Effects of the PPAR-β/δ agonist GW0742 during resuscitated porcine septic shock

Animal preparation

Anesthesia and surgical instrumentation have been described in detail previously [13–15]. Briefly, anesthesia was induced with atropine, propofol, and ketamine to allow endotracheal intubation and was maintained thereafter with pentobarbitone, buprenorphine, and pancuronium. Ventilator settings were fraction of inspired O₂ (FiO₂) 0.35, positive end-expiratory pressure (PEEP) 10 cmH₂O, tidal volume 8 mL·kg^-1, respiratory rate 10 to 12 breaths·min^-1 adjusted to maintain arterial PCO₂ = 35 to 40 mmHg, inspiratory (I)/expiratory (E) ratio 1:1.5, peak airway pressure <40 cmH₂O, and modified to I/E ratio 1:1 and PEEP 12 or 15 cmH₂O, respectively, if the ratio of arterial O₂ partial pressure (PaO₂)/FiO₂ is <300 or <200 mmHg [13–15]. The right jugular vein and carotid artery were exposed for the insertion of a central venous catheter sheath and the placement of a balloon-tipped pulmonary artery catheter to measure central venous (CVP), pulmonary arterial (MPAP), and pulmonary artery occlusion pressures (PAOP), and a thermistor-tipped arterial catheter for blood pressure (MAP) recording and transpulmonary single indicator thermodilution-cardiac output (CO) measurement. The right kidney and a femoral vein were surgically exposed, and a catheter was advanced into the inferior vena cava and manually guided into a right renal vein under visual control [14, 15]. A catheter in the urinary bladder allowed urine collection. Two tubes were placed through the abdominal wall for peritonitis induction. Ringer's solution was continuously infused as maintenance fluid (10 mL·kg^-1·h^-1). As needed, animals received hydroxyethyl starch to maintain cardiac filling pressures during surgery.

Experimental protocol

After instrumentation and an 8-hour recovery, baseline data were collected. Thereafter, the supernatant (3 mL·kg^-1) of 1.0 g·kg^-1 autologous feces incubated in 500 mL 0.9% saline for 12 h at 38°C was injected into the abdominal cavity via the abdominal drainage tubes. Hydroxyethyl starch (10 mL∙kg^-1∙h^-1, 5 mL∙kg^-1∙h^-1 if CVP or PAOP is >18 mmHg) allowed maintaining hyperdynamic hemodynamics. If necessary, norepinephrine was infused and titrated to maintain MAP at baseline values (no further increase if the heart rate is ≥160 min^-1 to avoid tachycardia-induced myocardial ischemia) [13–16]. Animals randomly received i.v. GW0742 (0.03 mg∙kg^-1, n = 10, body weight 71 (66 to 80) kg) or vehicle (DMSO; n = 12, body weight 75 (66 to 81) kg) at 6, 12, and 18 h after the induction of peritonitis. The same GW0742 dose attenuated renal dysfunction in murine endotoxic shock [2] and organ injury after kidney ischemia/reperfusion injury in diabetic rats [10]. The timing of the GW0742 administration was chosen, because10-day survival was doubled in mice injected with this dose at 6.5 and 12.5 h after cecal ligation and puncture-induced sepsis [2]. After additional data collection at 12 and 24 h after the induction of peritonitis, animals were sacrificed under deep anesthesia.

Measurements and calculations

Hemodynamics, gas exchange (calorimetric O₂ uptake and CO₂ production, arterial and mixed venous blood gases), glucose, lactate, creatinine, renal venous nitrite + nitrate (NO₂ ^- + NO₃ ^-), tumor necrosis factor-α (TNFα), and interleukin-6 (IL-6) concentrations were determined as described previously [13–16]. Endogenous glucose production and glucose oxidation were derived from plasma 1,2,3,4,5,6-¹³C₆-glucose and the mixed expiratory ¹³CO₂ isotope enrichment, respectively, during continuous glucose isotope infusion [13, 14]. Urinary and blood creatinine and Na⁺ levels were analyzed to calculate creatinine clearance and fractional Na⁺-excretion [9] together with blood neutrophil gelatinase-associated lipocalin (NGAL) [15]. At the end of the experiment, immediate post-mortem kidney tissue samples were analyzed for the expression of the inducible nitric oxide synthase (iNOS), heme oxygenase-1 (HO-1), and cleaved caspase-3 as well as for the activation of the nuclear transcription factor κB (NF-κB) as described in detail previously [15, 16]. Pyramid-shaped kidney specimens showing kidney cortex, medulla, renal papilla, and the corresponding renal calyx were dissected for histopathological examination, performed by an experienced pathologist (A.S.) blinded for the sample grouping [15]. Histopathological alterations were analyzed for the degree of 'glomerular tubularization’, dilatation of Bowman's space, and swelling of Bowman's capsule, cellular edema of the proximal tubule, distal tubular dilatation and elongation, tubular protein cylinders, and tubular necrosis as described in detail previously [15].

Immunohistochemistry allowed quantifying the formation of nitrotyrosine (rabbit anti-Nitrotyrosine, Millipore, Schwalbach, Germany) [16] and the expression of the PPAR-β/δ (rabbit anti-PPAR delta antibody #ab23673, Abcam plc, Cambridge, UK), the method being described in detail in the supplement. The latter was determined on formalin-fixed, paraffin-embedded kidney biopsies, which had been taken during surgical instrumentation both in FBM and young and healthy German Landswine in a previous study [15], as well as age-matched FBM pigs that had not been fed with the atherogenic diet (n = 5 in each group). Results are presented as mean densitometric sum red.

Statistical analysis

Data are presented as median (quartiles). After the exclusion of normal distribution using the Kolmogorov-Smirnoff test, differences within groups were analyzed by a Friedmann analysis of variance on ranks and a subsequent Dunn's test with Bonferroni correction. Inter-group differences were tested using a Mann–Whitney rank sum test.

Limitations of the study

Since we did not study the effect of GW0742 in young and healthy animals, we do not have direct evidence that the PPAR-β/δ down-regulation associated with the underlying hypercholesteremia and atherosclerosis rather than other potentially confounding factors contributed to our findings, e.g., the use of adult swine per se, the integration of standard intensive care procedures into the experimental design, the timing and dosing of the treatment, and/or the duration of the study. To our knowledge, GW0742 has only been studied in rodents. Nevertheless, GW501516, another highly specific PPAR-β/δ agonist with a chemical structure very close to that of GW0742 [1], attenuated endotoxin-induced NF-κB activation, and cytokine release not only in rodent [24–26] but also in human tissues [26, 27]. So far, a modulation of the PPAR-β/δ expression related to nutritional and/or metabolic interventions has only been studied in rodents as well [18, 19]. However, in swine, a similar diet-induced hypercholesteremia as in our experiments [28] was associated with a markedly reduced PPAR-α and PPAR-γ expression [29], coinciding with impaired glucose tolerance [28, 29], arterial hypertension [28], increased transaminase activities [29], and aggravated oxidative stress [28]. Treatment with resveratrol restored PPAR-α and PPAR-γ expression [29], going along with improved left heart perfusion and function during chronic myocardial ischemia [30, 31]. It could be argued that all rodent data on GW0742 were obtained in young animals, while we studied adult swine. In fact, ischemia/reperfusion injury and hemorrhagic shock induced PPAR-γ expression only in young but not in adult or old rodents [32, 33], which in turn impaired adaptive autophagy [33]. There are no data available on PPAR-β/δ expression under these conditions, but the above-mentioned studies on hypercholesteremia-induced PPAR-α and PPAR-γ down-regulation also originate from adult swine. Moreover, tissue PPAR-β/δ expression in kidneys from adult FBM swine that had not been fed with cholesterol-enriched diet was similar to that in young and healthy German Landswine (Figure  1B). We only studied the effect of 0.03 mg∙kg^-1 of GW0742. This dose was chosen because it attenuated kidney dysfunction in murine endotoxin- and cecal ligation and puncture-induced septic shock at plasma concentrations selectively activating the PPAR-β/δ without any cross-reactivity on the other PPAR isoforms [2]. Moreover, in rats, this dose reduced organ injury after kidney ischemia/reperfusion injury [10], and a ten times higher dose did not further influence myocardial ischemia/reperfusion injury [3]. It should be noted in this context that equipotent drug doses are usually much lower on a per kilogram basis in large species (e.g., swine, dogs) than in rodents [34]. FBM swine present with reduced creatinine clearance and moderate histological damage of the kidney already under baseline conditions [15]. Nevertheless, any pre-existing organ dysfunction most likely did not influence our results; due to fluid resuscitation and catecholamine infusion, creatinine clearance was comparable to that in young and healthy German Landswine undergoing the same protocol of resuscitated fecal peritonitis [13, 14].