Perfusate Biomarker Comparison During Renal Hypothermic and Normothermic Machine Perfusion: Do These Techniques Provide Similar Insights?

Abstract

Background: Hypothermic machine perfusion (HMP) and normothermic machine perfusion (NMP) are increasingly used in renal transplantation. Both techniques enable pretransplant organ viability assessment through biomarker measurements in the perfusion solution. This study examines similarities and differences in biomarker release during HMP and NMP, focusing on well-established biomarkers alongside functional markers in porcine and discarded human donor kidneys.

Methods: Discarded human donor kidneys (n = 25) underwent 4 h of oxygenated hypothermic machine perfusion (HMPO 2 ) and subsequently 4 h of NMP. Porcine kidneys were exposed to either minimal warm ischemia or 75 min of warm ischemia (n = 30 per group). Hereafter, kidneys were placed on HMPO 2 for 6 h followed by 6 h of NMP. Flow dynamics were recorded, and the biomarkers aspartate aminotransferase (ASAT), lactate dehydrogenase (LDH), N -acetyl-β-glucosaminidase, tissue inhibitor of metalloproteinases-2 (TIMP-2), and heart-type fatty acid-binding protein were measured longitudinally in the perfusates.

Results: For human kidneys, we found moderate to strong correlations between ASAT, LDH, TIMP-2, and heart-type fatty acid-binding protein content measured during HMPO 2 and the same biomarkers during NMP. In porcine kidneys, clear distinctions between ischemically damaged and healthy kidneys were observed in flow dynamics and content of ASAT, LDH, and TIMP-2 during both HMPO 2 and NMP.

Conclusions: Our findings suggest that biomarker release during HMPO 2 and NMP have similarities, indicating that some biomarkers might already be assessed during HMPO 2 . However, the predictive value of biomarkers in both techniques remains elusive. Additionally, NMP could provide important benefits over HMPO 2 , including functional assessment and reconditioning.

Graphical abstract

FIGURE 1.

Study design. Human deceased-donor kidneys, deemed unsuitable for transplantation, were preserved with either SCS or with HMP on arrival at our laboratory (A). Kidneys were placed on HMPO₂ and perfused for 4 h. Subsequently, kidneys were connected to an NMP device and perfused for another 6 h. To ensure a fair comparison, only the first 4 h of NMP were used in this study, aligning it with the total HMPO₂ duration. Porcine kidneys were randomized to sustain a MWI, or 75 min of WI (B). After bilateral nephrectomy, both kidneys were flushed with cold preservation solution. Subsequently, kidneys were connected to an HMPO₂ device and perfused for 6 h. Hereafter, kidneys were connected to an NMP circuit and perfused for an additional 6 h. HMP, hypothermic machine perfusion; HMPO₂, oxygenated HMP; MWI, minimal period of warm ischemia; NMP, normothermic machine perfusion; SCS, static cold storage.

FIGURE 2.

Hemodynamic alterations throughout ex vivo perfusion of discarded human kidneys. Considerable interindividual variability in perfusate flow and renal vascular resistance during both HMPO₂ (A and B) and NMP (C and D). Each line represents an individual kidney throughout the perfusion period (n = 25). HMPO₂, oxygenated hypothermic machine perfusion; NMP, normothermic machine perfusion.

FIGURE 3.

Biomarkers during ex vivo perfusion of discarded human kidneys. Absolute ASAT content during HMPO₂ (A) and NMP (B). Absolute LDH content during HMPO₂ (C) and NMP (D). Absolute H-FABP content during HMPO₂ (E) and NMP (F). Absolute NAG content during HMPO₂ (G) and NMP (H). Absolute TIMP-2 content during HMPO₂ (I) and NMP (J). Each line represents an individual kidney throughout the perfusion period (n = 25). ASAT, aspartate aminotransferase; H-FABP, heart-type fatty acid binding protein; HMPO₂, oxygenated hypothermic machine perfusion; LDH, lactate dehydrogenase; NAG, N-acetyl-β-glucosaminidase; NMP, normothermic machine perfusion; TIMP-2, tissue inhibitor of metalloproteinases-2.

FIGURE 4.

Spearman’s correlation between measured biomarkers during HMPO₂ and NMP of discarded human kidneys. ASAT (A), LDH (B), H-FABP (C), NAG (D), and TIMP-2 (E) correlations at 15, 120, and 240 min of perfusion. ASAT, aspartate aminotransferase; H-FABP, heart-type fatty acid binding protein; HMPO₂, oxygenated hypothermic machine perfusion; LDH, lactate dehydrogenase; NAG, N-acetyl-β-glucosaminidase; NMP, normothermic machine perfusion; r, Spearman correlation coefficient; TIMP-2, tissue inhibitor of metalloproteinases-2.

FIGURE 5.

Differences in flow dynamics and biomarkers between human kidneys with a moderate and high KDPI. Arterial flow during HMPO₂ (A) and NMP (B). Absolute ASAT content during HMPO₂ (C) and NMP (D). Absolute LDH content during HMPO₂ (E) and NMP (F). Absolute H-FABP content during HMPO₂ (G) and NMP (H). Absolute NAG content during HMPO₂ (I) and NMP (J). Absolute TIMP-2 content during HMPO₂ (K) and NMP (L). Absolute NAG content during HMPO₂ (I) and NMP (J). Median and interquartile range are shown. ASAT, aspartate aminotransferase; HMPO₂, oxygenated hypothermic machine perfusion; KDPI, kidney donor profile index; LDH, lactate dehydrogenase; H-FABP, heart-type fatty acid binding protein; NAG, N-acetyl-β-glucosaminidase; NMP, normothermic machine perfusion; ns, not significant; TIMP-2, tissue inhibitor of metalloproteinases-2.

FIGURE 6.

Hemodynamic alterations throughout ex vivo perfusion of porcine kidneys. Arterial flow and renal vascular resistance differences during both HMPO₂ (A and B) and NMP (C and D) of ischemically and healthy porcine kidneys. Means and SDs are shown. HMPO₂, oxygenated hypothermic machine perfusion; NMP, normothermic machine perfusion.

FIGURE 7.

Biomarker differences between ischemically damaged and healthy porcine kidneys during ex vivo perfusion. Absolute ASAT content during HMPO₂ (A) and NMP (B). Absolute LDH content during HMPO₂ (C) and NMP (D). Absolute NAG content during HMPO₂ (E) and NMP (F). Absolute TIMP-2 content during HMPO₂ (G) and NMP (H). Median and interquartile range are shown. ASAT, aspartate aminotransferase; HMPO₂, oxygenated hypothermic machine perfusion; LDH, lactate dehydrogenase; NAG, N-acetyl-β-glucosaminidase; NMP, normothermic machine perfusion; ns, not significant; TIMP-2, tissue inhibitor of metalloproteinases-2. *P ≤ 0.05, **P < 0.01, ***P < 0.001.

FIGURE 8.

Percentage difference of nonoverlapping AUCs of the minimal WI group and the 75 min WI group of porcine kidneys. Median and interquartile ranges are shown. ASAT, aspartate aminotransferase; AUC, area under the curve; HMPO₂, oxygenated hypothermic machine perfusion; LDH, lactate dehydrogenase; NAG, N-acetyl-β-glucosaminidase; NMP, normothermic machine perfusion; ns, not significant; TIMP-2, tissue inhibitor of metalloproteinases-2; WI, warm ischemia. *P ≤ 0.05, ***P < 0.001.