IL-6-mediated hepatocyte production is the primary source of plasma and urine neutrophil gelatinase–associated lipocalin during acute kidney injury
Authors: Nataliya I. Skrypnyk, Katja M. Gist, Kayo Okamura, John R. Montford, Zhiying You, Haichun Yang, Radu Moldovan, Evelyn Bodoni, Judith T. Blaine, Charles L. Edelstein, Danielle E. Soranno, Lara A. Kirkbride-Romeo, Benjamin R. Griffin, Chris Altmann, Sarah Faubel
Journal: Kidney International (2019)

Neutrophil gelatinase associated lipocalin (NGAL, Lcn2) is the most widely studied biomarker of acute kidney injury (AKI). Previous studies have demonstrated that NGAL is produced by the kidney and released into the urine and plasma. Consequently, NGAL is currently considered a tubule specific injury marker of AKI. However, the utility of NGAL to predict AKI has been variable suggesting that other mechanisms of production are present. IL-6 is a proinflammatory cytokine increased in plasma by two hours of AKI and mediates distant organ effects. Herein, we investigated the role of IL-6 in renal and extra-renal NGAL production. Wild type mice with ischemic AKI had increased plasma IL-6, increased hepatic NGAL mRNA, increased plasma NGAL, and increased urine NGAL; all reduced in IL-6 knockout mice. Intravenous IL-6 in normal mice increased hepatic NGAL mRNA, plasma NGAL and urine NGAL. In mice with hepatocyte specific NGAL deletion (Lcn2hep-/-) and ischemic AKI, hepatic NGAL mRNA was absent, and plasma and urine NGAL were reduced. Since urine NGAL levels appear to be dependent on plasma levels, the renal handling of circulating NGAL was examined using recombinant human NGAL. After intravenous recombinant human NGAL administration to mice, human NGAL in mouse urine was detected by ELISA during proximal tubular dysfunction, but not in pre-renal azotemia. Thus, during AKI, IL-6 mediates hepatic NGAL production, hepatocytes are the primary source of plasma and urine NGAL, and plasma NGAL appears in the urine during proximal tubule dysfunction. Hence, our data change the paradigm by which NGAL should be interpreted as a biomarker of AKI. Neutrophil gelatinase associated lipocalin (NGAL, Lcn2) is the most widely studied biomarker of acute kidney injury (AKI). Previous studies have demonstrated that NGAL is produced by the kidney and released into the urine and plasma. Consequently, NGAL is currently considered a tubule specific injury marker of AKI. However, the utility of NGAL to predict AKI has been variable suggesting that other mechanisms of production are present. IL-6 is a proinflammatory cytokine increased in plasma by two hours of AKI and mediates distant organ effects. Herein, we investigated the role of IL-6 in renal and extra-renal NGAL production. Wild type mice with ischemic AKI had increased plasma IL-6, increased hepatic NGAL mRNA, increased plasma NGAL, and increased urine NGAL; all reduced in IL-6 knockout mice. Intravenous IL-6 in normal mice increased hepatic NGAL mRNA, plasma NGAL and urine NGAL. In mice with hepatocyte specific NGAL deletion (Lcn2hep-/-) and ischemic AKI, hepatic NGAL mRNA was absent, and plasma and urine NGAL were reduced. Since urine NGAL levels appear to be dependent on plasma levels, the renal handling of circulating NGAL was examined using recombinant human NGAL. After intravenous recombinant human NGAL administration to mice, human NGAL in mouse urine was detected by ELISA during proximal tubular dysfunction, but not in pre-renal azotemia. Thus, during AKI, IL-6 mediates hepatic NGAL production, hepatocytes are the primary source of plasma and urine NGAL, and plasma NGAL appears in the urine during proximal tubule dysfunction. Hence, our data change the paradigm by which NGAL should be interpreted as a biomarker of AKI. Translational StatementAcute kidney injury (AKI) is the most common focus of inpatient consultation with nephrologists, and it is associated with increased morbidity and mortality. Current tools to identify and classify AKI are inadequate. Herein, we extensively characterize the production and renal handling of one of the most widely studied AKI biomarkers—neutrophil gelatinase–associated lipocalin (NGAL)—and demonstrate several new insights into its production and renal handling that may improve its accuracy to identify AKI and distinguish between pre-renal azotemia and acute tubular injury, which have major differences in the approach to clinical care. Given that we demonstrate that the major source of urine NGAL is the plasma and that urine NGAL increases during proximal tubule dysfunction, future studies should incorporate methods to account for plasma NGAL levels in patients with adjudicated pre-renal azotemia and acute tubular injury. Acute kidney injury (AKI) is the most common focus of inpatient consultation with nephrologists, and it is associated with increased morbidity and mortality. Current tools to identify and classify AKI are inadequate. Herein, we extensively characterize the production and renal handling of one of the most widely studied AKI biomarkers—neutrophil gelatinase–associated lipocalin (NGAL)—and demonstrate several new insights into its production and renal handling that may improve its accuracy to identify AKI and distinguish between pre-renal azotemia and acute tubular injury, which have major differences in the approach to clinical care. Given that we demonstrate that the major source of urine NGAL is the plasma and that urine NGAL increases during proximal tubule dysfunction, future studies should incorporate methods to account for plasma NGAL levels in patients with adjudicated pre-renal azotemia and acute tubular injury. Acute kidney injury (AKI) is a common complication in hospitalized patients, occurring in up to 20% of hospital admissions and 30% to 50% of intensive care unit admissions. AKI is an independent risk factor for death, with mortality rates as high as 50% in the intensive care unit.1Palevsky P.M. Zhang J.H. O'Connor T.Z. et al.Intensity of renal support in critically ill patients with acute kidney injury.New Engl J Med. 2008; 359: 7-20Crossref PubMed Scopus (1196) Google Scholar The development of biomarkers that can identify AKI early and more reliably than serum creatinine rise has been a translational research priority for over a decade with the premise that early recognition may lead to improved care and the development of new therapies.2American Society of NephrologyRenal research report.J Am Soc Nephrol. 2005; 16: 1886-1903Crossref PubMed Scopus (158) Google Scholar Neutrophil gelatinase–associated lipocalin (NGAL) (also known as lipocalin 2) is one of the most widely studied AKI biomarkers, having been tested in thousands of patients.3Nickolas T.L. Schmidt-Ott K.M. Canetta P. et al.Diagnostic and prognostic stratification in the emergency department using urinary biomarkers of nephron damage: a multicenter prospective cohort study.J Am Coll Cardiol. 2012; 59: 246-255Crossref PubMed Scopus (228) Google Scholar, 4Parikh C.R. Devarajan P. Zappitelli M. et al.Postoperative biomarkers predict acute kidney injury and poor outcomes after pediatric cardiac surgery.J Am Soc Nephrol. 2011; 22: 1737-1747Crossref PubMed Scopus (256) Google Scholar, 5Kim S. Kim H.J. Ahn H.S. et al.Is plasma neutrophil gelatinase-associated lipocalin a predictive biomarker for acute kidney injury in sepsis patients? A systematic review and meta-analysis.J Crit Care. 2016; 33: 213-223Crossref PubMed Scopus (27) Google Scholar NGAL has been considered an advantageous AKI biomarker6Schmidt-Ott K.M. Neutrophil gelatinase-associated lipocalin as a biomarker of acute kidney injury—where do we stand today?.Nephrol Dial Transplant. 2011; 26: 762-764Crossref PubMed Scopus (69) Google Scholar because it is reported to be a kidney-specific marker of AKI that is produced by the kidney during tubular injury.7Paragas N. Qiu A. Zhang Q. et al.The Ngal reporter mouse detects the response of the kidney to injury in real time.Nat Med. 2011; 17: 216-222Crossref PubMed Scopus (297) Google Scholar In addition, it is not influenced by systemic diseases, and it appears in the plasma and urine within 2 hours of injury after being released by injured tubules.8Mishra J. Dent C. Tarabishi R. et al.Neutrophil gelatinase-associated lipocalin (NGAL) as a biomarker for acute renal injury after cardiac surgery.Lancet. 2005; 365: 1231-1238Abstract Full Text Full Text PDF PubMed Scopus (1794) Google Scholar However, the performance of NGAL in predicting AKI has been variable, possibly because it is produced by cell types outside of the kidney or in response to other stimuli such as systemic inflammation and circulating cytokines.9Xiao X. Yeoh B.S. Vijay-Kumar M. Lipocalin 2: an emerging player in iron homeostasis and inflammation.Annu Rev Nutr. 2017; 37: 103-130Crossref PubMed Scopus (86) Google Scholar Interleukin (IL)-6 is a proinflammatory cytokine that increases within 2 hours in the plasma in patients and mice after AKI10Dennen P. Altmann C. Kaufman J. et al.Urine interleukin-6 is an early biomarker of acute kidney injury in children undergoing cardiac surgery.Crit Care. 2010; 14: R181Crossref PubMed Scopus (50) Google Scholar,11Klein C.L. Hoke T.S. Fang W.F. et al.Interleukin-6 mediates lung injury following ischemic acute kidney injury or bilateral nephrectomy.Kidney Int. 2008; 74: 901-909Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar and thus might play a role in NGAL production. In the present study, we investigated the role of IL-6 in extrarenal NGAL production during AKI. To determine the effect of IL-6 on plasma and urine NGAL levels during AKI, wild-type (WT) or IL-6–/– mice were subjected to sham (surgery/laparotomy), ischemic AKI (laparotomy with bilateral renal pedicle clamping), or BNx (laparotomy with removal of both kidneys). Normal mice were studied as a control for all 3 procedures. No kidney injury occurred after sham in either WT or IL-6–/–; kidney injury was similar after ischemic AKI in WT and IL-6–/– (as previously reported11Klein C.L. Hoke T.S. Fang W.F. et al.Interleukin-6 mediates lung injury following ischemic acute kidney injury or bilateral nephrectomy.Kidney Int. 2008; 74: 901-909Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar); blood urea nitrogen and plasma creatinine were similar in WT and IL-6–/– with BNx (Figure 1). Plasma IL-6 was increased in WT 4 hours after sham, AKI, and bilateral nephrectomy, and absent in IL-6–/–; plasma NGAL was increased in WT and reduced in IL-6–/– versus WT at both 4 and 24 hours after sham, AKI, and BNx; urine NGAL was increased 4 and 24 hours after sham and AKI in WT and reduced in IL-6–/– versus WT at both time points and in both conditions (Figure 2).Figure 2Plasma neutrophil gelatinase–associated lipocalin (NGAL) is reduced in interleukin (IL)-6–deficient (IL-6–/–) mice after sham (surgery alone), ischemic acute kidney injury (AKI), and bilateral nephrectomy (BNx) at 4 and 24 hours post-procedure. Plasma IL-6, plasma NGAL, and urine NGAL were determined in normal (unmanipulated) mice, and after sham, ischemic AKI, or BNx at 4 and 24 hours in wild-type (WT) and IL-6–/– mice. Plasma IL-6 was increased in WT mice, 4 and 24 hours after (a) sham, (b) AKI, and (c) BNx. Plasma NGAL was significantly reduced in IL-6–/– mice at 4 and 24 hours after (d) sham, (e) AKI, and (f) BNx. Urine NGAL was reduced after sham and ischemic AKI in IL-6–/– (g–j). n = 3 to 12 mice per group from 3 experiments. Results are expressed as mean ± SEM and analyzed by t test: WT versus IL-6–/– at the same time point (indicated above the bar) and versus normal WT (indicated below the bar: *P < 0.05; **P < 0.01, ***P < 0.0001). NS, nonsignificant.View Large Image Figure ViewerDownload Hi-res image Download (PPT) As a whole, these data demonstrate that IL-6 plays a key role in NGAL production after sham, ischemic AKI, and bilateral nephrectomy. To determine the sources of IL-6-mediated NGAL production after sham, AKI, and Bnx, NGAL mRNA levels were determined in the liver, kidney, lung, and spleen in WT and IL-6–/– (Figure 3). In the liver, NGAL mRNA levels were increased 4 and 24 hours after sham, AKI, and BNx, and they were significantly reduced at both time points and in all 3 conditions in IL-6–/–. In the kidney, NGAL mRNA increased at 4 and 24 hours post AKI and was not affected in IL-6–/–. In the lung and spleen, NGAL mRNA was minimally affected. To determine the sites of NGAL accumulation in organs, protein levels of NGAL were measured in the liver, kidney, lung, and spleen 4 and 24 hours after sham, AKI, and BNx, and immunofluorescence was examined 24 hours after ischemic AKI. Unlike mRNA, NGAL protein levels were elevated in all the organs after sham, AKI, and BNx and were reduced in IL-6–/– (Figure 4). NGAL localizes to both the proximal and distal tubule after AKI (Supplementary Figure S1). The relative contribution of organ production (mRNA) and accumulation (protein) of NGAL after ischemic AKI is graphically represented in Figure 5.Figure 5Relative neutrophil gelatinase–associated lipocalin (NGAL) production (mRNA) and accumulation (protein) after ischemic acute kidney injury (AKI) in wild-type (WT) and interleukin-6–deficient (IL-6–/–) mice. (a,b) NGAL production (mRNA) in liver, kidney, lung, and spleen is expressed as a percentage of organ production and demonstrates that the liver is the major organ source of NGAL production 4 and 24 hours post-AKI in WT mice after ischemic AKI; in IL-6–/– mice, the kidney is the major source of NGAL production. Thus, IL-6 is a mediator of hepatic, but not renal, NGAL production. (c,d) NGAL protein levels in the liver, kidney, lung, and spleen are expressed as a percentage of organ levels and demonstrate that after AKI, NGAL accumulates predominantly within the kidney, lung, and spleen (total protein was determined within organs; thus, organ accumulation does not distinguish free NGAL from NGAL contained within neutrophils). A similar distribution of NGAL accumulation was observed in IL-6–/– mice. (The sizes of the pie charts were adjusted to indicate relative production and accumulation between WT and IL-6–/– mice.) Differences in mRNA and protein highlight the fact that although the liver is the primary site of organ production, NGAL accumulation is widely distributed.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Altogether, the data in Figure 1, Figure 2, Figure 3, Figure 4 to 5 demonstrate that IL-6 contributes to hepatic NGAL production, which results in an increase in plasma NGAL levels in all 3 conditions tested: (sham) surgery, ischemic AKI, and BNx. Since the increase in plasma IL-6 corresponded to an increase in hepatic NGAL production after sham, ischemic AKI, and BNx, we hypothesized that circulating IL-6 was acting at the liver to induce NGAL production in hepatocytes. Given that hepatocytes contain the IL-6 receptor, a direct effect of IL-6 on hepatocytes is plausible. Therefore, to test whether circulating IL-6 mediates hepatic NGAL production, IL-6 was administered i.v. every hour for 3 hours to WT mice, and endpoints were determined 1 hour after the last i.v. injection (Figure 6). After IL-6 injection, hepatic NGAL production (mRNA) increased, as did plasma and urine NGAL protein levels. NGAL mRNA levels did not increase in the kidney, lung, or spleen. IL-6 did not affect kidney function; the plasma IL-6 level was increased in the IL-6-treated mice, as expected. Similarly, i.v. IL-6 to IL-6–/– with AKI increased liver NGAL mRNA levels and plasma NGAL, but not kidney, lung, or spleen NGAL mRNA levels (Supplementary Figure S2). Since the only source of IL-6 after i.v. injection is the plasma, these data demonstrate that circulating IL-6 mediates hepatic NGAL production that leads to increased plasma NGAL levels. To determine the onset of IL-6–mediated hepatic NGAL production, recombinant human IL-6 was added to human (HepG2) hepatocytes, and NGAL mRNA levels were determined in cells, and NGAL protein levels were determined in the supernatant at 1, 2, and 4 hours (Figure 7). NGAL mRNA levels significantly increased in the cells by 1 hour, and in the supernatant by 2 hours. The effect of dose and time on NGAL levels in response to IL-6 was assessed, in vitro, and duration of IL-6 exposure (24 hours) had a much greater effect on supernatant NGAL levels than did dose (Supplementary Figure S3). In vivo, the effect of various renal ischemia times on plasma IL-6 and NGAL levels was assessed and demonstrated that plasma IL-6 levels peaked at 4 hours, whereas plasma and urine NGAL levels peaked at 24 hours (Supplementary Figure S4). These data demonstrate that NGAL production persists up to 24 hours after IL-6 exposure. Many cytokines, including IL-6, mediate their effects through the phosphorylation and activation of the transcription factor STAT3. To assess the role of IL-6–mediated STAT3 activation after ischemic AKI, pSTAT3 was determined in nuclear and cytosolic fractions of the liver and kidney 4 and 24 hours after AKI in WT and IL-6–/– (Figure 8a–d). In the liver, but not the kidney, IL-6–/– had reduced pSTAT3 and absent translocation to the nucleus; in the liver in WT, translocation peaked at 24 hours, correlating with the peak of NGAL production. In vitro, vehicle, IL-6, or IL-6 plus Stattic (an inhibitor of STAT3 phosphorylation) was added to murine (AML12) or human (HepG2) hepatocytes. The increase in NGAL in the supernatant after IL-6 treatment was completely suppressed in the Stattic-treated group; IL-6 levels were not affected (Figure 8e–h). As a whole, these data demonstrate that STAT3 activation is required for IL-6–mediated NGAL production in hepatocytes. To confirm that the liver is the major source of NGAL production after AKI, ischemic AKI was performed in WT and Lcn2Hep–/– mice (Figure 9). Hepatocytes are specifically unable to produce NGAL in the Lcn2Hep–/– mice, whereas NGAL production capacity in all other cells remains intact.12Xu M.J. Feng D. Wu H. et al.Liver is the major source of elevated serum lipocalin-2 levels after bacterial infection or partial hepatectomy: a critical role for IL-6/STAT3.Hepatology. 2015; 61: 692-702Crossref PubMed Scopus (92) Google Scholar At 24 hours post AKI in Lcn2Hep–/–, plasma and urine NGAL levels were significantly reduced versus WT. NGAL mRNA was absent in the liver and not affected in the kidney, lung, or spleen. NGAL protein levels were significantly decreased in the liver, but not the kidney, lung, or spleen; kidney function/injury and plasma IL-6 levels were similar to those for WT. Given that NGAL is constitutively expressed in neutrophils,6Schmidt-Ott K.M. Neutrophil gelatinase-associated lipocalin as a biomarker of acute kidney injury—where do we stand today?.Nephrol Dial Transplant. 2011; 26: 762-764Crossref PubMed Scopus (69) Google Scholar we tested whether IL-6–/– mice might also have had lower plasma and urine NGAL levels due to an effect on neutrophils, by subjecting neutrophil-depleted mice to ischemic AKI (Figure 10). Neutrophil depleted mice with AKI had a similar level of kidney function/injury. Neutrophil depletion did not affect plasma NGAL, urine NGAL, or NGAL mRNA levels in the liver, kidney, lung, or spleen. Organ accumulation of NGAL was similar in the liver and kidney, but significantly reduced in the spleen (24 hours) and lung (4 and 24 hours). As lung neutrophil accumulation occurs after AKI,11Klein C.L. Hoke T.S. Fang W.F. et al.Interleukin-6 mediates lung injury following ischemic acute kidney injury or bilateral nephrectomy.Kidney Int. 2008; 74: 901-909Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar,13Andres-Hernando A. Okamura K. Bhargava R. et al.Circulating IL-6 upregulates IL-10 production in splenic CD4+ T cells and limits acute kidney injury-induced lung inflammation.Kidney Int. 2017; 91: 1057-1069Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar,14Altmann C. Ahuja N. Kiekhaefer C.M. et al.Early peritoneal dialysis reduces lung inflammation in mice with ischemic acute kidney injury.Kidney Int. 2017; 92: 365-376Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar the reduction in lung NGAL in the neutrophil-depleted mice likely reflects a reduction in lung neutrophil recruitment, and a similar mechanism may be at play within the spleen. Given that our data suggest that urine NGAL is predominantly derived from the plasma, we assessed the renal handling of circulating NGAL by determining the fate of i.v. administered recombinant human NGAL (hNGAL) in 6 different conditions, with varying degrees of renal impairment or tubular injury in which the glomerular filtration rate (GFR) was known (Figure 11). hNGAL is homologous to murine NGAL, and it is handled similarly by the kidney, but shows no cross-reactivity to murine NGAL by enzyme-linked immunosorbent assay and does not affect endogenous murine NGAL levels (data not shown). GFRs were as follows (percent of vehicle): (i) vehicle treatment: 100%; (ii) mild prerenal azotemia: 92%; (iii) severe pre-renal azotemia: 30%; (iv) proximal tubule (PT) injury due to maleic acid: 29%; (v) ischemic AKI: 1%; and (vi) BNx: 0% (not measured). Maleic acid is a toxin that specifically affects the PT and induces Fanconi syndrome.15Reynolds R. McNamara P.D. Segal S. On the maleic acid induced Fanconi syndrome: effects on transport by isolated rat kidney brushborder membrane vesicles.Life Sci. 1978; 22: 39-43Crossref PubMed Scopus (16) Google Scholar Plasma and urine hNGAL were determined 1 hour after i.v. injection (Figure 12). Plasma and urine levels of hNGAL were low in mice with normal kidney function. Plasma and urine hNGAL were similar to normal in mild pre-renal azotemia; plasma hNGAL was slightly, but significantly, higher in severe pre-renal azotemia, although urine hNGAL levels were similar to normal. In contrast, both plasma and urine hNGAL were elevated in PT injury and ischemic AKI, and plasma levels were increased in mice with BNx. In order to account for plasma levels and PT function in the assessment of urine excretion of NGAL, we calculated the fractional excretion of hNGAL (FE-hNGAL) as follows: FE-hNGAL = ([urine {hNGAL} × plasma creatinine]/[plasma h{NGAL} × urine creatinine]) × 100. FE-hNGAL was less than 1% in normal mice, mice with mild pre-renal azotemia, and mice with severe pre-renal azotemia, and it was greater than 20% in maleic acid–treated mice and mice with ischemic AKI. Endogenous (murine [m]) levels of plasma and urine NGAL, and FE-mNGAL were also determined, with results similar to those for hNGAL. Taken together, these data indicate that circulating NGAL is normally filtered, and then resorbed and metabolized/degraded by the PT. Excretion of the intact protein is not a normal mechanism of NGAL elimination, as urine levels were very low in mice with normal kidney function. With PT dysfunction, filtered NGAL is not resorbed and metabolized, and appears in the urine. FE-NGAL distinguishes between severe pre-renal azotemia and PT injury (maleic acid–treated) in mice with similar GFRs. NGAL is the most widely studied AKI biomarker, having been tested in thousands of patients for its potential to diagnose AKI early. Based on early studies demonstrating that NGAL is produced by the injured kidney and is then released into the urine and plasma,7Paragas N. Qiu A. Zhang Q. et al.The Ngal reporter mouse detects the response of the kidney to injury in real time.Nat Med. 2011; 17: 216-222Crossref PubMed Scopus (297) Google Scholar NGAL has been classified and studied specifically as a tubular injury biomarker.16Moledina D.G. Parikh C.R. Phenotyping of acute kidney injury: beyond serum creatinine.Semin Nephrol. 2018; 38: 3-11Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar In the present study, we systematically examined renal and extra-renal production of NGAL, the influence of the proinflammatory cytokine IL-6, and the renal handling of NGAL. The major novel findings of our report are that (i) IL-6 mediates hepatic NGAL production during AKI; (ii) the liver—not the kidney—is the primary source of both plasma and urine NGAL levels during AKI; and (iii) circulating NGAL appears in the urine during AKI associated with proximal tubular dysfunction. These data change the paradigm by which NGAL should be interpreted as a biomarker of AKI and extend the known role of IL-6 in the systemic response after AKI. To date, preclinical studies that have examined NGAL during AKI have focused on its role as an AKI biomarker7Paragas N. Qiu A. Zhang Q. et al.The Ngal reporter mouse detects the response of the kidney to injury in real time.Nat Med. 2011; 17: 216-222Crossref PubMed Scopus (297) Google Scholar,17Mishra J. Ma Q. Prada A. et al.Identification of neutrophil gelatinase-associated lipocalin as a novel early urinary biomarker for ischemic renal injury.J Am Soc Nephrol. 2003; 14: 2534-2543Crossref PubMed Scopus (1312) Google Scholar and in kidney function recovery.18Mori K. Lee H.T. Rapoport D. et al.Endocytic delivery of lipocalin-siderophore-iron complex rescues the kidney from ischemia-reperfusion injury.J Clin Invest. 2005; 115: 610-621Crossref PubMed Scopus (745) Google Scholar,19Mishra J. Mori K. Ma Q. et al.Amelioration of ischemic acute renal injury by neutrophil gelatinase-associated lipocalin.J Am Soc Nephrol. 2004; 15: 3073-3082Crossref PubMed Scopus (421) Google Scholar Independent of AKI, ongoing studies have clarified the systemic functions of NGAL in a variety of settings. In particular, it is now recognized that NGAL is part of the hepatic acute phase response (APR).12Xu M.J. Feng D. Wu H. et al.Liver is the major source of elevated serum lipocalin-2 levels after bacterial infection or partial hepatectomy: a critical role for IL-6/STAT3.Hepatology. 2015; 61: 692-702Crossref PubMed Scopus (92) Google Scholar The hepatic APR is characterized by the hepatic production of acute-phase proteins that collectively function to prepare the body to battle infection, minimize tissue injury, and initiate wound repair.20Jain S. Gautam V. Naseem S. Acute-phase proteins: As diagnostic tool.J Pharm Bioallied Sci. 2011; 3: 118-127Crossref PubMed Scopus (265) Google Scholar NGAL participates in the hepatic APR as a bactericidal agent via the sequestration of iron, and works in conjunction with other iron regulatory hepatic acute-phase proteins such as hepcidin,21Ganz T. Hepcidin and iron regulation, 10 years later.Blood. 2011; 117: 4425-4433Crossref PubMed Scopus (629) Google Scholar haptoglobin,22Boe A. Canosi U. Donini S. et al.Determination of haptoglobin expression in IL-6 treated HepG2 cells by ELISA and by RNA hybridization—evaluation of a quantitative method to measure IL-6.J Immunol Methods. 1994; 171: 157-167Crossref PubMed Scopus (9) Google Scholar and ferritin.23Kobune M. Kohgo Y. Kato J. et al.Interleukin-6 enhances hepatic transferrin uptake and ferritin expression in rats.Hepatology. 1994; 19: 1468-1475Crossref PubMed Scopus (80) Google Scholar Given that bacteria require iron for growth, iron sequestration is a key defense against infection. The important role of NGAL during infection is highlighted by studies demonstrating that NGAL knockout mice have increased susceptibility to infection.24Berger T. Togawa A. Duncan G.S. et al.Lipocalin 2-deficient mice exhibit increased sensitivity to Escherichia coli infection but not to ischemia-reperfusion injury.Proc Natl Acad Sci U S A. 2006; 103: 1834-1839Crossref PubMed Scopus (329) Google Scholar,25Chan Y.R. Liu J.S. Pociask D.A. et al.Lipocalin 2 is required for pulmonary host defense against Klebsiella infection.J Immunol. 2009; 182: 4947-4956Crossref PubMed Scopus (154) Google Scholar IL-6 is a major mediator of the hepatic APR and induces hepatocyte production of virtually all acute-phase proteins.26Gruys E. Toussaint M.J. Niewold T.A. et al.Acute phase reaction and acute phase proteins.J Zhejiang Univ Sci B. 2005; 6: 1045-1056Crossref PubMed Scopus (616) Google Scholar IL-6 production is triggered within minutes by numerous systemic insults such as surgery, trauma, and sepsis, as part of the systemic inflammatory response syndrome—which also occurs during AKI. In mice and patients with AKI, the plasma IL-6 level increases within 2 hours.27Hoke T.S. Douglas I.S. Klein C.L. et al.Acute renal failure after bilateral nephrectomy is associated with cytokine-mediated pulmonary injury.J Am Soc Nephrol. 2007; 18: 155-164Crossref PubMed Scopus (218) Google Scholar,28Liu K.D. Altmann C. Smits G. et al.Serum interleukin-6 and interleukin-8 are early biomarkers of acute kidney injury and predict prolonged mechanical ventilation in children undergoing cardiac surgery: a case-control study.Crit Care. 2009; 13: R104Crossref PubMed Scopus (132) Google Scholar The production and renal handling of IL-6 during AKI is well characterized. Notably, the kidney is not a major source of IL-6 production, as evidenced by the fact that plasma IL-6 increases after BNx.11Klein C.L. Hoke T.S. Fang W.F. et al.Interleukin-6 mediates lung injury following ischemic acute kidney injury or bilateral nephrectomy.Kidney Int. 2008; 74: 901-909Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar,27Hoke T.S. Douglas I.S. Klein C.L. et al.Acute renal failure after bilateral nephrectomy is associated with cytokine-mediated pulmonary injury.J Am Soc Nephrol. 2007; 18: 155-164Crossref PubMed Scopus (218) Google Scholar In contrast, the spleen and liver are the major organ sources of IL-6 in AKI,29Andres-Hernando A. Altmann C. Ahuja N. et al.Splenectomy exacerbates lung injury after ischemic acute kidney injury in mice.Am J Physiol Renal Physiol. 2011; 301: F907-F916Crossref PubMed Scopus (55) Google Scholar and the major cell source is the macrophage.30Altmann C. Andres-Hernando A. McMahan R.H. et al.Macrophages mediate lung inflammation in a mouse model of ischemic acute kidney injury.Am J Physiol Renal Physiol. 2012; 302: F421-F432Crossref PubMed Scopus (31) Goog