The association between hemoglobin levels and renal function parameters during normothermic machine perfusion: A retrospective cohort study using porcine kidneys.

BACKGROUND: Ex vivo normothermic machine perfusion (NMP) is a promising tool for assessing an isolated kidney prior to transplantation. However, there is no consensus on the perfusate's optimal oxygen-carrying capacity to support renal function. To investigate the association of hemoglobin levels with renal function parameters, a retrospective analysis of isolated, normothermically, perfused porcine kidneys was performed. METHODS: Between 2015 and 2021, a total of 228 kidneys underwent 4 h of NMP with perfusates that varied in hemoglobin levels. A generalized linear model was used to determine the association of hemoglobin levels with time-weighted means of renal function markers, such as fractional sodium excretion (FENa) and creatinine clearance (CrCl). Stratified by baseline hemoglobin level (<4.5, 4.5-6, or >6 mmol/L), these markers were modeled over time using a generalized linear mixed-effects model. All models were adjusted for potential confounders. RESULTS: Until a hemoglobin level of around 5 mmol/L was reached, increasing hemoglobin levels were associated with superior FENa and CrCl. Thereafter, this association plateaued. When hemoglobin levels were categorized, hemoglobin <4.5 mmol/L was associated with worse renal function. Hemoglobin levels were neither significantly associated with proteinuria during NMP nor with ATP levels at the end of NMP. Hemoglobin levels >6 mmol/L showed no additional benefits in renal function. CONCLUSION: In conclusion, we found an association between baseline hemoglobin levels and superior renal function parameters, but not injury, during NMP of porcine kidneys. Furthermore, we show that performing a retrospective cohort study of preclinical data is feasible and able to answer additional questions, reducing the potential use of laboratory animals.

Nitric oxide and long-term outcomes after kidney transplantation: Results of the TransplantLines cohort study.

Impaired endogenous nitric oxide (NO) production may contribute to graft failure and premature mortality in kidney transplant recipients (KTR). We investigated potential associations of 24-h urinary NOx (NO3- + NO2-) excretion (uNOx) with long-term outcomes. uNOx was determined by HPLC and GC-MS in 698 KTR and in 132 kidney donors before and after donation. Additionally, we measured urinary nitroso species (RXNO) by gas-phase chemiluminescence. Median uNOx was lower in KTR compared to kidney donors (688 [393-1076] vs. 1301 [868-1863] before donation and 1312 [982-1853] µmol/24 h after donation, P < 0.001). During median follow-up of 5.4 [4.8-6.1] years, 150 KTR died (61 due to cardiovascular disease) and 83 experienced graft failure. uNOx was inversely associated with all-cause mortality (HR per doubling of uNOx: 0.84 [95% CI 0.75-0.93], P < 0.001) and cardiovascular mortality (HR 0.78 [95% CI 0.67-0.92], P = 0.002). The association of uNOx with graft failure was lost when adjusted for renal function (HR per doubling of uNOx: 0.89 [95% CI 0.76-1.05], P = 0.17). There were no significant associations of urinary RXNO with outcomes. Our study suggests that KTR have lower NO production than healthy subjects and that lower uNOx is associated with a higher risk of all-cause and cardiovascular mortality.

Prolonged Organ Extraction Time Negatively Impacts Kidney Transplantation Outcome.

Main Problem: Following cold aortic flush in a deceased organ donation procedure, kidneys never reach the intended 0-4°C and stay ischemic at around 20°C in the donor's body until actual surgical retrieval. Therefore, organ extraction time could have a detrimental influence on kidney transplant outcome. Materials and Methods: We analyzed the association between extraction time and kidney transplant outcome in multicenter data of 5,426 transplant procedures from the Dutch Organ Transplantation Registry (NOTR) and 15,849 transplant procedures from the United Network for Organ Sharing (UNOS). Results: Extraction time was grouped per 10-min increment. In the NOTR database, extraction time was independently associated with graft loss [HR 1.027 (1.004-1.050); p = 0.022] and with DGF [OR 1.043 (1.021-1.066); p < 0.005]. An extraction time >80 min was associated with a 27.4% higher hazard rate of graft failure [HR 1.274 (1.080-1.502); p = 0.004] and such kidneys had 43.8% higher odds of developing DGF [OR 1.438, (1.236-1.673); p < 0.005]. In the UNOS database, increasing extraction times in DCD donors were associated with DGF [OR 1.036 (1.016-1.055); p < 0.005]. An extraction time >30 min was associated with 14.5% higher odds of developing DGF [OR 1.145 (1.063-1.233); p < 0.005]. Discussion: Prolonged kidney extraction time negatively influenced graft survival in Dutch donors and increased DGF risk in all deceased donor recipients.

Renal temperature reduction progressively favors mitochondrial ROS production over respiration in hypothermic kidney preservation.

BACKGROUND: Hypothermia, leading to mitochondrial inhibition, is widely used to reduce ischemic injury during kidney preservation. However, the exact effect of hypothermic kidney preservation on mitochondrial function remains unclear. METHODS: We evaluated mitochondrial function [i.e. oxygen consumption and production of reactive oxygen species (ROS)] in different models (porcine kidney perfusion, isolated kidney mitochondria, and HEK293 cells) at temperatures ranging 7-37 °C. RESULTS: Lowering temperature in perfused kidneys and isolated mitochondria resulted in a rapid decrease in oxygen consumption (65% at 27 °C versus 20% at 7 °C compared to normothermic). Decreased oxygen consumption at lower temperatures was accompanied by a reduction in mitochondrial ROS production, albeit markedly less pronounced and amounting only 50% of normothermic values at 7 °C. Consequently, malondialdehyde (a marker of ROS-induced lipid peroxidation) accumulated in cold stored kidneys. Similarly, low temperature incubation of kidney cells increased lipid peroxidation, which is due to a loss of ROS scavenging in the cold. CONCLUSIONS: Lowering of temperature highly affects mitochondrial function, resulting in a progressive discrepancy between the lowering of mitochondrial respiration and their production of ROS, explaining the deleterious effects of hypothermia in transplantation procedures. These results highlight the necessity to develop novel strategies to decrease the formation of ROS during hypothermic organ preservation.

H2S-Enriched Flush out Does Not Increase Donor Organ Quality in a Porcine Kidney Perfusion Model.

Kidney extraction time has a detrimental effect on post-transplantation outcome. This study aims to improve the flush-out and potentially decrease ischemic injury by the addition of hydrogen sulphide (H2S) to the flush medium. Porcine kidneys (n = 22) were extracted during organ recovery surgery. Pigs underwent brain death induction or a Sham operation, resulting in four groups: donation after brain death (DBD) control, DBD H2S, non-DBD control, and non-DBD H2S. Directly after the abdominal flush, kidneys were extracted and flushed with or without H2S and stored for 13 h via static cold storage (SCS) +/- H2S before reperfusion on normothermic machine perfusion. Pro-inflammatory cytokines IL-1b and IL-8 were significantly lower in H2S treated DBD kidneys during NMP (p = 0.03). The non-DBD kidneys show superiority in renal function (creatinine clearance and FENa) compared to the DBD control group (p = 0.03 and p = 0.004). No differences were seen in perfusion parameters, injury markers and histological appearance. We found an overall trend of better renal function in the non-DBD kidneys compared to the DBD kidneys. The addition of H2S during the flush out and SCS resulted in a reduction in pro-inflammatory cytokines without affecting renal function or injury markers.

Loss of Endothelial Glycocalyx During Normothermic Machine Perfusion of Porcine Kidneys Irrespective of Pressure and Hematocrit.

Normothermic machine perfusion (NMP) is a promising modality for marginal donor kidneys. However, little is known about the effects of NMP on causing endothelial glycocalyx (eGC) injury. This study aims to evaluate the effects of NMP on eGC injury in marginal donor kidneys and whether this is affected by perfusion pressures and hematocrits. Methods: Porcine slaughterhouse kidneys (n = 6/group) underwent 35 min of warm ischemia. Thereafter, the kidneys were preserved with oxygenated hypothermic machine perfusion for 3 h. Subsequently, 4 h of NMP was applied using pressure-controlled perfusion with an autologous blood-based solution containing either 12%, 24%, or 36% hematocrit. Pressures of 55, 75, and 95 mm Hg were applied in the 24% group. Perfusate, urine, and biopsy samples were collected to determine both injury and functional parameters. Results: During NMP, hyaluronan levels in the perfusate increased significantly (P < 0.0001). In addition, the positivity of glyco-stained glycocalyx decreased significantly over time, both in the glomeruli (P = 0.024) and peritubular capillaries (P = 0.003). The number of endothelial cells did not change during NMP (P = 0.157), whereas glomerular endothelial expression of vascular endothelial growth factor receptor-2 decreased significantly (P < 0.001). Microthrombi formation was significantly increased after NMP. The use of different pressures and hematocrits did not affect functional parameters during perfusion. Conclusions: NMP is accompanied with eGC and vascular endothelial growth factor receptor-2 loss, without significant loss of endothelial cells. eGC loss was not affected by the different pressures and hematocrits used. It remains unclear whether endothelial injury during NMP has harmful consequences for the transplanted kidney.

Real-time, multi-spectral motion artefact correction and compensation for laser speckle contrast imaging.

Laser speckle contrast imaging (LSCI) is so sensitive to motion that it can measure the movement of red blood cells. However, this extreme sensitivity to motion is also its pitfall as the clinical translation of LSCI is slowed down due to the inability to deal with motion artefacts. In this paper we study the effectiveness of a real-time, multi-spectral motion artefact correction and compensation by subduing an in vitro flow phantom and ex vivo porcine kidney to computer-controlled motion artefacts. On the in vitro flow phantom, the optical flow showed a good correlation with the total movement. This model results in a better signal-to-noise ratios for multiple imaging distances and the overestimation of perfusion was reduced. In the ex vivo kidney model, the perfusion overestimation was also reduced and we were still able to distinguish between ischemia and non-ischemia in the stabilized data whereas this was not possible in the non-stabilized data. This leads to a notably better perfusion estimation that could open the door to a multitude of new clinical applications for LSCI.

Real-time visualization of renal microperfusion using laser speckle contrast imaging.

SIGNIFICANCE: Intraoperative parameters of renal cortical microperfusion (RCM) have been associated with postoperative ischemia/reperfusion injury. Laser speckle contrast imaging (LSCI) could provide valuable information in this regard with the advantage over the current standard of care of being a non-contact and full-field imaging technique. AIM: Our study aims to validate the use of LSCI for the visualization of RCM on ex vivo perfused human-sized porcine kidneys in various models of hemodynamic changes. APPROACH: A comparison was made between three renal perfusion measures: LSCI, the total arterial renal blood flow (RBF), and sidestream dark-field (SDF) imaging in different settings of ischemia/reperfusion. RESULTS: LSCI showed a good correlation with RBF for the reperfusion experiment (0.94 ± 0.02; p < 0.0001) and short- and long-lasting local ischemia (0.90 ± 0.03; p < 0.0001 and 0.81 ± 0.08; p < 0.0001, respectively). The correlation decreased for low flow situations due to RBF redistribution. The correlation between LSCI and SDF (0.81 ± 0.10; p < 0.0001) showed superiority over RBF (0.54 ± 0.22; p < 0.0001). CONCLUSIONS: LSCI is capable of imaging RCM with high spatial and temporal resolutions. It can instantaneously detect local perfusion deficits, which is not possible with the current standard of care. Further development of LSCI in transplant surgery could help with clinical decision making.

Gasotransmitters in health and disease: a mitochondria-centered view.

Gasotransmitters fulfill important roles in cellular homeostasis having been linked to various pathologies, including inflammation and cardiovascular diseases. In addition to the known pathways mediating the actions of gasotransmitters, their effects in regulating mitochondrial function are emerging. Given that mitochondria are key organelles in energy production, formation of reactive oxygen species and apoptosis, they are important mediators in preserving health and disease. Preserving or restoring mitochondrial function by gasotransmitters may be beneficial, and mitigate pathogenetic processes. In this review we discuss the actions of gasotransmitters with focus on their role in mitochondrial function and their therapeutic potential.

Hydrogen sulphide-induced hypometabolism in human-sized porcine kidneys.

BACKGROUND: Since the start of organ transplantation, hypothermia-forced hypometabolism has been the cornerstone in organ preservation. Cold preservation showed to protect against ischemia, although post-transplant injury still occurs and further improvement in preservation techniques is needed. We hypothesize that hydrogen sulphide can be used as such a new preservation method, by inducing a reversible hypometabolic state in human sized kidneys during normothermic machine perfusion. METHODS: Porcine kidneys were connected to an ex-vivo isolated, oxygen supplemented, normothermic blood perfusion set-up. Experimental kidneys (n = 5) received a 85mg NaHS infusion of 100 ppm and were compared to controls (n = 5). As a reflection of the cellular metabolism, oxygen consumption, mitochondrial activity and tissue ATP levels were measured. Kidney function was assessed by creatinine clearance and fractional excretion of sodium. To rule out potential structural and functional deterioration, kidneys were studied for biochemical markers and histology. RESULTS: Hydrogen sulphide strongly decreased oxygen consumption by 61%, which was associated with a marked decrease in mitochondrial activity/function, without directly affecting ATP levels. Renal biological markers, renal function and histology did not change after hydrogen sulphide treatment. CONCLUSION: In conclusion, we showed that hydrogen sulphide can induce a controllable hypometabolic state in a human sized organ, without damaging the organ itself and could thereby be a promising therapeutic alternative for cold preservation under normothermic conditions in renal transplantation.