H. Thomas Lee

Endocytic delivery of lipocalin-siderophore-iron complex rescues the kidney from ischemia-reperfusion injury

Neutrophil gelatinase-associated lipocalin (Ngal), also known as siderocalin, forms a complex with iron-binding siderophores (Ngal:siderophore:Fe). This complex converts renal progenitors into epithelial tubules. In this study, we tested the hypothesis that Ngal:siderophore:Fe protects adult kidney epithelial cells or accelerates their recovery from damage. Using a mouse model of severe renal failure, ischemia-reperfusion injury, we show that a single dose of Ngal (10 microg), introduced during the initial phase of the disease, dramatically protects the kidney and mitigates azotemia. Ngal activity depends on delivery of the protein and its siderophore to the proximal tubule. Iron must also be delivered, since blockade of the siderophore with gallium inhibits the rescue from ischemia. The Ngal:siderophore:Fe complex upregulates heme oxygenase-1, a protective enzyme, preserves proximal tubule N-cadherin, and inhibits cell death. Because mouse urine contains an Ngal-dependent siderophore-like activity, endogenous Ngal might also play a protective role. Indeed, Ngal is highly accumulated in the human kidney cortical tubules and in the blood and urine after nephrotoxic and ischemic injury. We reveal what we believe to be a novel pathway of iron traffic that is activated in human and mouse renal diseases, and it provides a unique method for their treatment.

Association between Increases in Urinary Neutrophil Gelatinase–associated Lipocalin and Acute Renal Dysfunction after Adult Cardiac Surgery

Background:Acute renal dysfunction (ARD) and subsequent acute renal failure after cardiac surgery are associated with high mortality and morbidity. Early therapeutic or preventive intervention is hampered by the lack of an early biomarker for acute renal injury. Recent studies showed that urinary neutrophil gelatinase–associated lipocalin (NGAL or lipocalin 2) is up-regulated early (within 1–3 h) after murine renal injury and in pediatric ARD after cardiac surgery. The authors hypothesized that postoperative urinary NGAL concentrations are increased in adult patients developing ARD after cardiac surgery compared with patients without ARD. Methods:After institutional review board approval, 81 cardiac surgical patients were prospectively studied. Urine samples were collected immediately before incision and at various time intervals after surgery for NGAL analysis by quantitative immunoblotting. ARD was defined as peak postoperative serum creatinine increase by 50% or greater compared with preoperative serum creatinine. Results:Sixteen of 81 patients (20%) developed postoperative ARD, and the mean urinary NGAL concentrations in patients who developed ARD were significantly higher early after surgery (after 1 h: 4,195 ± 6,520 [mean ± SD] vs. 1,068 ± 2,129 ng/ml; P < 0.01) compared with patients who did not develop ARD. Mean urinary NGAL concentrations continued to increase and remained significantly higher at 3 and 18 h after cardiac surgery in patients with ARD. In contrast, urinary NGAL in patients without ARD decreased rapidly after cardiac surgery. Conclusions:Patients developing postoperative ARD had significantly higher urinary NGAL concentrations early after cardiac surgery. Urinary NGAL may therefore be a useful early biomarker of ARD after cardiac surgery. These findings may facilitate the early detection of acute renal injury and potentially prevent progression to acute renal failure.

Urinary Biomarkers in the Early Detection of Acute Kidney Injury after Cardiac Surgery

BACKGROUND AND OBJECTIVES Serum creatinine (Scr) does not allow for early diagnosis of acute kidney injury (AKI). The diagnostic utility of urinary kidney injury molecule-1 (KIM-1), N-acetyl-beta-D-glucosaminidase (NAG), and neutrophil gelatinase associated lipocalin (NGAL) was evaluated for the early detection of postoperative AKI in a prospective study of 90 adults undergoing cardiac surgery. Designs, setting, participants, & measurements: Urinary KIM-1, NAG, and NGAL were measured at 5 time points for the first 24 h after operation and normalized to the urinary creatinine concentration after cardiac surgery. Receiver-operating characteristic curves were generated and the areas under the curve (AUCs) compared for performance of biomarkers in detection of postoperative AKI. RESULTS Thirty-six patients developed AKI, defined as an increase in Scr of > or =0.3 mg/dl within 72 h after surgery. The AUCs for KIM-1 to predict AKI immediately and 3 h after operation were 0.68 and 0.65; 0.61 and 0.63 for NAG; and 0.59 and 0.65 for NGAL, respectively. Combining the three biomarkers enhanced the sensitivity of early detection of postoperative AKI compared with individual biomarkers: the AUCs for the three biomarkers combined were 0.75 and 0.78. The performance of combining biomarkers was even better among 16 early postoperative AKI patients with AUCs of 0.80 and 0.84, respectively. CONCLUSIONS The results of this study support that a combination of urinary biomarkers may allow for early detection of postoperative AKI after cardiac surgery before a rise in Scr.

Urinary Neutrophil Gelatinase-Associated Lipocalin and Acute Kidney Injury After Cardiac Surgery

BACKGROUND Neutrophil gelatinase-associated lipocalin (NGAL) is proposed as an early marker of kidney injury. We report the association of urinary NGAL with indexes of intraoperative renal hypoperfusion (cardiopulmonary bypass time and aortic cross-clamp time) and acute kidney injury (AKI) after adult cardiac surgery. STUDY DESIGN Diagnostic test accuracy. SETTING & PARTICIPANTS Adult cardiac surgical patients (n = 426) in a single center from 2004 to 2006. INDEX TEST Urinary NGAL immediately and 3, 18, and 24 hours after cardiac surgery, using an enzyme-linked immunosorbent assay. REFERENCE TEST OR OUTCOME Serum creatinine-based definition for AKI (increase in serum creatinine from preoperative values by >50% or >0.3 mg/dL within 48 hours). RESULTS Mean urinary NGAL level was 165 +/- 663 (SD) ng/mL preoperatively, peaked immediately after cardiac surgery at 1,490 +/- 102 ng/mL, and remained significantly higher 3, 18, and 24 hours after surgery. 85 patients (20%) developed AKI. Areas under the receiver operating characteristic curve for urinary NGAL immediately after and 3, 18, and 24 hours later as a predictor for AKI were 0.573 (95% confidence interval [CI], 0.506 to 0.640), 0.603 (95% CI, 0.533 to 0.674), 0.611 (95% CI, 0.544 to 0.679), and 0.584 (95% CI, 0.510 to 0.657), respectively. Urinary NGAL, but not serum creatinine, level correlated significantly with cardiopulmonary bypass and aortic cross-clamp times. Areas under receiver operating characteristic curves for cardiopulmonary bypass time and aortic cross-clamp time to predict AKI were 0.592 (95% CI, 0.518 to 0.666) and 0.593 (95% CI, 0.523 to 0.665), respectively. LIMITATIONS Limited sensitivity of changes in serum creatinine levels for kidney injury. CONCLUSIONS Urinary NGAL has limited diagnostic accuracy to predict AKI defined by change in serum creatinine after cardiac surgery.

Differential protective effects of Volatile anesthetics against renal ischemia-reperfusion injury in vivo

Background:Volatile anesthetics protect against cardiac ischemia–reperfusion injury via adenosine triphosphate–dependent potassium channel activation. The authors questioned whether volatile anesthetics can also protect against renal ischemia–reperfusion injury and, if so, whether cellular adenosine triphosphate–dependent potassium channels, antiinflammatory effects of volatile anesthetics, or both are involved. Methods:Rats were anesthetized with equipotent doses of volatile anesthetics (desflurane, halothane, isoflurane, or sevoflurane) or injectable anesthetics (pentobarbital or ketamine) and subjected to 45 min of renal ischemia and 3 h of reperfusion during anesthesia. Results:Rats treated with volatile anesthetics had lower plasma creatinine and reduced renal necrosis 24–72 h after injury compared with rats anesthetized with pentobarbital or ketamine. Twenty-four hours after injury, sevoflurane-, isoflurane-, or halothane-treated rats had creatinine (± SD) of 2.3 ± 0.7 mg/dl (n = 12), 1.8 ± 0.5 mg/dl (n = 6), and 2.4 ± 1.2 mg/dl (n = 6), respectively, compared with rats treated with pentobarbital (5.8 ± 1.2 mg/dl, n = 9) or ketamine (4.6 ± 1.2 mg/dl, n = 8). Among the volatile anesthetics, desflurane demonstrated the least reduction in plasma creatinine after 24 h (4.1 ± 0.8 mg/dl, n = 12). Renal cortices from volatile anesthetic–treated rats demonstrated reduced expression of intercellular adhesion molecule 1 protein and messenger RNA as well as messenger RNAs encoding proinflammatory cytokines and chemokines. Volatile anesthetic treatment reduced renal cortex myeloperoxidase activity and reduced nuclear translocation of proinflammatory nuclear factor &kgr;B. Adenosine triphosphate–dependent potassium channels are not involved in sevoflurane-mediated renal protection because glibenclamide did not block renal protection (creatinine: 2.4 ± 0.4 mg/dl, n = 3). Conclusion:Some volatile anesthetics confer profound protection against renal ischemia–reperfusion injury compared with pentobarbital or ketamine anesthesia by attenuating inflammation. These findings may have significant clinical implications for anesthesiologists regarding the choice of volatile anesthetic agents in patients subjected to perioperative renal ischemia.

A1 Adenosine Receptor Activation Inhibits Inflammation, Necrosis, and Apoptosis after Renal Ischemia-Reperfusion Injury in Mice

It was previously demonstrated that preischemic A(1) adenosine receptor (AR) activation protects renal function after ischemia-reperfusion (IR) injury in rats. The role of the A(1) AR in modulating inflammation, necrosis, and apoptosis in the kidney after IR renal injury was further characterized. C57BL/6 mice were subjected to 30 min of renal ischemia, with or without pretreatment with 1,3-dipropyl-8-cyclopentylxanthine or 2- chlorocyclopentyladenosine (selective A(1) AR antagonist and agonist, respectively). Plasma creatinine levels and renal inflammation, necrosis, and apoptosis were compared 24 h after renal injury. C57BL/6 mice that had been pretreated with the A(1) AR agonist demonstrated significantly improved renal function and reduced expression of inflammatory markers, necrosis, and apoptosis 24 h after IR injury. In contrast, C57BL/6 mice that had been pretreated with the A(1) AR antagonist demonstrated significantly worsened renal function and increased expression of inflammatory markers, necrosis, and apoptosis. In conclusion, it was demonstrated that endogenous and exogenous preischemic activation of the A(1) AR protects against IR injury in vivo, through mechanisms that reduce inflammation, necrosis, and apoptosis.

Pretreatment of Human Myocardium with Adenosine During Open Heart Surgery

Background: Depressed myocardial performance after cardiac surgery can be contributed to ischemic reperfusion injury (IRI) incurred during and following the cardiopulmonary bypass (CPB). Myocardial preconditioning (PC) achieved by brief ischemia and subsequent reperfusion appears to be a clinically useful method of improved cardiac protection during surgery involving CPB by retarding IRI. Based on animal studies, activation of cardiac adenosine (ADO) receptors prior to the prolonged ischemic period appears to mimic this PC phenomenon. Aims and Methods: We investigated whether the human myocardial PC can be mimicked with ADO in the setting of the coronary artery bypass graft (CABG). The specific proposed objective of this study was to determine whether ADO infusion just prior to starting the CPB can improve post‐CPB myocardial hemodynamics. Patients undergoing elective CABG with poor ventricular function (ejection fraction ± 30%), and with at least three‐vessel disease were selected for this study (n = 7 ADO, and n = 7 control). Results: Our results show that ADO infusion (250–350 μg/kg ± 10 min) just prior to CPB resulted in an immediately improved postbypass cardiac index (Cl) in the OR (Cl increase of 41.5%± 11.1% for ADO vs 9.7%± 6.0% for control, p < 0.05). Forty hours postoperatively in the intensive care unit, ADO patients had improved Cl (3.3 ± 0.2 L/min per m2 for ADO, vs 2.6 ± 0.1 L/min per m2 for control, p < 0.05), stroke volume index (35 ± 3 mL/m2 per beat for ADO, vs 26 ± 4 L/min per m2 for control, p < 0.05). ADO patients maintained lowered resting heart rate (90 ± 6 for ADO, vs 108 ± 4 for control, p < 0.05) 40 hours after the surgery. ADO patients also released significantly less CPK during the first 24 hours of the postoperative period. Conclusion: Based on these measurements, ADO pretreated patients had improved ventricular performance postoperatively. It also appears that ADO pretreatment results in lowered postoperative myocardial energy demand and less myocellular injury during CPB. To our knowledge, this is the first study to demonstrate that human myocardium can be hemodynamically improved with ADO pretreatment, and may be protected against IRI incurred during and following the CPB. We believe that a cardiac surgeon may now have the unique opportunity to confer myocardial protection during and after a cardiac surgical procedure.

Cytokines induce small intestine and liver injury after renal ischemia or nephrectomy

Patients with acute kidney injury (AKI) frequently suffer from extra-renal complications including hepatic dysfunction and systemic inflammation. We aimed to determine the mechanisms of AKI-induced hepatic dysfunction and systemic inflammation. Mice subjected to AKI (renal ischemia reperfusion (IR) or nephrectomy) rapidly developed acute hepatic dysfunction and suffered significantly worse hepatic IR injury. After AKI, rapid peri-portal hepatocyte necrosis, vacuolization, neutrophil infiltration and pro-inflammatory mRNA upregulation were observed suggesting an intestinal source of hepatic injury. Small intestine histology after AKI showed profound villous lacteal capillary endothelial apoptosis, disruption of vascular permeability and epithelial necrosis. After ischemic or non-ischemic AKI, plasma TNF-α, IL-17A and IL-6 increased significantly. Small intestine appears to be the source of IL-17A, as IL-17A levels were higher in the portal circulation and small intestine compared with the levels measured from the systemic circulation and liver. Wild-type mice treated with neutralizing antibodies against TNF-α, IL-17A or IL-6 or mice deficient in TNF-α, IL-17A, IL-17A receptor or IL-6 were protected against hepatic and small intestine injury because of ischemic or non-ischemic AKI. For the first time, we implicate the increased release of IL-17A from small intestine together with induction of TNF-α and IL-6 as a cause of small intestine and liver injury after ischemic or non-ischemic AKI. Modulation of the inflammatory response and cytokine release in the small intestine after AKI may have important therapeutic implications in reducing complications arising from AKI.

Acute kidney injury after hepatic ischemia and reperfusion injury in mice

Hepatic ischemia reperfusion (IR) is the leading cause of acute liver failure (ALF) during the perioperative period and patients with ALF frequently develop acute kidney injury (AKI). There is no effective therapy for AKI associated with ALF because pathomechanisms are incompletely characterized, in part due to the lack of an animal model. In this study, we characterize a novel murine model of AKI following hepatic IR. Mice subjected to ∼70% liver IR not only developed acute liver dysfunction, but also developed severe AKI 24 h after liver injury. Mice subjected to liver IR developed histological changes of acute tubular injury including focal proximal tubular cell necrosis involving the S3 segment, cortical tubular ectasia, focal tubular simplification and granular bile/heme cast formation. In addition, there was focal interstitial edema and hyperplasia of the juxtaglomerular apparatus. Inflammatory changes in the kidney after hepatic IR included neutrophil infiltration of the interstitium and upregulation of several proinflammatory mRNAs (tumor necrosis factor-α, keratinocyte-derived cytokine, monocyte chemotactic protein-1, macrophage inflammatory protein-2, intercellular adhesion molecule-1). In addition, marked renal endothelial cell apoptosis was detected involving peritubular interstitial capillaries, accompanied by increased renal vascular permeability. Finally, there was severe disruption of renal proximal tubule epithelial filamentous-actin. Our results show that AKI rapidly and reproducibly develops in mice after hepatic IR and is characterized by renal tubular necrosis, inflammatory changes and interstitial capillary endothelial apoptosis. Our murine model of AKI after liver injury closely mimics human AKI associated with ALF and may be useful in delineating the mechanisms and potential therapies for this common clinical condition.

Adenosine Generation and Signaling during Acute Kidney Injury

Acute kidney injury (AKI) is among the leading causes of morbidity and mortality in hospitalized patients. Particularly in the perioperative period, the most common cause of AKI is renal ischemia. At present, therapeutic modalities to prevent or treat AKI are extremely limited and the search for novel therapeutic interventions for ischemic AKI is an area of intense investigation. Recent studies implicate the endogenous signaling molecule, adenosine, in kidney protection from ischemia. As such, enzymatic production of adenosine from its precursor molecules ATP and AMP, and signaling events through adenosine receptors, play a critical role in attenuating renal inflammation and preserving kidney function during episodes of renal ischemia. Utilizing genetic mouse models with defects in adenosine generation or signaling provide strong evidence for the key role of extracellular adenosine in adapting renal tissues to limited oxygen availability and attenuating hypoxia-driven inflammation of the kidneys. Moreover, experimental therapeutics targeting individual adenosine receptors demonstrate strong prophylactic or therapeutic effects during murine AKI. If these experimental strategies can be translated into a clinical setting, adenosine receptor therapeutics may become an integral part in the prevention or treatment of AKI from renal ischemia.