Doxycycline Alters the Porcine Renal Proteome and Degradome during Hypothermic Machine Perfusion

Abstract

Ischemia-reperfusion injury (IRI) is a hallmark for tissue injury in donation after circulatory death (DCD) kidneys. The implementation of hypothermic machine perfusion (HMP) provides a platform for improved preservation of DCD kidneys. Doxycycline administration has shown protective effects during IRI. Therefore, we explored the impact of doxycycline on proteolytic degradation mechanisms and the urinary proteome of perfused kidney grafts. Porcine kidneys underwent 30 min of warm ischemia, 24 h of oxygenated HMP (control/doxycycline) and 240 min of ex vivo reperfusion. A proteomic analysis revealed distinctive clustering profiles between urine samples collected at T15 min and T240 min. High-efficiency undecanal-based N-termini (HUNTER) kidney tissue degradomics revealed significantly more proteolytic activity in the control group at T-10. At T240, significantly more proteolytic activity was observed in the doxycycline group, indicating that doxycycline alters protein degradation during HMP. In conclusion, doxycycline administration during HMP led to significant proteomic and proteolytic differences and protective effects by attenuating urinary NGAL levels. Ultimately, we unraveled metabolic, and complement and coagulation pathways that undergo alterations during machine perfusion and that could be targeted to attenuate IRI induced injury.

Keywords: degradomics; ischemia/reperfusion injury; machine perfusion; proteomics; renal transplantation.

Figure 1

Urine proteomics & kidney tissue degradomics experimental workflows. Porcine kidneys (n = 7 per group) were retrieved from a local abattoir and exposed to 30 min of warm ischemia, 24 h of oxygenated hypothermic machine perfusion (HMP) with or without the addition of doxycycline, and 4 h of ex vivo reperfusion for functionality assessment. Perfusate, tissue and urine samples were collected during HMP and ex vivo reperfusion for various analyses. Protein degradation was analyzed using renal cortex tissue collected before and after ex vivo reperfusion and using a HUNTER [26] degradomics workflow. Urinary proteomics was performed on collected ultra-filtrate during ex vivo reperfusion using a label-free quantitative proteomics workflow. Illustration is original and created using www.biorender.com (accessed on January 2022).

Figure 2

Urinary proteomic analysis of ex vivo reperfused kidneys revealed altered complement & coagulation, ECM and metabolic pathways. (a) Heat map and hierarchical clustering of proteins at T15, T60, T120, T180 and T240. (b) Heat map and hierarchical clustering of proteins in urine samples only at T15 (beginning) and T240 (end). Protein expression is clustered into A and B. (c) Gene ontology term enrichment analysis of proteins in cluster A. (d) Gene ontology term enrichment analysis of proteins in cluster B. (e) log2 Fold Change in abundance between DOXY and the control was plotted on the x-axis and the statistical significance of these changes on the y-axis as the −log10 (p-value). DOXY: doxycycline treated.

Figure 3

Renal tissue degradomics during ex vivo reperfusion modulated by doxycycline. (a) Heat map and hierarchical clustering of degradation products across all tissue samples. (b) Represents the log2 fold change of degradation products between DOXY and the control group pre-reperfusion (T-10). (c) Represents the log2 fold change of degradation products between DOXY and the control group post-reperfusion (T240). (d) Represents a KEGG pathways analysis of degradation products identified as significantly downregulated and (e) upregulated pre-reperfusion. (f) Represents a KEGG pathways analysis of degradation products identified as significantly upregulated post-reperfusion. DOXY; doxycycline treated.

Figure 4

Kidney degradome dynamics during ex vivo reperfusion. (a) Log2 Fold Change in abundance between post (T240) and pre (T-10) reperfusion was plotted on the x-axis and the statistical significance of these changes on the y-axis as the -log10 (p-value). Significantly downregulated degradation products (blue). Significantly upregulated degradation products (red). (b) String cluster and KEGG pathway analysis of degradation products significantly downregulated after reperfusion. (c) String clustering and KEGG pathway analysis of degradation products significantly upregulated after reperfusion.

Figure 5

Renal function during hypothermic machine perfusion and ex vivo reperfusion. (a) Arterial flow during hypothermic machine perfusion (HMP) shown in mL/min/100 gr. of kidney weight over time. (b) LDH levels in perfusate in U/L during HMP. (c) Fractional sodium excretion shown in % during reperfusion. (d) ATP levels in cortex tissue shown in pmol per ug protein at time point: HMP0h, HMP24h or pre-reperfusion, 120 min into reperfusion and 240 min into reperfusion or post-reperfusion. (e) Oxygen consumption shown in mL O₂/min/100 gr. of kidney weight during reperfusion. (f) Gelatinase activity after 240 min of reperfusion. (g) Absolute accumulative urinary protein levels in mg. (h) Absolute accumulative urinary NGAL levels in pg. (i) delta LDH levels in perfusate in U/L during reperfusion. (j) delta ASAT levels in perfusate in U/L during reperfusion. * = p < 0.05. Data shown as mean ± SEM. DOXY; doxycycline treated.