#1446 PCSK9 inhibitor addition during hypothermic perfusion protects kidney from ischemic damage in a rat model of donation after circulatory death
Authors: Grignano Maria Antonietta , Gregorini Marilena , Esposito Pasquale , Barisione Chiara , Verzola Daniela , Rumeo Noemi , Picciotto Daniela , Ivaldo Caterina , Malabarba Stefano , Islami Tefik , Viazzi Francesca , Rampino Teresa 
Published: May 2024

Abstract

Background and Aims
PCSK9 inhibitors (PCSK9i) are a new class of cholesterol-lowering drugs that is gaining market share in treating hypercholesterolemia because of their effectiveness in lowering LDL cholesterol and cardiovascular risk, even in patients with kidney disease.
In addition to the well-known effect of inhibiting the LDL receptor catabolism, new pharmacological properties are being discovered.
Experimental cellular and animal models have showed that PCSK9i reduce apoptosis, pyroptosis and in acute myocardial infarction decrease the infarct area, suggesting their protective role in tissue ischemia. However, so far PCSK9i effect on renal ischemic damage has not yet been studied.
The aim of this study was to evaluate the effects of PEP 2-8(PCSK9i) addition during hypothermic perfusion (HP) on ischemic kidney damage (ID) in a rat model of donation after circulatory death (DCD).

Method
n. 15 Sprague-Dawley rats were used as DCD donors. Briefly after a midline laparotomy, the left and right retroperitoneal renal areas were exposed, and lumbar arteries were isolated and sectioned; subsequently, the renal arteries and veins were isolated. After 30 min of warm ischemia, induced by aorta clamping, left and right nephrectomies were completed with preservation of the renal hilums.
Then the kidneys were perfused with Perf-Gen Solution (100 ml) (Institut Georges Lopez, Lissieu, France) or Perf-Gen solution (100 ml) supplemented with 3ug (10ug/kg) of PEP 2-8. Continuous HP was performed for 6 h at 4 °C.
DCD right and left kidneys of each rat were randomized to the following experimental groups:
CTRL group: n = 15 DCD rat kidneys perfused with Perf-Gen
PEP group: n = 15 DCD rat kidneys perfused with Pef-Gen supplemented with PEP 2-8.
Effluent fluid was collected at the beginning (T0) and at the end (T6h) of HP. Glucose and potassium levels were measured in the effluent fluid at T0 and T6h.
At T6h the kidneys were splitted into three aliquots; one was fixed in 10% formalin for morphological studies, and the other two were stored in trizol for gene expression studies and at −80 °C for biochemical assays respectively.
Tubular ischemic damage (TID) was evaluated at T6 by scoring all tubules observed in at least 10 non-consecutive high-powered fields, as described by Paller (Paller Score).
NOX- 4 gene expression was studied by RT-PCR assay. Tissue LDH and ATP levels were measured at T6h.

Results
PEP 2-8 treated kidneys showed a lower TID score compared to CTRL (PEP 107.0 IQR 90.75-116.0 vs CTRL 134.5 IQR 131.3-155.3 p &lt; 0.005) (Fig. 1). The percentage of tubules with score zero (normal tubules) was higher in PEP than CTRL (PEP 29.0% range % 24.2-39.5 vs CTRL 9.5% range % 5.5-12.0, p &lt; 0.001). The percentage of tubules with necrosis was lower in PEP than CTRL (PEP 5.5% range % 3.2-10.5 vs CTRL 12.5% range % 9.0-15.7, p &lt; 0.005).
Expression of NOX-4 gene, known inducer of apoptosis, was lower in PEP compared to CTRL (p &lt; 0.05)
Tissue ATP content was higher in PEP than CTRL (PEP 0.031 ± 0.01 vs CTRL 0.025 ± 0.006 p &lt; 0.05). In tissue samples at T6 LDH was lower in PEP than CTRL (PEP 554.8 ± 164.7 vs CTRL 742.4 ± 256.7 p &lt; 0.05) conversely the potassium release in effluent was significant only in CTRL (T6 vs T0 p &lt; 0.01), according to TID score results.
During HP, PEP 2-8 treated kidneys released more glucose in the effluent than CTRL kidneys (PEP 35.4 IQR 12.3-82.9 vs CTRL 12.3 IQR 7.1-70.5 p &lt; 0.01).

Conclusion
Our preliminary results show that PEP 2-8 added to Perf-Gen solution during HP protects kidneys from ID, preserving energy metabolism in rat DCD model. To the best of our knowledge, this is the first evidence of protective role of PCSK9 inhibition in pre-transplant renal conditioning and potentially it could be used in conditioning other organs (liver, lung). However, further studies are necessary to understand the mechanisms underlying PCSK9i effect on energy metabolism and neoglucogenesis.