Key Findings: To our knowledge, this study is the first to comprehensively define changes in organ-specific AP activity and expression induced by CPB/DHCA, as well as changes in tissue-level AP activity with the infusion of exogenous BiAP. In control infant pigs not exposed to CPB/DHCA (anesthesia with mechanical ventilation only), the highest total AP activity was found in kidneys, followed by the intestines and liver with lower activity in the lungs and heart. TNAP mRNA was the dominant isoenzyme expressed in the kidneys and liver, while the jejunum and ileum predominantly expressed the intestinal isoenzyme. Exposure to CPB/DHCA resulted in a decrease in kidney AP activity but an increase in lung AP activity, with no significant changes in total AP activity in the other organs assessed. We found no evidence that kidney AP mRNA expression was altered by CPB/DHCA, suggesting that decreased tissue AP activity was due to enzyme loss rather than decreased transcription. In contrast, CPB/DHCA resulted in increased expression of TNAP mRNA in the lung, likely leading to the increased tissue AP activity. The ileum demonstrated a mixed picture, with increased expression of its predominant AP isoenzyme (iAP) following exposure to CPB/DHCA, but no change in total tissue AP activity, suggesting a combination of increased production with ongoing AP loss. Treatment of the piglets undergoing CPB/DHCA with the highest dose of BiAP resulted in increased total AP activity in kidney and liver only. BiAP infusion did not appear to significantly alter AP mRNA expression in any organ.
Organ-specific AP activity and expression in healthy controls: AP is used routinely as a biomarker for liver and bone disease and most clinicians are familiar with these two sources of AP. It is less commonly appreciated that AP is a ubiquitous enzyme that is conserved from bacteria to humans and is present in most organs, including kidney, intestines, and lung in addition to liver and bone.[16, 50-54] Relatively little is known regarding the biology of AP at the tissue level or its organ-specific role/response to disease. In this study, we demonstrate that the highest tissue AP activity is found not in the liver but instead in the kidneys and small intestine (bone was not tested), with intermediate activity in the liver and colon and lower activity in the thoracic organs. These findings validate early studies in large animals,[55, 56] which similarly found the highest levels of AP activity in the kidneys and intestines. As expected, the specific isoenzyme expressed differed between these organs, with the ileum and jejunum largely expressing the intestinal isoform and the kidneys expressing the tissue nonspecific isoform. This expression pattern is also consistent with prior studies in small and large animals as well as human studies and supports the utility of pigs as a large animal model of AP biology.
The biologic role for high AP expression in the kidneys and intestines in healthy animals has not been fully elucidated. In both of these organs AP localizes primarily to the epithelial surfaces: the proximal tubules of the kidneys[57] and the microvilli along the apical surface of intestinal enterocytes.[58, 59] IAP is expressed throughout the intestines, with the highest levels in the duodenum and lower levels in the colon.[60] High concentrations of iAP are found in apical vesicles that can secrete functional iAP into the intestinal lumen.[60, 61] Recent studies of AP in the intestines point towards a protective role against LPS and other microbacterial products, consistent with their brush border localization.[16, 60, 62, 63] AP may also have a role in absorption of nutrients from the intestinal lumen.[16, 60, 62, 64, 65] The biologic role of epithelial AP in healthy kidneys is less clear.[66] TNAP is the predominant isoform and is located in the S1, S2, and S3 segments of the proximal tubule with a small amount of iAP present in the S3 segment as well.[66-68] Preclinical data suggest that AP contributes to maintenance of inorganic phosphate homeostasis[64] and may lead to adenosine production and regulation of renovascular tone.[69]
Intestinal AP activity and expression following CPB/DHCA: In this study, we found that exposure to CPB/DHCA resulted in increased ileal expression of iAP mRNA with a similar trend in the colon. We did not find similar changes in the jejunum, suggesting that watershed areas of the intestines may be more highly affected. The increase in mRNA expression was not associated with an increase in AP activity, indicating either concurrent loss of iAP protein or a delay between transcription and translation. Prior studies in murine models of ischemia-reperfusion and colitis demonstrate significant decreases in iAP activity with intestinal injury. Jejunal AP activity decreases after superior mesenteric artery/vein clamping.[53, 54] Decreased intestinal AP activity has also been shown in models of colitis[70] and necrotizing enterocolitis.[71] iAP loss is thought to increase susceptibility to injury and worsen intestinal barrier function, both of which are improved with interventions to increase intestinal AP activity.[70-72] Contrary to our findings, in both vascular clamping and colitis, loss of iAP activity was not balanced by an increase in iAP mRNA expression.[54, 70] Several possibilities could explain this difference, including severity of injury (prolonged vascular clamping compared to the more translationally relevant DHCA), location of sampling (jejunum vs ileum), and complexity of the injury model and the subsequent tissue response. Further studies are needed to better understand the mechanism and time course of these changes in intestinal AP as well as their relationship to injury severity.
Renal AP activity and expressionfollowing CPB/DHCA: Our group has previously published that kidney tissue AP activity is significantly lower in piglets exposed to CPB/DHCA compared to anesthesia controls and that BiAP infusion increases renal AP activity.[49] Here we find that kidney AP mRNA expression is unaffected by CPB/DHCA, indicating that the decrease in renal tissue AP activity is secondary to loss of tissue AP without a concurrent increase in production. Both preclinical models and human studies have demonstrated loss of renal AP activity in a variety of pathologies including ischemia, interstitial nephritis, and obstructive uropathy.[73-75] Preclinical murine models of ischemia-reperfusion with therapeutic AP administration demonstrated improved histologic injury scores and increased cortical paO2,[76] potentially via increased adenosine signaling.[28, 32, 77, 78] To our knowledge, though, only one study has examined AP mRNA expression from kidney tissue. Contrary to our findings in CPB/DHCA, Kapojos et al found increased mesangial cell AP mRNA expression following TNFα stimulation in vitro.[79] The authors did not find similar increases in AP expression in other forms of kidney pathology such as nephritis and acute graft rejection. It is possible that upregulation of AP in the kidney is relatively unique to lipopolysaccharide/TNFα stimulation. Alternatively, the level of early cortical injury induced by CPB/DHCA may limit the ability of proximal tubule and glomerular cells to increase AP production until recovery has occurred.
Lung AP activity and expression following CPB/DHCA: The lung was the only organ we studied where exposure to CPB/DHCA resulted in an increase in both AP activity and TNAP expression, a novel finding that has not been previously reported to our knowledge. Other disease models have used AP as a biomarker of lung injury,[80] but only recently have research efforts focused on understanding the importance of AP in lung pathology. TNAP lines the epithelial/mucosal surface of the lung with a predominance in the lower airways.[51] It is produced primarily by type 2 alveolar cells, although neutrophils may contribute.[52] Lipopolysaccharide and extracellular adenine nucleotides appear to be the most promising candidate targets of pulmonary epithelial AP under pathologic conditions. Intratracheal or intraperitoneal instillation of LPS increases neutrophil production of AP.[81] Ambroxol, a bronchial expectorant known to release AP-containing surfactant particles, increases lung tissue AP and leads to decreased pulmonary and serum LPS following intratracheal instillation of LPS.[19] Extracellular adenine nucleotides are released into the alveolar space during pulmonary infection or other pathologic conditions such as ventilator induced lung injury. There they act as danger-associated molecular patterns, leading to immune activation, capillary leak, and decreased surfactant production.[82] Low levels of extracellular adenine nucleotides in the alveoli are cleared sequentially by CD39 and CD73 to adenosine, but under pathologic conditions these enzymes become saturated[82] and TNAP becomes the primary enzyme responsible for clearance to adenosine.[51] Adenosine production in turn promotes anti-inflammatory signaling through pulmonary purinergic receptors and may also promote healing following acute lung injury.[83] Based on our findings, increased pulmonary production of AP may be part of the host response to the ischemia-reperfusion injury associated with CPB/DHCA. Further studies are required to confirm the primary cell type responsible for this increase in AP production and to understand the clinical importance of increased lung tissue AP.
Effects of AP infusion on tissue AP activity: To our knowledge, changes in tissue-level AP activity following BiAP infusion have not previously been reported. Infusion of our highest dose of BiAP to piglets undergoing CPB/DHCA resulted in a significant increase of total AP activity in kidneys and liver only. Lower doses of AP did not significantly change tissue AP activity in any organ. BiAP infusion did not appear to substantially alter TNAP mRNA expression in any organ. Our high dose regimen used a similar bolus dosing to previous adult human studies (75U/kg vs 67.5U/kg) but a higher continuous infusion (25U/kg/hr vs 5.5-8.3U/kg/hr),[38, 39, 84] which was needed to significantly raise circulating AP activity. While these human studies did not evaluate tissue-level changes, Peters et al did evaluate tissue distribution of a recombinant chimeric human AP molecule (human intestinal AP with the crown replaced by human placental AP) in adult minipigs.[85] Using iodine-125 labeled versions of this recombinant AP, the group demonstrated substantial delivery to the liver, supporting our findings of delivery of BiAP to the liver following CPB/DHCA. Contrary to our study, the authors did not find substantially more delivery to the kidneys compared to the other organs tested. It is possible that there are differences in the organ-specific delivery based on pediatric vs adult animals, infusion dose, disease type, or exact AP molecule administered. Alternatively, we cannot rule out the possibility that infusion of exogenous AP helps preserve native AP activity in the kidneys. This action could potentially occur through systemic dephosphorylation of toxic molecules resulting in less renal tubular epithelial exposure and subsequent decreased tubular epithelial injury. Continued studies are needed to clarify the exact tissue distribution of BiAP following CPB/DHCA.
Limitations: Our study has several limitations. First is the small sample size. Swine models are useful for the study of complex physiology like cardiac surgery and CPB-induced organ injury[86] but have inherently greater variability than murine or cell based models. Therefore, it is possible that we were underpowered to detect more subtle differences in specific organs (type 2 error) that may have been identified with a larger sample size. To maintain study feasibility, we were only able to study a single, acute postoperative time point. Future studies should include serial time points throughout the window of postoperative critical illness (24-48 hours) in order to determine ongoing changes in AP biology and their association with postoperative organ injury. In any animal model, there is the possibility that the metabolism of and physiologic response to a novel therapeutic may not adequately replicate similar responses in humans. We chose to use a porcine model due to the similarities in both the physiologic response to CPB and drug metabolism.[87-92] However, staged clinical trials in children and adults undergoing cardiac surgery are needed to definitively prove both the safety and efficacy of this therapy. While it is possible to differentiate among human AP isoenzymes/isoforms,[46] the previously published lack of available antibodies to exogenous BiAP and native non-human AP makes this differentiation challenging in animal models.[71] Therefore we cannot determine what proportion of changes in AP activity are directly due to BiAP delivery versus prevention of native AP depletion. Use of alternative measurement techniques such as mass spectrometry may be needed to measure changes in specific isoenzymes and to better track organ-specific drug delivery. Finally, this study was not designed to evaluate the impact of these tissue-level changes in AP. Future studies are warranted to evaluate the effects of native and exogenous AP on tissue inflammation and injury.