Kimberley E. Wever

Ischemic preconditioning in the animal kidney, a systematic review and meta-analysis.

Ischemic preconditioning (IPC) is a potent renoprotective strategy which has not yet been translated successfully into clinical practice, in spite of promising results in animal studies. We performed a unique systematic review and meta-analysis of animal studies to identify factors modifying IPC efficacy in renal ischemia/reperfusion injury (IRI), in order to enhance the design of future (clinical) studies. An electronic literature search for animal studies on IPC in renal IRI yielded fifty-eight studies which met our inclusion criteria. We extracted data for serum creatinine, blood urea nitrogen and histological renal damage, as well as study quality indicators. Meta-analysis showed that IPC reduces serum creatinine (SMD 1.54 [95%CI 1.16, 1.93]), blood urea nitrogen (SMD 1.42 [95% CI 0.97, 1.87]) and histological renal damage (SMD 1.12 [95% CI 0.89, 1.35]) after IRI as compared to controls. Factors influencing IPC efficacy were the window of protection (<24 hu200a=u200aearly vs. ≥24 hu200a=u200alate) and animal species (rat vs. mouse). No difference in efficacy between local and remote IPC was observed. In conclusion, our findings show that IPC effectively reduces renal damage after IRI, with higher efficacy in the late window of protection. However, there is a large gap in study data concerning the optimal window of protection, and IPC efficacy may differ per animal species. Moreover, current clinical trials on RIPC may not be optimally designed, and our findings identify a need for further standardization of animal experiments.

Remote ischaemic preconditioning by brief hind limb ischaemia protects against renal ischaemia-reperfusion injury: the role of adenosine

BACKGROUNDnRemote ischaemic preconditioning (RIPC) is a strategy to protect a target organ against ischaemia-reperfusion injury (IRI) by inducing short-term ischaemia/reperfusion (I/R) in a remote organ. RIPC of the kidney by temporary limb occlusion would be a safe, inexpensive and noninvasive method to prevent renal damage in, e.g., transplantation and aortic surgery. We investigated whether brief hind limb occlusion can protect against renal IRI and whether this protection is adenosine dependent.nnnMETHODSnRats underwent either no RIPC, unilateral RIPC or bilateral RIPC. The preconditioning stimulus was either continuous (12/12 I/R) or fractionated (three times 4/4 I/R). After the last reperfusion period, we induced 25 ischaemia in the right kidney.nnnRESULTSnAfter 24 h of reperfusion, renal function was improved by 30-60% in both bilateral RIPC groups and in the fractionated unilateral group. Renal tubule damage and kidney injury molecule-1 expression were reduced in three of four RIPC groups. Treatment with the adenosine receptor blocker 8-(p-sulfophenyl)theophylline had no effect on fractionated or continuous RIPC.nnnCONCLUSIONSnBrief hind limb ischaemia induces protection against renal IRI, which makes this a promising strategy to prevent renal IRI in a clinical setting. Bilateral RIPC was more effective than unilateral RIPC, and this protection occurs via an adenosine-independent mechanism.

The Usefulness of Systematic Reviews of Animal Experiments for the Design of Preclinical and Clinical Studies

The question of how animal studies should be designed, conducted, and analyzed remains underexposed in societal debates on animal experimentation. This is not only a scientific but also a moral question. After all, if animal experiments are not appropriately designed, conducted, and analyzed, the results produced are unlikely to be reliable and the animals have in effect been wasted. In this article, we focus on one particular method to address this moral question, namely systematic reviews of previously performed animal experiments. We discuss how the design, conduct, and analysis of future (animal and human) experiments may be optimized through such systematic reviews. In particular, we illustrate how these reviews can help improve the methodological quality of animal experiments, make the choice of an animal model and the translation of animal data to the clinic more evidence-based, and implement the 3Rs. Moreover, we discuss which measures are being taken and which need to be taken in the future to ensure that systematic reviews will actually contribute to optimizing experimental design and thereby to meeting a necessary condition for making the use of animals in these experiments justified.

Regarding "Remote and local ischemic preconditioning equivalently protects rat skeletal muscle mitochondrial function during experimental aortic cross-clamping".

OBJECTIVEnThis study investigated whether remote (rIPC) and local ischemic preconditioning (IPC) similarly limit skeletal muscle dysfunction induced by aortic cross-clamping.nnnMETHODSnRats were divided in three groups: the sham-operated control group (C) underwent surgery without clamping. The ischemia-reperfusion group (IR) had 3 hours of ischemia induced by aortic clamping and collateral vessels ligation, followed by 2 hours of reperfusion. The IPC group had, before prolonged ischemia, three bouts of 10 minutes of ischemia and 10 minutes of reperfusion on the right hind limb. Thus, right hind limbs had local IPC and left hind limbs had rIPC. Complexes I, II, III, and IV activities of the mitochondrial respiratory chain of the gastrocnemius muscle were measured using glutamate-malate (V(max)), succinate (V(succ)), and N,N,N,N-tetramethyl-p-phenylenediamine dihydrochloride (TMPD)-ascorbate (V(TMPD)). Expressions of genes involved in apoptosis (Bax, Bcl-2) and antioxidant defense (superoxide dismutase 1 [SOD 1], SOD2, glutathione peroxidase [GPx]) were determined by quantitative real-time polymerase chain reaction. Glutathione was also measured.nnnRESULTSnRight and left hind limb mitochondrial functions were similar in controls and after IR. IR reduced V(max) (-21.2%, 6.6 ± 1 vs 5.2 ± 1 μmol O(2)/min/g dry weight, P = .001), V(succ) (-22.2%, P = .032), and V(TMPD) (-22.4%, P = .033), and increased Bax (63.4%, P = .020) and Bax/Bcl-2 ratio (+84.6%, P = .029). SODs and GPx messenger RNA were not modified, but glutathione tended to be decreased after IR. Local IPC and rIPC counteracted similarly these deleterious effects, restoring mitochondrial maximal oxidative capacities and normalizing Bax, the Bax/Bcl-2 ratio, and glutathione.nnnCONCLUSIONSnRemote ischemic preconditioning protection against IR injury is equivalent to that achieved by local IPC. It might deserve a broader use in clinical practice.nnnCLINICAL RELEVANCEnAcute and chronic ischemia induce mitochondrial dysfunction in human skeletal muscles, and improving muscle mitochondrial function improves subjects’ status. Compared with local ischemic preconditioning (IPC), remote IPC (rIPC) appears easier to perform and is safer for the vessel and territory involved in ischemic injury. This study demonstrates that the muscle protection afforded by rIPC is equivalent to that achieved by IPC. Acknowledging that IPC procedures should be specifically adapted to patient characteristics to be successful, our results support a broader use of rIPC in the setting of vascular surgery.

Diannexin protects against renal ischemia reperfusion injury and targets phosphatidylserines in ischemic tissue

Renal ischemia/reperfusion injury (IRI) frequently complicates shock, renal transplantation and cardiac and aortic surgery, and has prognostic significance. The translocation of phosphatidylserines to cell surfaces is an important pro-inflammatory signal for cell-stress after IRI. We hypothesized that shielding of exposed phosphatidylserines by the annexin A5 (ANXA5) homodimer Diannexin protects against renal IRI. Protective effects of Diannexin on the kidney were studied in a mouse model of mild renal IRI. Diannexin treatment before renal IRI decreased proximal tubule damage and leukocyte influx, decreased transcription and expression of renal injury markers Neutrophil Gelatinase Associated Lipocalin and Kidney Injury Molecule-1 and improved renal function. A mouse model of ischemic hind limb exercise was used to assess Diannexin biodistribution and targeting. When comparing its biodistribution and elimination to ANXA5, Diannexin was found to have a distinct distribution pattern and longer blood half-life. Diannexin targeted specifically to the ischemic muscle and its affinity exceeded that of ANXA5. Targeting of both proteins was inhibited by pre-treatment with unlabeled ANXA5, suggesting that Diannexin targets specifically to ischemic tissues via phosphatidylserine-binding. This study emphasizes the importance of phosphatidylserine translocation in the pathophysiology of IRI. We show for the first time that Diannexin protects against renal IRI, making it a promising therapeutic tool to prevent IRI in a clinical setting. Our results indicate that Diannexin is a potential new imaging agent for the study of phosphatidylserine-exposing organs in vivo.

Remote Ischemic Preconditioning To Reduce Contrast-Induced Nephropathy: A Randomized Controlled Trial.

BACKGROUNDnDespite the increasing use of pre- and post-hydration protocols and low osmolar instead of high osmolar iodine containing contrast media, the incidence of contrast induced nephropathy (CIN) is still significant. There is evidence that contrast media cause ischemia reperfusion injury of the renal medulla. Remote ischemic preconditioning (RIPC) is a non-invasive, safe, and low cost method to reduce ischemia reperfusion injury. The aim of this study is to investigate whether RIPC, as an adjunct to standard preventive measures, reduces contrast induced acute kidney injury in patients at risk of CIN.nnnMETHODSnThe RIPCIN study is a multicenter, single blinded, randomized controlled trial in which 76 patients at risk of CIN received standard hydration combined with RIPC or hydration with sham preconditioning. RIPC was applied by four cycles of 5 min ischemia and 5 min reperfusion of the forearm. The primary outcome measure was the change in serum creatinine from baseline to 48 to 72 hours after contrast administration.nnnRESULTSnWith regard to the primary endpoint, no significant effect of RIPC was found. CIN occurred in four patients (2 sham and 2 RIPC). A pre-defined subgroup analysis of patients with a Mehran risk score ≥11, showed a significantly reduced change in serum creatinine from baseline to 48 to 72 hours in patients allocated to the RIPC group (Δ creatinine -3.3 ± 9.8 μmol/L) compared with the sham group (Δ creatinine +17.8 ± 20.1 μmol/L).nnnCONCLUSIONnRIPC, as an adjunct to standard preventive measures, does not improve serum creatinine levels after contrast administration in patients at risk of CIN according to the Dutch guideline. However, the present data indicate that RIPC might have beneficial effects in patients at a high or very high risk of CIN (Mehran score ≥ 11). The RIPCIN study is registered at: http://www.controlled-trials.com/ISRCTN76496973.

Humoral signalling compounds in remote ischaemic preconditioning of the kidney, a role for the opioid receptor

BACKGROUNDnRenal ischaemia-reperfusion injury (IRI) is a common clinical problem associated with significant mortality and morbidity. One strategy to reduce this damage is remote ischaemic preconditioning (RIPC), in which brief ischaemia of a limb protects the kidney against a prolonged ischaemic insult. The mechanism of renal RIPC has not yet been elucidated. Here, we address the gap in our understanding of renal RIPC signalling, using a rat model of renal IRI and RIPC by brief hind limb ischaemia.nnnMETHODSnRats were treated with either no RIPC, RIPC+vehicle or RIPC+ an inhibitor or antagonist of one of the following candidate signalling molecules: noradrenalin, cannabinoids, glucocorticoids, inducible nitric oxide synthase, calcitonin gene-related peptide, ganglion-mediated signalling, haem oxygenase and free radicals. Subsequently, the animals underwent 25 min of renal ischaemia and 2 days of reperfusion, after which renal function and damage were assessed.nnnRESULTSnRIPC by three 4 min cycles of hind limb ischaemia effectively reduced renal IRI. Pre-treatment with the opioid receptor antagonist naloxone completely blocked this protective effect, when compared with animals treated with RIPC+vehicle; serum creatinine and urea increased (307.8±43.7 versus 169.5±16.7 µmol/L and 42.2±4.9 versus 27.6±2.2 mmol/L, respectively), as did the renal histological damage (score 4.2±0.7 versus 2.8±0.5) and expression of kidney injury molecule-1 (KIM-1; relative-fold increase in mRNA expression 164±18 versus 304±33). All other antagonists were without effect.nnnCONCLUSIONSnRenal RIPC by brief hind limb ischaemia may be the result of endorphin release from the hind limb. The importance of opioid signalling in renal RIPC provides vital clues for its successful translation to the clinical setting.

Determinants of the Efficacy of Cardiac Ischemic Preconditioning: A Systematic Review and Meta-Analysis of Animal Studies

Background Ischemic preconditioning (IPC) of the heart is a protective strategy in which a brief ischemic stimulus immediately before a lethal ischemic episode potently limits infarct size. Although very promising in animal models of myocardial infarction, IPC has not yet been successfully translated to benefit for patients. Objective To appraise all preclinical evidence on IPC for myocardial infarction and identify factors hampering translation. Methods and results Using systematic review and meta-analysis, we identified 503 animal studies reporting infarct size data from 785 comparisons between IPC-treated and control animals. Overall, IPC reduced myocardial infarction by 24.6% [95%CI 23.5, 25.6]. Subgroup analysis showed that IPC efficacy was reduced in comorbid animals and non-rodents. Efficacy was highest in studies using 2–3 IPC cycles applied <45 minutes before myocardial infarction. Local and remote IPC were equally effective. Reporting of study quality indicators was low: randomization, blinding and a sample size calculation were reported in 49%, 11% and 2% of publications, respectively. Conclusions Translation of IPC to the clinical setting may be hampered by the observed differences between the animals used in preclinical IPC studies and the patient population, regarding comorbidity, sex and age. Furthermore, the IPC protocols currently used in clinical trials could be optimized in terms of timing and the number of ischemic cycles applied. In order to inform future clinical trials successfully, future preclinical studies on IPC should aim to maximize both internal and external validity, since poor methodological quality may limit the value of the preclinical evidence.

Low-pressure pneumoperitoneum during laparoscopic donor nephrectomy to optimize live donors' comfort.

Nowadays, laparoscopic donor nephrectomy (LDN) has become the gold standard to procure live donor kidneys. As the relationship between donor and recipient loosens, it becomes of even greater importance to optimize safety and comfort of the surgical procedure. Low‐pressure pneumoperitoneum has been shown to reduce pain scores after laparoscopic cholecystectomy. Live kidney donors may also benefit from the use of low pressure during LDN. To evaluate feasibility and efficacy to reduce post‐operative pain, we performed a randomized blinded study. Twenty donors were randomly assigned to standard (14 mmHg) or low (7 mmHg) pressure during LDN. One conversion from low to standard pressure was indicated by protocol due to lack of progression. Intention‐to‐treat analysis showed that low pressure resulted in a significantly longer skin‐to‐skin time (149 ± 86 vs. 111 ± 19 min), higher urine output during pneumoperitoneum (23 ± 35 vs. 11 ± 20 mL/h), lower cumulative overall pain score after 72 h (9.4 ± 3.2 vs. 13.5 ± 4.5), lower deep intra‐abdominal pain score (11 ± 3.3 vs. 7.5 ± 3.1), and a lower cumulative overall referred pain score (1.8 ± 1.9 vs. 4.2 ± 3). Donor serum creatinine levels, complications, and quality of life dimensions were not significantly different. Our data show that low‐pressure pneumoperitoneum during LDN is feasible and may contribute to increase live donors’ comfort during the early post‐operative phase.

How systemic inflammation modulates adenosine metabolism and adenosine receptor expression in humans in vivo

Objective:Adenosine modulates inflammation and prevents associated organ injury by activation of its receptors. During sepsis, the extracellular adenosine concentration increases rapidly, but the underlying mechanism in humans is unknown. We aimed to determine the changes in adenosine metabolism and signaling both in vivo during experimental human endotoxemia and in vitro. Design:We studied subjects participating in three different randomized double-blind placebo-controlled trials. In order to prevent confounding by the different pharmacological interventions in these trials, analyses were performed on data of placebo-treated subjects only. Setting:Intensive care research unit at the Radboud University Nijmegen Medical Center. Subjects:In total, we used material of 24 healthy male subjects. Interventions:Subjects received 2 ng/kg Escherichia coli endotoxin (lipopolysaccharide) intravenously. Measurements and Main Results:Following experimental endotoxemia, endogenous adenosine concentrations increased. Expression of 5’ectonucleotidase messenger RNA was upregulated (p = .01), whereas adenosine deaminase messenger RNA was downregulated (p = .02). Furthermore, both adenosine deaminase and adenosine kinase activity was significantly diminished (both p ⩽ .0001). A2a and A2b receptor messenger RNA expression was elevated (p = .02 and p = .04, respectively), whereas messenger RNA expression of A1 and A3 receptors was reduced (both, p = .03). In vitro, lipopolysaccharide dose-dependently attenuated the activity of both adenosine deaminase and adenosine kinase (both p ⩽ .0001). Conclusions:Adenosine metabolism and signaling undergo adaptive changes during human experimental endotoxemia promoting higher levels of adenosine thereby facilitating its inflammatory signaling.