The Relationship Between Ischemia-reperfusion Injury (IRI) And Kidney Function

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PART Ⅰ: Metformin Preconditioning and Postconditioning to Reduce lschemia Reperfusion Injury in an lsolated Ex Vivo Rat and Porcine Kidney Normothermic Machine Perfusion Model

Tobias M.Huink,Leonie H.Venemal,Rene A.Posma,Nynke J.de Vries, Andrie C.Westerkampl,& et al.

WHAT IS THECURRENT KNOWLEDGE ON THE TOPIC?

lschemia-reperfusion injury (IRI) negatively affects outcomes of kidney transplantation, whereas metformin has been proposed to attenuate IRI beyond its glucose-lowering actions. Machine perfusion provides a platform to add protective agents, like metformin, prior to or during reperfusion to decrease IRI. Previous data showed that metformin preconditioning was able to improve hepatobiliary function during normothermic machine perfusion and subsequent transplantation of rat livers.

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WHAT QUESTION DID THIS STUDY ADDRESS?

In this study, potential beneficial effects of preconditioning and postconditioning with metformin on IRI were evaluated in different isolated ex vivo normothermic machine perfusion models using rat and porcine kidneys.

WHAT DOES THIS STUDY ADD TO OUR KNOWLEDGE?

We show that both metformin preconditioning and postconditioning reduced rat and porcine kidney injury, but that the improvements were minor. It remains unknown whether addition of metformin during machine perfusion results in improved organ quality after transplantation. Moreover, we show that the use of in vivo toxic concentrations of metformin during machine perfusion was not associated with cellular damage, indicating that it seems to be safe to use these concentrations during ex vivo perfusion of kidneys.

HOW MIGHT THIS CHANGE CLINICAL PHARMACOLOGY OR TRANSLATIONAL SCIENCE?

Being widely implemented， machine perfusion is an ideal platform for pharmacological interventions to diminish or prevent IRI, and to test therapeutics in isolated ex vivo organs. As the effects of metformin were minor, it remains unclear whether it should be used as a renoprotective agent prior to transplantation. Our results do not necessarily advocate the use of metformin as an intervention prior to kidney transplantation, but future research is required to investigate whether metformin in combination with other potential treatments might be beneficial.

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RESULTS

Perfusion parameters

The IVR remained constant during NMP in the rat model, without significant differences between the treatment groups(Figure S1a).No significant differences were found in the porcine kidney groups regarding IVR(Figure S1b). Kidney function parameters

No significant differences in total creatinine clearance were observed between all treatment groups in the rat study (Figure 2a). In the porcine study, total creatinine clearance did not differ between metformin-treated kidneys and controls (Figure 2b), although there was a tendency toward lower creatinine clearance in the metformin-treated group (P=0.07).

Metformin-pretreated rats of whom kidneys were perfused with 30 mg/L metformin had a significantly lower urine production compared with rats pretreated with metformin without subsequent perfusion with metformin (P= 0.02), and rats pretreated with oral saline whose kidneys were postconditioned with 30 mg/L metformin (P = 0.04). Postconditioning with 300 mg/L metformin did not yield any significant difference in urine production, irrespective of preconditioning conditions. Compared with controls, total urine production in porcine kidneys was significantly lower in met-formintreated kidneys(P=0.004; Figure 2c,d).

In the rat study, total protein excretion was lower in metformin preconditioned kidneys without subsequent perfusion with metformin than the control group(P=0.001). No differences in total protein excretion were observed between all other experimental groups (Figure 2e).

During NMP of porcine kidneys, no significant differences in urinary protein excretion were found between the metformin and control group (Figure 2f).

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Injury markers

Metformin preconditioned rats whose kidneys were not perfused with metformin had lower LDH release than controls (P= 0.005). Metformin preconditioned rats whose kidneys subsequently were perfused with 30 mg/L metformin had lower LDH release than controls(P=0.01) and had a lower LDH release than saline preconditioned rats whose kidneys were perfused with 30 mg/L metformin (P=0.04). Perfusion with 300 mg/L metformin did not result in decreased LDH release (Figure 3a). Total LDH release during NMP did not differ between metformin treated porcine kidneys and controls (Figure 3b).

Both preconditioning and postconditioning with metformin was not associated with a significant difference in the total amount of released ASAT in the rat study (Figure 3c). Likewise, no differences between metformin-treated kidneys and con-trols in ASAT levels were found in porcine kidneys(Figure 3d).

Morphological signs of ischemia reperfusion injury Compared with controls, tubular necrosis was significantly reduced in kidneys that were perfused with 300 mg/L metformin, independent of saline(P=0.02)or metformin (P=0.02)preconditioning. Metformin preconditioned rats whose kidneys were perfused with 300 mg/L metformin had less tubular necrosis compared with metformin preconditioned rats without subsequent metformin perfusion (P=0.01) and metforminpreconditioned rats of whose kidneys were perfused with 30 mg/L metformin (P=0.02; Figure 3e).

Proximal tubular cell vacuolation was significantly reduced in saline preconditioned rats whose kidneys were perfused with 300 mg/L metformin compared with saline preconditioned rats whose kidneys were perfused with 30 mg/L metformin (P=0.05). Vacuolation was also significantly lower in metformin preconditioned kidneys perfused with 300 mg/L metformin compared with metformin preconditioned kidneys without metformin perfusion (P=0.02)(Figure 3g). No statistical differences regarding edema formation were seen in the rat study (Figure 3f).No differences in morphological signs of IRI were observed at the end of NMP in porcine kidneys (Figure 3h).

Figure 3 Injury markers in perfusion fluid and morphological injury in tissue after normothermic machine perfusion. Total markers of injury were calculated as the area under the curve. Total amount of lactate dehydrogenase (LDH) (a, b) and aspartate aminotransferase (ASAT) (c, d) in the perfusate. Tissue was scored on proximal tubular cell necrosis (ranging from mild to severe: 1–5), edema (ranging from mild to severe: 1–4), and proximal tubular cell vacuolation (ranging from mild to severe: 1–4) in the rat study, respectively (e–g). Signs of ischemia reperfusion injury in porcine kidneys were scored (h). Data are presented as mean ± standard error of the mean. Groups contain 5–7 kidneys. *P < 0.05, **P < 0.01, compared with all other groups.

Gene expression

Relative mRNA expression of genes involved in the regulation of the vascular tone was evaluated in both rat and porcine kidneys. Compared with controls, the expression of EDN-1 was significantly decreased in rat kidneys perfused with 300 mg/L metformin, regardless of preconditioning with saline (P=0.002) or metformin (P=0.005;Figure 4a). Compared with saline pretreated rats whose kidneys were perfused with 30 mg/L metformin, eNOS expression was significantly decreased in controls (P=0.04)and saline preconditioned rats whose kidneys were perfused with 300 mg/L metformin (P=0.01; Figure 4b). No differences regarding EDN-1 or eNOS expression were found in the porcine kidney study (Figure 4h). KLF-2 expression was not different between groups in both the rat and porcine kidney study (Figure 4c,h).

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Moreover, genes involved in endothelial activation were evaluated. The vWF expression did not differ between treat-ment groups in both rat and porcine kidneys(Figure 4d,). Compared with controls, gene expression of VCAM-1 was significantly decreased in rat kidneys perfused with 30 mg/L metformin irrespective of preconditioning with saline (P=0.002) or metformin (P=0.03). Saline pretreated rats whose kidneys were perfused with 30 mg/L metformin had lower VCAM-1 expression than metformin preconditioned rats whose kidneys were also perfused with 30 mg/L metformin (P=0.04). Saline preconditioned rats whose kidneys were perfused with 300 mg/L metformin had significantly higher VCAM-1 expression than saline preconditioned rats whose kidneys were perfused with 30 mg/L metformin (P=0.002).On the other hand, metformin preconditioned rats whose kidneys were perfused with 300 mg/L metformin had significantly higher VCAM-1 expression than controls (P = 0.03; Figure 4e).

Compared with metformin preconditioned rats whose kidneys were perfused with 300 mg/L metformin,L-6 gene expression was decreased in metformin preconditioned rats whose kidneys were not perfused with metformin (P=0.03), and metformin-preconditioned rats whose kidneys were perfused with 30 mg/L metformin (P=0.004; Figure 4f).

HSP-70 expression in rat kidneys was not different between treatment groups (Figure 4g).Expression of vWF, VCAM-1, and IL-6 was not significantly different between experimental groups within the porcine kidney study (Figure 4i).However, HSP-70 was upregulated in metformin treated porcine kidneys (P=0.03; Figure 4l).

Figure 4 Relative mRNA expression of genes involved in the regulation of vascular tone, including vasoconstriction (endothelin 1 (EDN-1)), vasodilatation (endothelial nitric oxide synthase (eNOS)), and laminar flow shear stress (Krüppel-like factor 2 (KLF-2)) in both the rat (respectively, a–c) and porcine study (h). Genes involved in the activation of the endothelium, playing a role in platelet (von Willebrand factor (vWF)) and leukocyte adhesion (vascular cell adhesion molecule 1 (VCAM-1)), and inflammatory processes (IL-6) were assessed in rat (respectively, d–f) and porcine kidneys (i). Heat-shock protein 70 (HSP-70) expression was evaluated as well in both rat and porcine kidneys (respectively, g, i). Data are presented as mean ± standard error of the mean. Groups contain 5–7 kidneys. *P < 0.05, **P < 0.01.

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