Mihwa Kim

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 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.

Ischemic Preconditioning Provides Both Acute and Delayed Protection against Renal Ischemia and Reperfusion Injury in Mice

Acute as well as delayed ischemic preconditioning (IPC) provides protection against cardiac and neuronal ischemia reperfusion (IR) injury. This study determined whether delayed preconditioning occurs in the kidney and further elucidated the mechanisms of renal IPC in mice. Mice were subjected to IPC (four cycles of 5 min of ischemia and reperfusion) and then to 30 min of renal ischemia either 15 min (acute IPC) or 24 h (delayed IPC) later. Both acute and delayed renal IPC provided powerful protection against renal IR injury. Inhibition of Akt but not extracellular signal-regulated kinase phosphorylation prevented the protection that was afforded by acute IPC. Neither extracellular signal-regulated kinase nor Akt inhibition prevented protection that was afforded by delayed renal IPC. Pretreatment with an antioxidant, N-(2-mercaptopropionyl)-glycine, to scavenge free radicals prevented the protection that was provided by acute but not delayed renal IPC. Inhibition of protein kinase C or pertussis toxin-sensitive G-proteins attenuated protection from both acute and delayed renal IPC. Delayed renal IPC increased inducible nitric oxide synthase (iNOS) as well as heat-shock protein 27 synthesis, and the renal protective effects of delayed preconditioning were attenuated by a selective inhibitor of iNOS (l-N(6)[1-iminoethyl]lysine). Moreover, delayed IPC was not observed in iNOS knockout mice. Both acute and delayed IPC were independent of A(1) adenosine receptors (AR) as a selective A(1)AR antagonist failed to block preconditioning and acute and delayed preconditioning occurred in mice that lacked A(1)AR. Therefore, this study demonstrated that acute or delayed IPC provides renal protection against IR injury in mice but involves distinct signaling pathways.

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.

α2-Adrenergic agonists protect against radiocontrast-induced nephropathy in mice

Radiocontrast nephropathy (RCN) is a common clinical problem for which there is no effective therapy. Utilizing a murine model, we tested the hypothesis that alpha(2)-adrenergic receptor agonists (clonidine and dexmedetomidine) protect against RCN induced with iohexol (a nonionic low-osmolar radiocontrast). C57BL/6 mice were pretreated with saline, clonidine, or dexmedetomidine before induction of RCN. Some mice were pretreated with yohimbine (a selective alpha(2)-receptor antagonist) before saline, clonidine, or dexmedetomidine administration. alpha(2)-Agonist-treated mice had reduced plasma creatinine, renal tubular necrosis, renal apoptosis, and renal cortical proximal tubule vacuolization 24 h after iohexol injection. Yohimbine reversed the protective effects of clonidine and dexmedetomidine pretreatment. Injection of iohexol resulted in a rapid ( approximately 90 min) fall of renal outer medullary blood flow. Clonidine and dexmedetomidine pretreatment significantly attenuated this perfusion decrease without changing systemic blood pressure. To determine whether proximal tubular alpha(2)-adrenergic receptors mediate the cytoprotective effects, we treated cultured human proximal tubule (HK-2) cells and rat pulmonary microvascular endothelial cells with iohexol after vehicle, clonidine, or dexmedetomidine pretreatment. Iohexol caused a direct dose-dependent reduction of HK-2 and rat pulmonary microvascular endothelial cell viability, but alpha(2)-agonists failed to preserve the viability of both cell types. We conclude that alpha(2)-adrenergic receptor agonists protect mice against RCN by preserving outer medullary renal blood flow. As alpha(2)-agonists are widely utilized during the perioperative period, our findings may have significant clinical relevance to improving outcomes following radiocontrast exposure.

Urinary neutrophil gelatinase-associated lipocalin as a marker of acute kidney injury after orthotopic liver transplantation

BACKGROUND Urinary neutrophil gelatinase-associated lipocalin (NGAL) is a novel, sensitive and specific biomarker that is rapidly released after kidney injury. It predicts acute kidney injury (AKI) in multiple clinical scenarios. We hypothesized that urinary NGAL can predict AKI after liver transplantation. METHODS Urine was collected in 92 patients undergoing liver transplantation (18 living-related and 74 deceased) before surgery, after reperfusion of the liver graft and then 3, 18 and 24 h later. NGAL was analyzed with enzyme-linked immunosorbent assay and corrected for dilution/concentration by calculating urinary NGAL/urine creatinine ratios. AKI was defined by Risk-Injury-Failure-Loss-Endstage stage kidney disease (RIFLE)-risk criteria (increase of serum creatinine by >50%). RESULTS Urinary NGAL/urine creatinine ratio was low prior to surgery and increased immediately after reperfusion, peaked 3 h later and remained elevated at 18 and 24 h. Urinary NGAL/urine creatinine ratios were higher in patients with post-operative (post-OP) AKI defined by RIFLE--risk criteria 3 and 18 h after reperfusion. The area under the curve of the receiver operator characteristics curve of urinary NGAL/urine creatinine ratio to predict AKI was 0.800 (95% CI: 0.732-0.869, P < 0.0001) 3 h and 0.636 (95% CI: 0.551-0.720, P < 0.005) 18 h after reperfusion. CONCLUSIONS We conclude that urinary NGAL/urine creatinine ratio is able to predict post-OP AKI 3 and 18 h after transplantation with good discrimination.

Kidney-specific reconstitution of the A1 adenosine receptor in A1 adenosine receptor knockout mice reduces renal ischemia-reperfusion injury.

Genetic deletion of the adenosine A1 receptor (A1AR) increased renal injury following ischemia-reperfusion injury suggesting that receptor activation is protective in vivo. Here we tested this hypothesis by expressing the human-A(1)AR in A(1)AR knockout mice. Renal ischemia-reperfusion was induced in knockout mice 2 days after intrarenal injection of saline or a lentivirus encoding enhanced green fluorescent protein (EGFP) or EGFP-human-A(1)AR. We found that the latter procedure induced a robust expression of the reporter protein in the kidneys of knockout mice. Mice with kidney-specific human-A(1)AR reconstitution had significantly lower plasma creatinine, tubular necrosis, apoptosis, and tubular inflammation as evidenced by decreased leukocyte infiltration, pro-inflammatory cytokine, and intercellular adhesion molecule-1 expression in the kidney following injury compared to mice injected with saline or the control lentivirus. Additionally, there were marked disruptions of the proximal tubule epithelial filamentous (F)-actin cytoskeleton in both sets of control mice upon renal injury, whereas the reconstituted mice had better preservation of the renal tubule actin cytoskeleton, which co-localized with the human-A(1)ARs. Consistent with reduced renal injury, there was a significant increase in heat shock protein-27 expression, also co-localizing with the preserved F-actin cytoskeleton. Our findings suggest that selective expression of cytoprotective A(1)ARs in the kidney can attenuate renal injury.

Paneth cell-derived interleukin-17A causes multiorgan dysfunction after hepatic ischemia and reperfusion injury.

Hepatic ischemia and reperfusion (IR) injury is a major clinical problem that leads to frequent extrahepatic complications including intestinal dysfunction and acute kidney injury (AKI). In this study we aimed to determine the mechanisms of hepatic IR‐induced extrahepatic organ dysfunction. Mice subjected to 60 minutes of hepatic IR not only developed severe hepatic injury but also developed significant AKI and small intestinal injury. Hepatic IR induced small intestinal Paneth cell degranulation and increased interleukin‐17A (IL‐17A) levels in portal vein plasma and small intestine. We also detected increased levels of IL‐17A messenger RNA (mRNA) and protein in Paneth cells after hepatic IR with laser capture dissection. IL‐17A‐neutralizing antibody treatment or genetic deletion of either IL‐17A or IL‐17A receptors significantly protected against hepatic IR‐induced acute liver, kidney, and intestinal injury. Leukocyte IL‐17A does not contribute to organ injury, as infusion of wildtype splenocytes failed to exacerbate liver and kidney injury in IL‐17A‐deficient mice after hepatic IR. Depletion of Paneth cell numbers by pharmacological (with dithizone) or genetic intervention (SOX9 flox/flox Villin cre+/− mice) significantly attenuated intestinal, hepatic, and renal injury following liver IR. Finally, depletion of Paneth cell numbers significantly decreased small intestinal IL‐17A release and plasma IL‐17A levels after liver IR. Conclusion: Taken together, the results show that Paneth cell‐derived IL‐17A plays a critical role in hepatic IR injury and extrahepatic organ dysfunction. Modulation of Paneth cell dysregulation may have therapeutic implications by reducing systemic complications arising from hepatic IR. (HEPATOLOGY 2011;)

Increased Incidence of Acute Kidney Injury with Aprotinin Use during Cardiac Surgery Detected with Urinary NGAL

Background: Use of aprotinin has been associated with acute kidney injury after cardiac surgery. Neutrophil gelatinase-associated lipocalin (NGAL) is a novel, very sensitive marker for renal injury. Urinary NGAL may be able to detect renal injury caused by aprotinin. This study determined if the use of aprotinin is associated with an increased incidence of acute kidney injury and increased levels of urinary NGAL. Methods: In this prospective, observational study 369 patients undergoing cardiac surgery were enrolled. 205 patients received aprotinin and 164 received epsilon amino-caproic acid intraoperatively. Urinary NGAL was measured before and immediately after cardiac surgery and 3, 18 and 24 h later. The association of aprotinin use with the incidence of acute kidney injury (increase of serum creatinine >0.5 mg/dl) and NGAL levels was determined using logistic and linear regression models. Results: 51 of 205 patients (25%) who received aprotinin developed acute kidney injury compared to 19 of 164 patients (12%) who received epsilon amino-caproic acid (p = 0.0013). Aprotinin use was associated with a two-fold higher risk of acute kidney injury when adjusted for potential confounders (age, Parsonnet score, preoperative serum creatinine, cardiopulmonary bypass and cross-clamp times; multiple logistic regression: OR = 2.164; CI (95%) = 1.102 to 4.249; p = 0.0249. Urinary NGAL was 19 times higher immediately after cardiopulmonary bypass and 18 times higher 3 h later in patients who had received aprotinin (postoperative: 19.23; CI (95%) = 12.60 to 29.33; p < 0.0001; 3 h post-cardiopulmonary bypass 18.67; CI (95%) = 11.45 to 30.43; p < 0.0001). Conclusions: Postoperative urinary NGAL – a novel marker for renal injury – is increased in cardiac surgical patients receiving aprotinin compared to patients receiving epsilon amino-caproic acid. These results further support the hypothesis that aprotinin may cause renal injury. The substantial rise of urinary NGAL associated with aprotinin use may in part be due to aprotinin blocking the uptake of NGAL by megalin/gp330 receptors in the proximal tubules.