PART Ⅱ: Metformin Preconditioning And Postconditioning To Reduce Lschemia Reperfusion Injury in An Lsolated Ex Vivo Rat And Porcine Kidney Normothermic Machine Perfusion Model
Mar 26, 2022
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Tobias M.Huink,Leonie H.Venemal,Rene A.Posma,Nynke J.de Vries, Andrie C.Westerkampl,& et al.
INTRODUCTION
Kidney transplantation is the treatment of choice for patients with end-stage renal disease. Unfortunately, demand is higher than the availability of organs.2 This shortage has resulted in increased use of suboptimal quality organs from donation after circulatory death (DCD) donors.5 Kidneys from DCD donors are more susceptible to ischemia-reperfusion injury(IRl), which is linked to significantly higher incidences of delayed graft function, affecting both short-term and long-term outcome.45 IRI is caused by the impairment of blood flow and subsequent reoxygenation, which results in the generation of reactive oxygen species (ROS), initiating a cascade of detrimental cellular responses.67 The severity of ischemia correlates strongly with early renal graft failure after kidney transplantation and contributes to increased morbidity. Therefore, warm ischemia (W) during organ retrieval and implantation, and cold ischemic time during preservation, respectively, should be kept to a minimum. Machine perfusion strategies can play a pivotal role in decreasing IRI by supplying oxygen during ischemic phases in organ donation. Furthermore, it provides a platform to add possible protective agents prior to reperfusion (preconditioning) or during reperfusion (post-conditioning) and is a suitable method to assess short-term renal function and injury.9-11
The biguanide metformin is the most used oral antihyperglycemic drug to treat patients with type 2 diabetes. The mechanism of action and the pleiotropic effects of metformin primarily result from the inhibition of complex 1 within the mitochondrial respiratory chain.12-14 Therefore, metformin may attenuate IRI beyond its glucose-lowering actions and has been proposed as an organ-protective agent during circumstances in which IRI occurs, such as transplantation.15,16
The aim of this study was to evaluate the potential beneficial effects of preconditioning and postconditioning with metformin on IRI in two different isolated ex vivo normothermic machine perfusion (NMP) models using rat and porcine kidneys.
NOURISH KIDNEY: DRAGON HERB CISTANCHE
METHODS
Rat study
Animals. Male Lewis rats weighing 270-300 g were used (Harlan Laboratories, Boxmeer, The Netherlands). The Institutional Animal Care and Use Committee of the University of Groningen approved the study protocol (DEC6708C). The animals received care according to the Dutch Law on Animal Experiments, following the National Institute of Health's Principles of Laboratory Animal Care.
Experimental design, organ retrieval, and preservation. A total of 31 rats were divided into 6 groups (n=5-6 per group;Figure 1).In all groups,15minutesof W and24hours of cold preservation was used to induce ischemic injury. Preconditioning was performed by administering 300 mg/kg metformin(1,1-dimethylbiguanide hydrochloride; Sigma-Aldrich, St. Louis, MO) dissolved in saline (0.9% NaCl) or saline alone through oral gavage 12 and 2 hours before nephrectomy. Saline, 30 mg/L metformin or 300 mg/L metformin was added to the perfusate as postconditioning agent. The concentration of 300 mg/kg body weight was chosen, as this dose resulted in serum metformin concentrations that correspond to those found in humans during maintenance metformin therapy.17
The procurement of kidneys was performed with minor changes to the DCD procedure, as described previously. 18 In short, rats were anaesthetized with 2–5% isoflurane and laparotomy was performed via a midline incision. Anticoagulation, using 500 IU heparin (Leo Pharma, Ballerup, Denmark), was administered via the dorsal penile vein. After 15 minutes of WI, nephrectomy of the left kidney was performed. The renal artery and ureter were cannulated. The kidney was flushed in situ with 10 mL saline (37°C) and 5 mL 4°C University of Wisconsin (UW) Cold Storage Solution (Bridge to Life, Columbia, SC). Once removed, kidneys were flushed with 5 mL UW once again and stored in UW for 24 hours at 4°C.
Normothermic machine perfusion. The rat NMP method has been described extensively earlier.18 In brief, NMP was performed for 90 minutes using a roller pump (Ismatec ISM404, Zürich, Switzerland). The perfusion pressure was set at 102 mmHg, controlled at the renal artery. In the control group, the perfusion fluid consisted of 100 mL William’s Medium E supplemented with 30 mmol/L HEPES, 50 g/L albumin, and 7 mmol/L creatinine (all Sigma-Aldrich). In the experimental groups, either 30 or 300 mg/L metformin was added to the perfusate. The perfusion fluid was oxygenated with 95% oxygen and 5% carbon dioxide with a flow of 0.5 L/ min. The temperature of the perfusion fluid was maintained at 37°C using a water bath and heat exchanger (Julabo, Seelbach, Germany). The flow was recorded every 10 minutes, using a calibrated flow sensor (ME1PXN Inline, Transonic Systems, Ithaca, NY). After NMP, biopsies of the kidneys were submerged immediately in 4% formaldehyde or snap-frozen in liquid nitrogen and subsequently stored at −80°C.
Figure 1 Schematic representation of the experimental groups. Groups contain 5–7 kidneys. HMP, hypothermic machine perfusion; NMP, normothermic machine perfusion; WIT, warm ischemia time.
Porcine study
Animals. Kidneys from female Dutch Landrace pigs were collected from an abattoir. The animals were stunned and exsanguinated according to local standard procedures. During exsanguination, approximately 1 L of blood was collected in a container containing 25,000 IU unfractionated heparin (Leo Pharma). Because slaughterhouse waste material was used, no animal ethics committee approval was required.
Experimental design, organ retrieval, and preservation. To induce ischemic injury, 30 minutes of WI was used. After this period, the kidney was flushed with 180 mL saline at 4°C. Immediately after the flush, a cortical biopsy was taken (Invivo, Best, The Netherlands) and stored in 4% buffered formaldehyde. To accommodate transport from the abattoir to the laboratory and as a preservation technique, the renal artery was cannulated and the kidneys were attached to a pulsatile pressure-controlled hypothermic machine perfusion (HMP) setup (Kidney Assist Transport; Organ Assist, Groningen, The Netherlands). The kidneys were perfused at 4°C using 500 mL UW Machine Perfusion Solution for 3 hours (Bridge to Life, London, UK) with or without the addition of 2 mg metformin, with a mean arterial pressure of 25 mmHg. Oxygen (100%) was supplied to the oxygenator (Hilite LT1000; Medos Medizintechnik AG, Stolberg, Germany)with a fixed flow rate of 0.1 L/min.
Normothermic machine perfusion. The kidneys were reperfused using an ex vivo NMP setup for 4 hours that was described previously. In our study, increasing doses of metformin or saline were added using an infusion pump. In short, after HMP, the renal artery wascannulated and flushed. The ureter was cannulated for urine collection.Afterward, the kidney was weighed, and a biopsy was taken and stored in 4% buffered formaldehyde for further analysis.Subsequently, the kidneys were placed in an ex vivo pressure-controlled NMP circuit. NMP was performed with 500 mL leucocyte depleted,(BioR 02 plus; Fresenius Kabi, Bad Homburg, Germany) autologous, oxygenated (carbogen,flow 0.5L/min)blood for 4 hours at a mean arterial pressure of 80 mmHg. Other compounds added to the perfusion fluid are provided in Table S1. In the experimental group, metformin dissolved in Ringer's lactate solution (20 mg/mL;Baxter, Utrecht, The Netherlands) was infused using an infusion pump controlled by custommade software in which infusion profiles could be defined(Alaris; CareFusion, Rolle, Switzerland).Every 30 minutes during NMP, the infusion speed was increased according to a prespecified schedule (Table S2).This schedule was based on human pharmacokinetic data indicating that metformin clearance is four times higher than the creatinine clearance.19 In the control group, kidneys were perfused without the addition of metformin. For each experiment, the flow was recorded using the clampon flow probe (ME7PXL; Transonic Systems), which was attached to the tubing close to the renal artery. This flow probe has been calibrated for the tubing used.Every 15 minutes during NMP, urine was collected and replaced with a corresponding volume of Ringer's lactate solution, which was also recorded. The temperature of the perfusion fluid was maintained at 37°C using an integrated heat exchanger, connected to a water bath (Julabo).Blood and urine samples were taken after 15 and 60 minutes and every following hour for biochemical analyses. When NMP was finished, biopsies were taken and stored in 4% buffered formaldehyde, or snap-frozen and stored at-80℃ for further analysis.
IMPROVE KIDNEY FUNCTION CISTANCHE SUPPLEMENT
Both studies
Biochemical analyses. Perfusate and urine samples were centrifuged (1,300 g for 10 minutes in the rat model and 1,000 g for 12 minutes in the porcine model, respectively, both at 4°C) and the supernatant was stored at -80°C. Lactate dehydrogenase (LDH), aspartate aminotransferase (ASAT), and creatinine were determined in perfusate, and creatinine in urine, respectively, by the Laboratory Center of the University Medical Center Groningen using standard biochemical analyses. The amount ofprotein excreted in rat urine was measured using a Pierce BCA Protein Assay(Thermo Fisher Scientific, Waltham, MA),whereas total protein concentration in porcine urine was measured using standard biochemical analyses at the Laboratory Center.
Realtime quantitative polymerase chain reaction. Realtime polymerase chain reaction (PCR) was carried out according to standard procedures on the Tagman Applied Biosystems 7900HT RealTime PCR system, as described previously.20Amplification of gene fragments involved in the regulation of vascular tone (endothelin 1 (EDN-1,encoding for ET-1); endothelial nitric oxide synthase (eNOS); and Krüppel-like factor 2 (KLF-2), endothelial activation (Von Willebrand factor (wWF);vascular cell adhesion molecule 1(VCAM-1) and IL-6), and heat-shock protein 70(HSP-70) was done with primer sets listed in Table S3.In short, total RNA was extracted from kidney sections using TRlzol (Life Technologies, Gaithersburg, MD). The cDNA obtained from rats was used as an internal reference during PCR to test primer efficiency. Gene expression was normalized with the mean of β-actin mRNA content. Results were expressed as 2-△ACT, where the CT value represents the difference between cycle threshold values.
Figure 2 Perfusion and renal function parameters assessed during and after normothermic machine perfusion of rat and porcine kidneys. Total creatinine clearance of rat (a) and porcine (b) kidneys. Total urine production (c, d) and total protein excreted in the urine (e, f) of rat and porcine kidneys, respectively. Data are presented as mean ± standard error of the mean. Groups contain 5–7 kidneys. *P < 0.05 and **P < 0.01.
Morphological scoring. Biopsies stored in 4%formaldehyde were embedded in paraffin and were cut into 4 μm slices. Coupes were subsequently stained with periodic acid Schiff and 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).21 Histopathological assessment was done blinded by two researchers. An independent clinical pathologist with extensive experience in assessing histopathological signs of injury after machine perfusion of rat and porcine organs validated the scores.
Calculations. Intrarenal vascular resistance (IVR) was calculated by dividing the mean arterial pressure by flow (expressed in mmHg/mL min). Creatinine clearance was calculated to estimate glomerular filtration rate using the following equation:[creatinine in urine]* urine flow/[creatinine in perfusate].
Statistical analysis. All data are expressed as the mean± standard error of the mean. When comparing two groups at a single time point, differences were assessed using an unpaired Student's t-test.Differences for total urine production, morphological scores, and gene expression were tested using analysis of variance. All statistical tests are two-tailed and P≤ 0.05 was considered statistically significant. SPSS Statistics version 23(IBM, Armonk, NY)was used for all analyses. The area under the curve (AUC)was calculated according to the trapezoid rule, and was used to approximate the total creatinine clearance, the total amount of protein excreted in the urine, and the total levels of ASAT and LDH in the perfusate.
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