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.

Impact of device variability and protocol differences on kidney function during normothermic machine perfusion: A comparative study using porcine and human kidneys.

INTRODUCTION: A growing interest in renal normothermic machine perfusion (NMP) has resulted in more clinically available perfusion devices. While all perfusion systems have the same aim, there are significant differences in their circuits, pumps, sensors, and software. Therefore, our objective was to assess the impact of different perfusion protocols and devices on kidney function and perfusion parameters during NMP. METHODS: Porcine kidneys were subjected to 30 min of warm ischemia, 24 h of static cold storage, and subsequently perfused for 6 h using (1) the Kidney Assist (KA) machine with a pressure of 75 mm Hg, (2) the KA device incorporating several adjustments and a pressure of 85 mm Hg (modified KA), or (3) the Perlife (PL) perfusion device (n = 4). Consecutively, discarded human kidneys were perfused using the KA or modified KA (n = 3) protocol. RESULTS: The PL group quickly reached the device's upper flow limit and consequently received a significantly lower pressure compared to the KA groups. The arterial pO2 was significantly lower in the PL group. Yet, hemoglobin concentration increased over time, and oxygen consumption was significantly higher compared to the KA groups. Fractional sodium excretion was significantly lower in the PL group. Tissue ATP levels, urine production, and creatinine clearance rates did not differ between groups. In human kidneys, the modified KA group showed significantly lower vascular resistance, higher oxygen delivery, and lower levels of lactate in the perfusate compared to the KA group. CONCLUSIONS: This study shows that perfusion characteristics and kidney function are significantly influenced by the perfusion protocol and the device and its settings during normothermic machine perfusion and therefore should be interpreted with caution.

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.

Metformin Preconditioning and Postconditioning to Reduce Ischemia Reperfusion Injury in an Isolated Ex Vivo Rat and Porcine Kidney Normothermic Machine Perfusion Model.

Metformin may act renoprotective prior to kidney transplantation by reducing ischemia-reperfusion injury (IRI). This study examined whether metformin preconditioning and postconditioning during ex vivo normothermic machine perfusion (NMP) of rat and porcine kidneys affect IRI. In the rat study, saline or 300 mg/kg metformin was administered orally twice on the day before nephrectomy. After 15 minutes of warm ischemia, kidneys were preserved with static cold storage for 24 hours. Thereafter, 90 minutes of NMP was performed with the addition of saline or metformin (30 or 300 mg/L). In the porcine study, after 30 minutes of warm ischemia, kidneys were preserved for 3 hours with oxygenated hypothermic machine perfusion. Subsequently, increasing doses of metformin were added during 4 hours of NMP. Metformin preconditioning of rat kidneys led to decreased injury perfusate biomarkers and reduced proteinuria. Postconditioning of rat kidneys resulted, dose-dependently, in less tubular cell necrosis and vacuolation. Heat shock protein 70 expression was increased in metformin-treated porcine kidneys. In all studies, creatinine clearance was not affected. In conclusion, both metformin preconditioning and postconditioning can be done safely and improved rat and porcine kidney quality. Because the effects are minor, it is unknown which strategy might result in improved organ quality after transplantation.

Increasing metformin concentrations and its excretion in both rat and porcine ex vivo normothermic kidney perfusion model.

INTRODUCTION: Metformin can accumulate and cause lactic acidosis in patients with renal insufficiency. Metformin is known to inhibit mitochondria, while renal secretion of the drug by proximal tubules indirectly requires energy. We investigated whether addition of metformin before or during ex vivo isolated normothermic machine perfusion (NMP) of porcine and rat kidneys affects its elimination. RESEARCH DESIGN AND METHODS: First, Lewis rats were pretreated with metformin or saline the day before nephrectomy. Subsequently, NMP of the kidney was performed for 90 min. Metformin was added to the perfusion fluid in one of three different concentrations (none, 30 mg/L or 300 mg/L). Second, metformin was added in increasing doses to the perfusion fluid during 4 hours of NMP of porcine kidneys. Metformin concentration was determined in the perfusion fluid and urine by liquid chromatography-tandem mass spectrometry. RESULTS: Metformin clearance was approximately 4-5 times higher than creatinine clearance in both models, underscoring secretion of the drug. Metformin clearance at the end of NMP in rat kidneys perfused with 30 mg/L was lower than in metformin pretreated rats without the addition of metformin during perfusion (both p≤0.05), but kidneys perfused with 300 mg/L trended toward lower metformin clearance (p=0.06). Creatinine clearance was not different between treatment groups. During NMP of porcine kidneys, metformin clearance peaked at 90 min of NMP (18.2±13.7 mL/min/100 g). Thereafter, metformin clearance declined, while creatinine clearance remained stable. This observation can be explained by saturation of metformin transporters with a Michaelis-Menten constant (95% CI) of 23.0 (10.0 to 52.3) mg/L. CONCLUSIONS: Metformin was secreted during NMP of both rat and porcine kidneys. Excretion of metformin decreased under increasing concentrations of metformin, which might be explained by saturation of metformin transporters rather than a self-inhibitory effect. It remains unknown whether a self-inhibitory effect contributes to metformin accumulation in humans with longer exposure times.

Intraoperative Fluid Restriction is Associated with Functional Delayed Graft Function in Living Donor Kidney Transplantation: A Retrospective Cohort Analysis.

BACKGROUND: In 2016 we observed a marked increase in functional delayed graft function (fDGF) in our living donor kidney transplantation (LDKT) recipients from 8.5% in 2014 and 8.8% in 2015 to 23.0% in 2016. This increase coincided with the introduction of a goal-directed fluid therapy (GDFT) protocol in our kidney transplant recipients. Hereupon, we changed our intraoperative fluid regimen to a fixed amount of 50 mL/kg body weight (BW) and questioned whether the intraoperative fluid regimen was related to this increase in fDGF. METHODS: a retrospective cohort analysis of all donors and recipients in our LDKT program between January 2014-February 2017 (n = 275 pairs). RESULTS: Univariate analysis detected various risk factors for fDGF. Dialysis dependent recipients were more likely to develop fDGF compared to pre-emptively transplanted patients (p < 0.001). Recipients developing fDGF received less intraoperative fluid (36 (25.9-50.0) mL/kg BW vs. 47 (37.3-55.6) mL/kg BW (p = 0.007)). The GDFT protocol resulted in a reduction of intraoperative fluid administration on average by 850 mL in total volume and 21% in mL/kg BW compared to our old protocol (p < 0.001). In the unadjusted analysis, a higher intraoperative fluid volume in mL/kg BW was associated with a lower risk for the developing fDGF (OR 0.967, CI (0.941-0.993)). After adjustment for the confounders, prior dialysis and the use of intraoperative noradrenaline, the relationship of fDGF with fluid volume was still apparent (OR 0.970, CI (0.943-0.998)). CONCLUSION: Implementation of a GDFT protocol led to reduced intraoperative fluid administration in the LDKT recipients. This intraoperative fluid restriction was associated with the development of fDGF.