Beyond Being a Biomarker: Lipocalin-2/NGAL as a Facilitator for Protective Drug Action in Hypoxic Kidney Injury

Abstract

In the present issue of Acta Physiologica [1], an international consortium of investigators reports that the iron-transporter glycoprotein lipocalin-2 (LCN2), originally identified in neutrophils and named neutrophil gelatinase-associated lipocalin, abbreviated NGAL or 24p3, may protect the kidneys from ischemia–reperfusion injury. The findings suggest that LCN2 protects the kidneys by restoring the sensitivity of soluble guanylate cyclase (sGC) to drug activators in afferent glomerular arterioles through a receptor-mediated mechanism. LCN2 is produced and released by several tissues including adipose tissue, liver, kidneys, and neutrophils. It is categorized as an acute-phase protein that is upregulated during inflammatory states. LCN2 is widely used in clinical and experimental settings as an early biomarker in acute kidney injury and for the staging of chronic kidney disease.

The study by Zhao et al. [1] is an elegant follow-up study on a series of independent observations dating 10–20 years back, which include a study by authors from 2016 [2]. A consistent kidney-protective effect of exogenous LCN2 was found in preclinical kidney ischemia-injury models, including a kidney transplantation model. The study in Acta shows ex vivo with murine, isolated kidney microvessels, that LCN2 mitigates excessive microvascular resistance through restoring vascular smooth sGC sensitivity towards activator drugs. The sensitivity is typically lost by more severe prolonged hypoxia. Soluble GCs can be oxidized to the heme-free form, apo-sGC, and the authors confirm that apo-sGC cannot be activated by the endogenous agonist nitric oxide (NO). The class of sGC activator drugs is unique and different from sGC stimulators since they can overcome this state and activate apo-sGC independently of NO to increase target cell cyclic guanosine monophosphate (cGMP) production even under detrimental oxidative stress. Zhao et al. [1] show that LCN2 restores sensitivity of the kidney afferent arterioles towards sGC activators dependent on iron. The effect is found in arterioles subjected to hypoxia ex vivo after isolation and in arterioles subjected to hypoxia “in situ” in transplanted kidneys before microdissection and testing. The conclusion is that by delivering ferric iron bound to LCN2 (holo-LCN2) to arterioles, this oxidizes sGC, which restores sensitivity to activator drugs. The study corroborates that LCN2 may be a direct, extracellular, signaling molecule that indirectly protects vascular smooth muscle suffering from prolonged ischemic insults in the kidneys (Figure 1).

What is the mechanism? LCN2 binds hydrophobic microbial siderophores, which are small molecules that bacteria produce to sequester iron from their environment. The acute phase reactant LCN2 is thereby bacteriostatic since iron is a vital nutrient for many microbes. Deletion of LCN2 increases susceptibility to Escherichia coli infections in mice [3]. LCN2 connects iron metabolism and immune responses in conditions with infection but, as shown by Zhao et al. [1], also in hypoxic injuries where protective signaling is conferred by LCN2 only in its iron-loaded form. Holo-LCN2 binds ferric iron, which is generally considered oxidizing due to its ability to accept electrons. Hydrolysis of ferric-iron complexes often involves a reduction step to facilitate iron release. Zhao et al. [1] hypothesize that LCN2, in its iron carrying capacity, may oxidize soluble guanylyl cyclase, which by itself is detrimental for enzyme function but favors the action of the class of sGC activators—drugs that are effective at sGC only in the oxidized state (Figure 1). This ability could prove applicable in kidney transplantation. Here, as in other pathological states with longer duration of hypoxia, the sensitivity toward sGC activators is reduced. In the authors' previous study, using a mouse model, administration of exogenous LCN2 was found to ameliorate acute rejection, suggesting that LCN2 could enhance graft survival and overall kidney function post-transplantation [2].

The study leaves some open questions subject to further debate and investigation (Figure 1).

First, we do not know if the sensitivity of arterioles in the present setting outside and within kidneys after prolonged ischemia is hampered toward direct stimulators of sGC like NO and, for example, vericiguat since the authors do not test it directly but use the agent ODQ (1H-[1,2,4]oxadiazolo-[4, 3-a]quinoxalin-1-one) as a surrogate for this condition. Such lower sensitivity was the case in renal medullary microvessels in a previous study by the authors' group [4].

Second, how does the polar, glycoprotein LCN2 with a molecular weight ~25 kDa reach intracellular sGC? Authors propose that the multiligand receptor megalin/LRP-2 mediates cellular uptake of LCN2 in afferent arterioles, which would align well with previous studies observing rapid uptake of LCN2 into proximal tubular cells rich in megalin. There is sparse evidence for expression of megalin in kidney vasculature, and although the authors show a positive immunoblot, the proposal is not tested by intervention, and it remains debatable, also with known alternative routes for LCN2 uptake, for example, heparan sulphate proteoglycans [5].

Third, how does LCN2 exert the effect on sGC? Authors exclude that this occurs through cGMP because the second messenger does not increase in concentration in the perfusate from isolated kidneys. Since cGMP was not determined in arterioles or kidney tissue and cell-permeable cGMP variants were not applied, this cannot be ruled out and remains a possibility (Figure 1).

The protection by LCN2 in ischemia–reperfusion injury in kidneys appears to be a consistent finding across laboratories, but at the same time, there is not a uniform protective effect of LCN2 in kidneys. While mice with deletion of the LCN2 gene have no major difference in renal graft injury [2] such mice display less, and not more, renal injury in models with proteinuria and direct chemical injury [6], in experimental diabetic nephropathy [7] and in chronic kidney disease models [8]. A significant confounder is whether endogenous LCN2 appears in its iron-carrying holo-form, which is thought to deliver iron to damaged cells, or in its oxidized form, apo-LCN2, that deprives cells of iron. One reason for discrepant findings could therefore relate to iron, which can be detrimental in excessive amounts leading to oxidative stress and kidney damage and be equally important in appropriate quantities for cellular function and recovery. There is much data showing that LCN2 is an acute and sensitive indicator for detecting kidney graft rejection, reflecting early tubular injury [9].

As an intriguing perspective, the present findings indicate that renal resistance vessels can be made sensitive to relevant vasodilator drugs by LCN2 after prolonged ischemic hypoxia. Thus, rather than LCN2 being a new wonder drug, the studies imply the donation of iron or heme groups to oxidize sGC is a potential pathway to restore pharmacologically vascular function after ischemia. The use of LCN2 could be one therapeutic strategy to manipulate iron chelation and control supplementation. The data lend further support to the view that balancing iron is important to optimize outcomes in patients experiencing acute and prolonged renal ischemia and bring LCN2 and the renal vasculature into focus.

The author declares no conflicts of interest.