Magnetic resonance imaging as a noninvasive adjunct to conventional assessment of functional differences between kidneys in vivo and during ex vivo normothermic machine perfusion.

Normothermic machine perfusion (NMP) is increasingly considered for pretransplant kidney quality assessment. However, fundamental questions about differences between in vivo and ex vivo renal function, as well as the impact of ischemic injury on ex vivo physiology, remain unanswered. This study utilized magnetic resonance imaging (MRI), alongside conventional parameters to explore differences between in vivo and ex vivo renal function and the impact of warm ischemia on a kidney's behavior ex vivo. Renal MRI scans and samples were obtained from living pigs (n = 30) in vivo. Next, kidney pairs were procured and exposed to minimal, or 75 minutes of warm ischemia, followed by 6 hours of hypothermic machine perfusion. Both kidneys simultaneously underwent 6-hour ex vivo perfusion in MRI-compatible NMP circuits to obtain multiparametric MRI data. Ischemically injured ex vivo kidneys showed a significantly altered regional blood flow distribution compared to in vivo and minimally damaged organs. Both ex vivo groups showed diffusion restriction relative to in vivo. Our findings underscore the differences between in vivo and ex vivo MRI-based renal characteristics. Therefore, when assessing organ viability during NMP, it should be considered to incorporate parameters beyond the conventional functional markers that are common in vivo.

Ex-Vivo Kidney Perfusion With Hemoglobin-Based Oxygen Carriers, Red Blood Cells, or No Oxygen Carrier.

INTRODUCTION: Normothermic machine perfusion (NMP) of donor kidneys provides the opportunity to assess and improve organ viability prior to transplantation. This study explored the necessity of an oxygen carrier during NMP and whether the hemoglobin-based oxygen carrier (HBOC-201) is a suitable alternative to red blood cells (RBCs). METHODS: Porcine kidneys were perfused with a perfusion solution containing either no-oxygen carrier, RBCs, or HBOC-201 for 360 min at 37°C. RESULTS: Renal flow and resistance did not differ significantly between groups. NMP without an oxygen carrier showed lower oxygen consumption with higher lactate and aspartate aminotransferase levels, indicating that the use of an oxygen carrier is necessary for NMP. Cumulative urine production and creatinine clearance in the RBC group were significantly higher than in the HBOC-201 group. Oxygen consumption, injury markers, and histology did not differ significantly between these two groups. However, methemoglobin levels increased to 45% after 360 min in the HBOC-201 group. CONCLUSIONS: We conclude that HBOC-201 could be used as an alternative for RBCs, but accumulating methemoglobin levels during our perfusions indicated that HBOC-201 is probably less suitable for prolonged NMP. Perfusion with RBCs, compared to HBOC-201, resulted in more favorable renal function during NMP.

Renal cortex microperfusion evaluated by laser speckle contrast imaging in an ex vivo perfused kidney model-A proof-of-concept study.

BACKGROUND: Validated quantitative biomarkers for assessment of renal graft function during normothermic machine perfusion (NMP) conditions are lacking. The aim of this project was to quantify cortex microperfusion during ex vivo kidney perfusion using laser speckle contrast imaging (LSCI), and to evaluate the sensitivity of LSCI when measuring different levels of renal perfusion. Furthermore, we aimed to introduce LSCI measurements during NMP in differentially damaged kidneys. METHODS: Eleven porcine kidneys were nephrectomized and perfused ex vivo. Cortex microperfusion was simultaneously monitored using LSCI. First, a flow experiment examined the relationship between changes in delivered renal flow and corresponding changes in LSCI-derived cortex microperfusion. Second, renal cortical perfusion was reduced stepwise by introducing a microembolization model. Finally, LSCI was applied for measuring renal cortex microperfusion in kidneys exposed to minimal damage or 2 h warm ischemia (WI). RESULTS: Cortex microperfusion was calculated from the LSCI-obtained data. The flow experiment resulted in relatively minor changes in cortex microperfusion compared to the pump-induced changes in total renal flow. Based on stepwise injections of microspheres, we observed different levels of cortex microperfusion that correlated with administrated microsphere dosages (r2 = 0.95-0.99). We found no difference in LSCI measured cortex microperfusion between the kidneys exposed to minimal damage (renal cortex blood flow index, rcBFI = 2090-2600) and 2 h WI (rcBFI = 2189-2540). CONCLUSIONS: Based on this preliminary study, we demonstrated the feasibility of LSCI in quantifying cortex microperfusion during ex vivo perfusion. Furthermore, based on LSCI-measurements, cortical microperfusion was similar in kidneys exposed to minimal and 2 h WI.

In a real-life setting, risk factors, coronary artery calcium score, and coronary stenosis at computed tomography angiography are associated with major adverse cardiovascular events and all-cause mortality among kidney transplant candidates.

Kidney failure is associated with an increased risk of cardiovascular disease and death. This single-center, a retrospective study evaluated the association between risk factors, coronary artery calcium score (CACS), coronary computed tomography angiography (CTA), major adverse cardiovascular events (MACEs), and all-cause mortality in kidney transplant candidates. Data on clinical risk factors, MACE, and all-cause mortality were collected from patient records. A total of 529 kidney transplant candidates were included (median follow-up of 4.7 years). CACS was evaluated in 437 patients and CTA in 411. Both the presence of ≥3 risk factors, CACS of ≥400, as well as multiple-vessel stenoses or left main artery disease predicted MACE (hazard ratio, 2.09; [95% confidence interval, 1.35-3.23]; 4.65 [2.20-9.82]; 3.70 [1.81-7.57]; 4.90 [2.40-10.01]) and all-cause mortality (harad ratio, 4.44; [95% confidence interval, 2.54-7.76]; 4.47 [2.22-9.02]; 2.82 [1.34-5.94]; 5.41 [2.81-10.41]) in univariate analyses. Among patients eligible for CACS and CTA (n = 376), only CACS and CTA were associated with both MACE and all-cause mortality. In conclusion, risk factors, CACS, and CTA provide information on the risk of MACE and mortality in kidney transplant candidates. An additional value of CACS and CTA compared with risk factors was observed for the prediction of MACE in a subpopulation undergoing both CACS and CTA.

Proteinuria is accompanied by intratubular complement activation and apical membrane deposition of C3dg and C5b-9 in kidney transplant recipients.

Proteinuria predicts accelerated decline in kidney function in kidney transplant recipients (KTRs). We hypothesized that aberrant filtration of complement factors causes intraluminal activation, apical membrane attack on tubular cells, and progressive injury. Biobanked samples from two previous studies in albuminuric KTRs were used. The complement-activation split products C3c, C3dg, and soluble C5b-9-associated C9 neoantigen were analyzed by ELISA in urine and plasma using neoepitope-specific antibodies. Urinary extracellular vesicles (uEVs) were enriched by lectin and immunoaffinity isolation and analyzed by immunoblot analysis. Urine complement excretion increased significantly in KTRs with an albumin-to-creatinine ratio of ≥300 mg/g compared with <30 mg/g. Urine C3dg and C9 neoantigen excretion correlated significantly to changes in albumin excretion from 3 to 12 mo after transplantation. Fractional excretion of C9 neoantigen was significantly higher than for albumin, indicating postfiltration generation. C9 neoantigen was detected in uEVs in six of the nine albuminuric KTRs but was absent in non-albuminuric controls (n = 8). In C9 neoantigen-positive KTRs, lectin affinity enrichment of uEVs from the proximal tubules yielded signal for iC3b, C3dg, C9 neoantigen, and Na+-glucose transporter 2 but only weakly for aquaporin 2. Coisolation of podocyte markers and Tamm-Horsfall protein was minimal. Our findings show that albuminuria is associated with aberrant filtration and intratubular activation of complement with deposition of C3 activation split products and C5b-9-associated C9 neoantigen on uEVs from the proximal tubular apical membrane. Intratubular complement activation may contribute to progressive kidney injury in proteinuric kidney grafts.NEW & NOTEWORTHY The present study proposes a mechanistic coupling between proteinuria and aberrant filtration of complement precursors, intratubular complement activation, and apical membrane attack in kidney transplant recipients. C3dg and C5b-9-associated C9 neoantigen associate with proximal tubular apical membranes as demonstrated in urine extracellular vesicles. The discovery suggests intratubular complement as a mediator between proteinuria and progressive kidney damage. Inhibitors of soluble and/or luminal complement activation with access to the tubular lumen may be beneficial.

Outcomes following SARS-CoV-2 infection in individuals with and without solid organ transplantation-A Danish nationwide cohort study.

The risk of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, hospitalization and death, and the effects of SARS-CoV-2 vaccines in solid organ transplant recipients (SOTRs) is still debated. We performed a nationwide, population-based, matched cohort study, including all Danish SOTRs (n = 5184) and a matched cohort from the general population (n = 41 472). Cox regression analyses were used to calculate incidence rate ratios (IRRs). SOTRs had a slightly increased risk of SARS-CoV-2 infection and were vaccinated earlier than the general population. The overall risk of hospital contact with COVID-19, severe COVID-19, need for assisted respiration, and hospitalization followed by death was substantially higher in SOTRs (IRR: 32.8 95%CI [29.0-37.0], 9.2 [6.7-12.7], 12.5 [7.6-20.8], 12.4 [7.9-12.7]). The risk of hospitalization and death after SARS-CoV-2 infection decreased substantially in SOTRs after the emergence of the Omicron variant (IRR: 0.45 [0.37-0.56], 0.17 [0.09-0.30]). Three vaccinations reduced the risk of SARS-CoV-2 infection only marginally compared to two vaccinations, but SOTRs with three vaccinations had a lower risk of death (IRR: 022 [0.16-0.35]). We conclude that SOTRs have a risk of SARS-CoV-2 infection comparable to the general population, but substantially increased the risk of hospitalization and death following SARS-CoV-2 infection. A third vaccination only reduces the risk of SARS-CoV2 infection marginally, but SOTRs vaccinated 3 times have reduced mortality.

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.

A comparison of four established GFR formulas to estimate measured GFR and changes in GFR in adult kidney transplant recipients.

The accurate assessment of glomerular filtration rate (GFR) is important in the follow-up of kidney transplant recipients in order to identify graft dysfunction. A number of formulas have been proposed to calculate GFR from endogenous plasma markers such as creatinine or cystatin C since measuring GFR using exogenous markers is troublesome. This study compares and evaluates the ability of four different GFR formulas to estimate kidney graft function and to detect changes in GFR in kidney transplant recipients. The study included patients from the prospective, multicenter CONTEXT trial in kidney transplant recipients. GFR was measured using plasma clearance of 51Cr-EDTA and estimated using the MDRD, CKD-EPI Creatinine, CKD-EPI Cystatin C and CKD-EPI Cystatin C + Creatinine equations at three (n = 83) and twelve (n = 65) months post-transplantation. For each formula mean bias, precision, and accuracy were evaluated. The MDRD equation had the lowest mean bias (0.2 ml/min/1.73 m2), whereas the CKD-EPI Cystatin C + Creatinine equation had the highest precision (8 ml/min/1.73 m2). Accuracy at three months were similar for all equations (P30 > 80%) except for the CKD-EPI Cystatin C equation, which performed poorer (P30 = 55%). None of the formulas evaluated avoided misclassification of changes in GFR. The most optimal combination of precision and accuracy suggests the use of CKD-EPI Creatinine + Cystatin C equation in kidney transplant recipients.

Corticomedullary shunting after ischaemia and reperfusion in the porcine kidney?

BACKGROUND: Renal perfusion may redistribute from cortex to medulla during systemic hypovolaemia and after renal ischaemia for other reasons, but there is no consensus on this matter. We studied renal perfusion after renal ischaemia and reperfusion. METHODS: Renal perfusion distribution was examined by use of 153Gadolinium-labeled microspheres (MS) after 2 h (hrs) and 4 h ischaemia of the pig kidney followed by 4 h of reperfusion. Intra-arterial injected MS are trapped in the glomeruli in renal cortex, which means that MS are not present in the medulla under normal physiological conditions. RESULTS: Visual evaluation after reperfusion demonstrated that MS redistributed from the renal cortex to the medulla in 6 out of 16 pigs (38%) subjected to 4 h ischaemia and in one out of 18 pigs subjected to 2 h ischaemia. Central renal uptake of MS covering the medullary/total renal uptake was significantly higher in kidneys subjected to 4 h ischaemia compared with pigs subjected to 2 h ischaemia (69 ± 5% vs. 63 ± 1%, p < 0.001), and also significantly higher than in the contralateral kidney (69 ± 5% vs. 63 ± 2%, p < 0.001). Analysis of blood and urine demonstrated no presence of radioactivity. CONCLUSION: The study demonstrated the presence of MS in the renal medulla in response to renal ischaemia and reperfusion suggesting that severe ischaemia and reperfusion of the pig kidney leads to opening of functional shunts bypassing glomeruli.

Dosing Limitation for Intra-Renal Arterial Infusion of Mesenchymal Stromal Cells.

The immunomodulatory and regenerative properties of mesenchymal stromal cells (MSCs) make MSC therapy a promising therapeutic strategy in kidney disease. A targeted MSC administration via the renal artery offers an efficient delivery method with limited spillover to other organs. Although local administration alleviates safety issues with MSCs in systemic circulation, it introduces new safety concerns in the kidneys. In a porcine model, we employed intra-renal arterial infusion of ten million allogenic adipose tissue-derived MSCs. In order to trigger any potential adverse events, a higher dose (hundred million MSCs) was also included. The kidney function was studied by magnetic resonance imaging after the MSC infusion and again at two weeks post-treatment. The kidneys were assessed by single kidney glomerular filtration rate (skGFR) measurements, histology and inflammation, and fibrosis-related gene expression. None of the measured parameters were affected immediately after the administration of ten million MSCs, but the administration of one hundred million MSCs induced severe adverse events. Renal perfusion was reduced immediately after MSC administration which coincided with the presence of microthrombi in the glomeruli and signs of an instant blood-mediated inflammatory reaction. At two weeks post-treatment, the kidneys that were treated with one hundred million MSCs showed reduced skGFR, signs of tissue inflammation, and glomerular and tubular damage. In conclusions, the intra-renal administration of ten million MSCs is well-tolerated by the porcine kidney. However, higher concentrations (one hundred million MSCs) caused severe kidney damage, implying that very high doses of intra-renally administered MSCs should be undertaken with caution.

Mesenchymal stromal cell treatment of donor kidneys during ex vivo normothermic machine perfusion: A porcine renal autotransplantation study.

Normothermic machine perfusion (NMP) of injured kidneys offers the opportunity for interventions to metabolically active organs prior to transplantation. Mesenchymal stromal cells (MSCs) can exert regenerative and anti-inflammatory effects in ischemia-reperfusion injury. The aims of this study were to evaluate the safety and feasibility of MSC treatment of kidneys during NMP using a porcine autotransplantation model, and examine potential MSC treatment-associated kidney improvements up to 14 days posttransplant. After 75 min of kidney warm ischemia, four experimental groups of n = 7 underwent 14 h of oxygenated hypothermic machine perfusion. In three groups this was followed by 240 min of NMP with infusion of vehicle, 10 million porcine, or 10 million human adipose-derived MSCs. All kidneys were autotransplanted after contralateral nephrectomy. MSC treatment did not affect perfusion hemodynamics during NMP or cause adverse effects at reperfusion, with 100% animal survival. MSCs did not affect plasma creatinine, glomerular filtration rate, neutrophil gelatinase-associated lipocalin concentrations or kidney damage assessed by histology during the 14 days, and MSCs retention was demonstrated in renal cortex. Infusing MSCs during ex vivo NMP of porcine kidneys was safe and feasible. Within the short posttransplant follow-up period, no beneficial effects of ex vivo MSC therapy could be demonstrated.

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.

Organ-specific metabolic profiles of the liver and kidney during brain death and afterwards during normothermic machine perfusion of the kidney.

We investigated metabolic changes during brain death (BD) using hyperpolarized magnetic resonance (MR) spectroscopy and ex vivo graft glucose metabolism during normothermic isolated perfused kidney (IPK) machine perfusion. BD was induced in mechanically ventilated rats by inflation of an epidurally placed catheter; sham-operated rats served as controls. Hyperpolarized [1-13 C]pyruvate MR spectroscopy was performed to quantify pyruvate metabolism in the liver and kidneys at 3 time points during BD, preceded by injecting hyperpolarized[1-13 C]pyruvate. Following BD, glucose oxidation was measured using tritium-labeled glucose (d-6-3H-glucose) during IPK reperfusion. Quantitative polymerase chain reaction and biochemistry were performed on tissue/plasma. Immediately following BD induction, lactate increased in both organs (liver: eµd 0.21, 95% confidence interval [CI] [-0.27, -0.15]; kidney: eµd 0.26, 95% CI [-0.40, -0.12]. After 4 hours of BD, alanine production decreased in the kidney (eµd 0.14, 95% CI [0.03, 0.25], P < .05). Hepatic lactate and alanine profiles were significantly different throughout the experiment between groups (P < .01). During IPK perfusion, renal glucose oxidation was reduced following BD vs sham animals (eµd 0.012, 95% CI [0.004, 0.03], P < .001). No differences in enzyme activities were found. Renal gene expression of lactate-transporter MCT4 increased following BD (P < .01). In conclusion, metabolic processes during BD can be visualized in vivo using hyperpolarized magnetic resonance imaging and with glucose oxidation during ex vivo renal machine perfusion. These techniques can detect differences in the metabolic profiles of the liver and kidney following BD.

Subclinical effects of remote ischaemic conditioning in human kidney transplants revealed by quantitative proteomics.

BACKGROUND: Remote ischaemic conditioning (RIC) is currently being explored as a non-invasive method to attenuate ischaemia/reperfusion injuries in organs. A randomised clinical study (CONTEXT) evaluated the effects of RIC compared to non-RIC controls in human kidney transplants. METHODS: RIC was induced prior to kidney reperfusion by episodes of obstruction to arterial flow in the leg opposite the transplant using a tourniquet (4 × 5 min). Although RIC did not lead to clinical improvement of transplant outcomes, we explored whether RIC induced molecular changes through precision analysis of CONTEXT recipient plasma and kidney tissue samples by high-resolution tandem mass spectrometry (MS/MS). RESULTS: We observed an accumulation of muscle derived proteins and altered amino acid metabolism in kidney tissue proteomes, likely provoked by RIC, which was not reflected in plasma. In addition, MS/MS analysis demonstrated transient upregulation of several acute phase response proteins (SAA1, SAA2, CRP) in plasma, 1 and 5 days post-transplant in RIC and non-RIC conditions with a variable effect on the magnitude of acute inflammation. CONCLUSIONS: Together, our results indicate sub-clinical systemic and organ-localised effects of RIC.

Goal-Directed Fluid Therapy Does Not Improve Early Glomerular Filtration Rate in a Porcine Renal Transplantation Model.

BACKGROUND: Insufficient fluid administration intra- and postoperatively may lead to delayed renal graft function (DGF), while fluid overload increases the risk of heart failure, infection, and obstipation. Several different fluid protocols have been suggested to ensure optimal fluid state. However, there is a lack of evidence of the clinical impact of these regimens. This study aimed to determine whether individualized goal-directed fluid therapy (IGDT) positively affects the initial renal function compared to a high-volume fluid therapy (HVFT) and to examine the effects on renal endothelial glycocalyx, inflammatory and oxidative stress markers, and medullary tissue oxygenation. The hypothesis was that IGDT improves early glomerular filtration rate (GFR) in pigs subjected to renal transplantation. METHODS: This was an experimental randomized study. Using a porcine renal transplantation model, animals were randomly assigned to receive IGDT or HVFT during and until 1 hour after transplantation from brain-dead donors. The kidneys were exposed to 18 hours of cold ischemia. The recipients were observed until 10 hours after reperfusion, which included GFR measured as clearance of chrom-51-ethylendiamintetraacetat (Cr-EDTA), animal weight, and renal tissue oxygenation by fiber optic probes. The renal expression of inflammatory and oxidative stress markers as well as glomerular endothelial glycocalyx were analyzed in the graft using polymerase chain reaction (PCR) technique and immunofluorescence. RESULTS: Twenty-eight recipient pigs were included for analysis. We found no evidence that IGDT improved early GFR compared to HVFT (P = .45), while animal weight increased more in the HVFT group (a mean difference of 3.4 kg [1.96-4.90]; P < .0001). A better, however nonsignificant, preservation of glomerular glycocalyx (P = .098) and significantly lower levels of the inflammatory marker cyclooxygenase 2 (COX-2) was observed in the IGDT group when compared to HVFT. COX-2 was 1.94 (1.50-2.39; P = .012) times greater in the HVFT group when compared to the IGDT group. No differences were observed in outer medullary tissue oxygenation or oxidative stress markers. CONCLUSIONS: IGDT did not improve early GFR; however, it may reduce tissue inflammation and could possibly lead to preservation of the glycocalyx compared to HVFT.

Hyperpolarised 13C-MRI metabolic and functional imaging: an emerging renal MR diagnostic modality.

Magnetic resonance imaging (MRI) is a well-established modality for assessing renal morphology and function, as well as changes that occur during disease. However, the significant metabolic changes associated with renal disease are more challenging to assess with MRI. Hyperpolarized carbon-13 MRI is an emerging technique which provides an opportunity to probe metabolic alterations at high sensitivity by providing an increase in the signal-to-noise ratio of 20,000-fold or more. This review will highlight the current status of hyperpolarised 13C-MRI and its translation into the clinic and how it compares to metabolic measurements provided by competing technologies such as positron emission tomography (PET).

High-sensitivity Troponin T in hemodialysis patients: a randomized placebo-controlled sub-study investigating angiotensin-II-blockade, variation over time and associations with clinical outcome.

BACKGROUND: Troponin T (TnT) is a well-known risk factor for negative outcome in hemodialysis (HD) patients, but little is known about variation over time, and the impact of clinical and dialysis specific factors. This study investigated the effect of angiotensin II receptor blockade (ARB), short and long-term variation in TnT and associations with clinical parameters. METHODS: In this analysis based on the SAFIR-cohort (Clinical Trials ID: NCT00791830) 81 HD patients were randomized double-blind for placebo (n = 40) or angiotensin II receptor blocker (ARB) treatment (n = 41) with irbesartan (150-300 mg) and followed for 12 months with six serial measurements of TnT using a high-sensitivity assay. RESULTS: Fifty-four patients (67%) completed follow-up. Baseline TnT-medians (min-max) were (placebo/ARB): 45(14-295)/46(10-343) ng/L. ARB-treatment did not significantly affect mean TnT-levels over the 12-month study period. Median week-to-week and one-year TnT-variation (5th-95th-percentile range) using all samples regardless of intervention were: 0(- 14-10) ng/L (week-to-week) and 3(- 40-71) ng/L (12 months). Median TnT-amplitude, capturing the change from the lowest to the highest TnT-value observed during the one-year study period was 38% or 20.5 ng/L. Median ratios with 95% limits of agreement were: 1.00(0.73-1.37); P = 0.92 (1 week/baseline; n = 77) and 1.07(0.52-2.25); P = 0.19 (12 months/baseline; n = 54). Baseline TnT was positively correlated with diabetes, ultrafiltration volume, arterial stiffness, change in intradialytic total peripheral resistance and N-terminal pro b-type natriuretic peptide (NT-proBNP) and negatively correlated with hematocrit, residual renal function and change in intradialytic cardiac output. High baseline TnT was associated with a higher risk of admission and cardiovascular (CV) events during follow-up. Increase in TnT over time (ΔTnT = 12-months-baseline) was significantly associated with increase in left ventricular (LV) mass and NT-proBNP and decrease in LV ejection fraction and late intradialytic stroke volume. ΔTnT was not significantly associated with admissions, CV or intradialytic hypotensive events during follow-up. Admissions were significantly more likely with a high (TnT-amplitude> 20.5 ng/L) than a low TnT-amplitude. Peaks in TnT were less frequent in aspirin-treated patients. CONCLUSION: ARB-treatment had no significant effect on TnT-levels. Week-to-week variation was generally low, yet over 12 months individual patients had considerable TnT fluctuations. Rise in TnT over time was significantly correlated with markers of cardiac deterioration. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT00791830 . Date of registration: November 17, 2008. EudraCT no: 2008-001267-11.

Early Immunological Effects of Ischemia-Reperfusion Injury: No Modulation by Ischemic Preconditioning in a Randomised Crossover Trial in Healthy Humans.

Ischemic preconditioning (IPC) has been protective against ischemia-reperfusion injury (IRI), but the underlying mechanism is poorly understood. We examined whether IPC modulates the early inflammatory response after IRI. Nineteen healthy males participated in a randomised crossover trial with and without IPC before IRI. IPC and IRI were performed by cuff inflation on the forearm. IPC consisted of four cycles of five minutes followed by five minutes of reperfusion. IRI consisted of twenty minutes followed by 15 min of reperfusion. Blood was collected at baseline, 0 min, 85 min and 24 h after IRI. Circulating monocytes, T-cells subsets and dendritic cells together with intracellular activation markers were quantified by flow cytometry. Luminex measured a panel of inflammation-related cytokines in plasma. IRI resulted in dynamic regulations of the measured immune cells and their intracellular activation markers, however IPC did not significantly alter these patterns. Neither IRI nor the IPC protocol significantly affected the levels of inflammatory-related cytokines. In healthy volunteers, it was not possible to detect an effect of the investigated IPC-protocol on early IRI-induced inflammatory responses. This study indicates that protective effects of IPC on IRI is not explained by direct modulation of early inflammatory events.

Infusing Mesenchymal Stromal Cells into Porcine Kidneys during Normothermic Machine Perfusion: Intact MSCs Can Be Traced and Localised to Glomeruli.

Normothermic machine perfusion (NMP) of kidneys offers the opportunity to perform active interventions, such as the addition of mesenchymal stromal cells (MSCs), to an isolated organ prior to transplantation. The purpose of this study was to determine whether administering MSCs to kidneys during NMP is feasible, what the effect of NMP is on MSCs and whether intact MSCs are retained in the kidney and to which structures they home. Viable porcine kidneys were obtained from a slaughterhouse. Kidneys were machine perfused during 7 h at 37 °C. After 1 h of perfusion either 0, 105, 106 or 107 human adipose tissue derived MSCs were added. Additional ex vivo perfusions were conducted with fluorescent pre-labelled bone-marrow derived MSCs to assess localisation and survival of MSCs during NMP. After NMP, intact MSCs were detected by immunohistochemistry in the lumen of glomerular capillaries, but only in the 107 MSC group. The experiments with fluorescent pre-labelled MSCs showed that only a minority of glomeruli were positive for infused MSCs and most of these glomeruli contained multiple MSCs. Flow cytometry showed that the number of infused MSCs in the perfusion circuit steeply declined during NMP to approximately 10%. In conclusion, the number of circulating MSCs in the perfusate decreases rapidly in time and after NMP only a small portion of the MSCs are intact and these appear to be clustered in a minority of glomeruli.

Identification of differential gene expression patterns in human arteries from patients with chronic kidney disease.

Uremia accelerates atherosclerosis, but little is known about affected pathways in human vasculature. This study aimed to identify differentially expressed arterial transcripts in patients with chronic kidney disease (CKD). Global mRNA expression was estimated by microarray hybridization in iliac arteries ( n = 14) from renal transplant recipients and compared with renal arteries from healthy living kidney donors ( n = 19) in study 1. Study 2 compared nonatherosclerotic internal mammary arteries (IMA) from five patients with elevated plasma creatinine levels and age- and sex-matched controls with normal creatinine levels. Western blotting and immunohistochemistry for selected proteins were performed on a subset of study 1 samples. Fifteen gene transcripts were significantly different between the two groups in study 1 [fold changes (FC) > 1.05 and false discovery rates (FDR) < 0.005]. Most upregulated mRNAs associated with cellular signaling, apoptosis, TNFα/NF-κB signaling, smooth muscle contraction, and 10 other pathways were significantly affected. To focus attention on genes from genuine vascular cells, which dominate in IMA, concordant deregulated genes in studies 1 and 2 were examined and included 23 downregulated and eight upregulated transcripts (settings in study 1: FC > 1.05 and FDR < 0.05; study 2: FC > 1.2 and P < 0.2). Selected deregulated gene products were investigated at the protein level, and whereas HIF3α confirmed mRNA upregulation, vimentin showed upregulation in contrast to the mRNA results. We conclude that arteries from CKD patients display change in relatively few sets of genes. Many were related to differentiated vascular smooth muscle cell phenotype. These identified genes may contribute to understanding the development of arterial injury among patients with CKD.