a setting that recapitulates the dimensions and biomechanics of human coronary arteries. OBJECTIVES: This study sought to analyze the effect of pressure on coronary atherosclerosis and explore the underlying mechanisms. METHODS: Using inflatable suprarenal aortic cuffs, we increased mean arterial pressure by >30 mm Hg in the cephalad body part of wild-type and hypercholesterolemic proprotein convertase subtilisin kexin type 9 (PCSK9)D374Y Yucatan minipigs for >1 year. Caudal pressures remained normal. RESULTS: Under hypercholesterolemic conditions in PCSK9D374Y transgenic minipigs, cephalad hypertension accelerated coronary atherosclerosis to almost 5-fold with consistent development of fibroatheromas that were sufficiently large to cause stenosis on computed tomography angiography. This was caused by local pressure forces, because vascular beds shielded from hypertension, but exposed to the same humoral factors, showed no changes in lesion formation. The same experiment was conducted under normocholesterolemic conditions in wild-type minipigs to examine the underlying mechanisms. Hypertension produced clear changes in the arterial proteome with increased abundance of mechanical strength proteins and reduced levels of infiltrating plasma macromolecules. This was paralleled by increased smooth muscle cells and increased intimal accumulation of low-density lipoproteins in the coronary arteries. CONCLUSIONS: Increased pressure per se facilitates coronary atherosclerosis. Our data indicate that restructuring of the artery to match increased tensile forces in hypertension alters the passage of macromolecules and leads to increased intimal accumulation of low-density lipoproteins.

No effect of remote ischaemic conditioning on inflammation in a porcine kidney transplantation model.

MAIN PROBLEM: Delayed graft function after kidney transplantation is associated with decreased graft survival and increased patient mortality but the pathogenesis is poorly understood. Remote ischaemic conditioning (rIC) may prevent delayed graft function by an anti-inflammatory effect. In a porcine model of transplantation from adults to children, we investigated the inflammatory response in the transplanted kidney and the effect of rIC. METHODS: Kidneys were recovered from brain dead donor pigs(63kg) and transplanted into two groups of recipient pigs(15kg) after 22h of cold ischaemia. Recipients were randomised to either: rIC (n=8) performed before the 10-h reperfusion period or no-rIC (n=8). Non-transplanted kidneys from eight brain dead pigs served as controls. RESULTS: Compared to controls, transplantation increased the number of apoptotic cells, macrophages and neutrophils in the kidney. After transplantation, IL-10 levels increased and IL-6 levels decreased in the kidney, whereas levels of TNF-α and IL-8 were not affected. A significant rise in plasma IL-1ß and IL-6 was observed in the recipients after transplantation. Plasma IL-10 was not affected by transplantation and TNF-α and IL-8 were below detection limit. No effect of rIC was found with regards to cell infiltration or cytokine production. CONCLUSION: Renal transplantation elicits an inflammatory response in the kidney manifested as apoptotic cell death, macrophage and neutrophil infiltration, and an anti-inflammatory cytokine response 10h after transplantation. This response was not modified by rIC.

Improved GFR and renal plasma perfusion following remote ischaemic conditioning in a porcine kidney transplantation model.

Delayed graft function (DGF) complicates approximately 25% of kidney allografts donated after brain death (DBD). Remote ischaemic conditioning (rIC) involves brief, repetitive, ischaemia in a distant tissue in connection with ischaemia/reperfusion in the target organ. rIC has been shown to induce systemic protection against ischaemic injuries. Using a porcine kidney transplantation model with donor (63 kg) recipient (15 kg) size mismatch, we investigated the effects of recipient rIC on early renal plasma perfusion and GFR. Brain death was induced in donor pigs (n = 8) and kidneys were removed and kept in cold storage until transplantation. Nephrectomized recipient pigs were randomized to rIC (n = 8) or non-rIC (n = 8) with one kidney from the same donor in each group. rIC consisted of 4 × 5 min clamping of the abdominal aorta. GFR was significantly higher in the rIC group compared with non-rIC (7.2 ml/min vs. 3.4 ml/min; ΔGFR = 3.7 ml/min, 95%-CI: 0.3-7.2 ml/min, P = 0.038). Renal plasma perfusion in both cortex and medulla measured by dynamic contrast-enhanced magnetic resonance imaging (MRI) was significantly higher over time in the rIC group compared with non-rIC. This experimental study demonstrated a positive effect of rIC on early graft perfusion and function in a large animal transplantation model.

Early outcome in renal transplantation from large donors to small and size-matched recipients - a porcine experimental model.

Kidney transplantation from a large donor to a small recipient, as in pediatric transplantation, is associated with an increased risk of thrombosis and DGF. We established a porcine model for renal transplantation from an adult donor to a small or size-matched recipient with a high risk of DGF and studied GFR, RPP using MRI, and markers of kidney injury within 10 h after transplantation. After induction of BD, kidneys were removed from ∼63-kg donors and kept in cold storage for ∼22 h until transplanted into small (∼15 kg, n = 8) or size-matched (n = 8) recipients. A reduction in GFR was observed in small recipients within 60 min after reperfusion. Interestingly, this was associated with a significant reduction in medullary RPP, while there was no significant change in the size-matched recipients. No difference was observed in urinary NGAL excretion between the groups. A significant higher level of HO-1 mRNA was observed in small recipients than in donors and size-matched recipients indicating cortical injury. Improvement in early graft perfusion may be a goal to improve short- and long-term GFR and avoid graft thrombosis in pediatric recipients.

Microdialysis for detection of renal ischemia after experimental renal transplantation.

PURPOSE: We designed an experimental renal transplantation model and evaluated microdialysis as a detector of induced postoperative ischemia, a feared complication that when caused by vascular thrombosis most often causes renal graft loss. MATERIALS AND METHODS: Two microdialysis catheters were placed in the left kidney in 16 pigs, including 1 superficially in the renal cortex and 1 fixed on the renal capsule. Two-hour baseline measurements were made at steady state, after which the kidney was removed and subjected to warm and cold ischemia. It was subsequently re-anastomosed end to end in situ and reperfused for 5 hours. Pigs were then randomized into a total renal artery occlusion and a control group. RESULTS: At baseline there were no changes in local metabolites (glucose, glutamate, glycerol and lactate) and no significant difference between the groups. Glycerol increased 4-fold in each group during cold ischemia but there were no pivotal alterations in other metabolites. After kidney reperfusion glycerol decreased and all metabolites were in steady state after 1 hour. At 30 minutes after postoperative ischemia was introduced there were significant increases in all kidneys in ischemia vs steady state reperfusion levels of cortical lactate, glutamate, glycerol and the lactate-to-glucose ratio (each rank sum test p <0.001). No metabolic changes were seen in controls. CONCLUSIONS: Microdialysis detected significant metabolic changes after postoperative ischemia in pigs with experimental renal transplantation, while no metabolic changes were observed in controls. In the future microdialysis may become a valuable tool for postoperative observation of transplanted kidneys, most probably with major impact on early graft survival.