Renal stress in vivo in real-time--visualised by the NGAL reporter mouse

Acute kidney injury (AKI) is a serious syndrome in hospitalized patients of epidemic dimension with many patients dying of AKI or its complications [1, 2]. There is a large body of literature showing the importance of novel biomarkers of AKI [3--9]. Such markers are central to early diagnosis for timely treatment and potentially cure of AKI. In clinical practice, the currently applied renal biomarkers including serum creatinine, urea and urinary output reflect loss of renal function. These markers delay the diagnosis of AKI by 24–48 h, thereby they may contribute to the lack of specific therapies for AKI despite huge efforts during the last decades. Neutrophil gelatinase-associated lipocalin (NGAL) is a quite novel and already intensely investigated renal biomarker indicating early structural damage. The source of NGAL and mechanisms of its expression remained elusive until the January issue of Nature Medicine, where a Technical Report was published [10], describing how NGAL expression is monitored by bioluminescence and fluorescence in experimental models of AKI (ischaemia-reperfusion, application of cisplatin or lipid A). In a first step, a heterozygous diffusion reporter mouse was created that expresses luciferase2 and mCherry (Luc2/mC) under the control of the NGAL promoter. This promoter is responsive to toxic stimuli. Polymerase chain reaction was used to genotype the NGAL-Luc2/mC double fusion reporters. NGAL gene and protein expression in vivo was quantified by photon emission detection and in parallel by measuring urinary NGAL (uNGAL) levels. This model allows detection of NGAL expression in ‘real-time’ and Paragas et al. [10] visualised this in the kidneys after various insults, thus, gaining insight into the kinetic, organ and cell specificity of NGAL expression in response to renal injury. The most striking findings in response to different challenges were (i) after subjection to uni- and bilateral ischaemia/ reperfusion of the kidney and injection of luciferin, the operated kidney exhibited NGAL-Luc2/mC activity early and in a dose-dependent manner, (ii) intraperitoneal infusion of lipid A as a sepsis model induced NGAL-Luc2/mC bioluminescence in the kidney, liver, lung, spleen and trachea, (iii) NGAL expression was nuclear factor (NF)-kappaB-regulated and (iv) ischaemia-reperfusion in wild-type NGAL-Luc2/mC kidneys transplanted to NGAL−/− knockout animals, identified uNGAL originating from the kidneys and neutrophils not contributing to NGAL secretion to a significant extent. Major findings were also that damaged epithelia of the thick ascending limb (TAL) of Henle and the collecting ducts (CDs) are the main source of NGAL, whereas uninjured epithelia in these tubule segments and the proximal tubules did not express NGAL. Pharmacological interventions that targeted infections were able to decrease NGAL expression, whereas a volume depletion model did not render elevated NGAL expression levels. The innovative in vivo mouse model permits a continuous surveillance of organ damage without invasive procedures. The data are more sensitive and accurate than changes of serum creatinine values, the standard biochemical marker of AKI. Using such mice for toxicity monitoring, e.g. in drug development, may replace elaborate urine and plasma collections [11]. In this regard, the NGAL luminescent mouse strain also allows for a rigorous monitoring of potentially nephroprotective substances. What are further advancements? The results suggest a new paradigm showing the quantitative linkage between cellular stress, expression of NGAL in the kidney and excretion of NGAL via the urine. From the findings of this work, it may be deduced that novel biomarkers should be generally subjected to a proof-of-concept study, where the cellular origin of protein expression should be tracked in vivo besides the mere determination of protein concentrations in biofluids like the urine. Proximal tubular cells appear to be extremely sensitive to stress, and as a result, these cells undergo necrosis and apoptosis first following ischaemia/reperfusion. In recent years, there has been a recognition of cellular changes in TAL and CD cells following different types of insults, such as cold ischaemia (e.g. in renal transplants), in hypoxic though functioning kidneys and with drug toxicity [12]. One of the important findings of the highlighted study is that cells in the distal nephron are activated to express NGAL within a few hours following damage, suggesting that the distal nephron is responsive early on, before other nephron segments may be affected. The underlying mechanisms remain to be discovered. Interestingly, a two compartment model may be developed that is schematically depicted in Figure 1. In this model, proximal and distal nephron segments fulfil different functions regarding synthesis and secretion of NGAL. Reabsorption and degradation may be the preponderant mechanism of protein turnover in the proximal nephron segments, whereas secretion of NGAL in the distal segments may indicate cellular stress in this compartment. Importantly, urine NGAL expression might be a sign of the earliest stage of AKI, where clinically no abnormalities are present. Nonetheless, (i) the ‘systemic pool’ of serum NGAL (sNGAL) conceivably originates not only from the kidneys but also from extrarenal sources, (ii) rising sNGAL (from tubular backleak or extrarenal source) during AKI may reflect reduced glomerular filtration rate given its molecular weight of 25 kDa and (iii) though the major source of uNGAL is the distal nephron, a small fraction may come from the systemic pool, escaping proximal tubular reabsorption due to proximal tubular injury. Model of sNGAL reabsorption and uNGAL excretion. sNGAL is reabsorbed in the proximal tubule under normal conditions and during AKI. Rising sNGAL may (i) indicate its origin being from tubular backleak or an extrarenal source or (ii) reflect reduced glomerular filtration rate. Though the major source of uNGAL is the distal nephron, a small fraction may come from the ‘systemic pool’, escaping proximal tubular reabsorption due to proximal tubular injury. The authors confirmed previous clinical and preclinical findings [13, 14] with uNGAL not increasing after volume depletion, thus indicating that NGAL is an important clinical tool to discriminate patients with ‘pre-renal azotaemia’ from those with AKI and therefore ‘true’ structural damage. Using bioimaging of organs and isolated kidney cells, in situ hybridisation, immunostaining and reciprocal cross-transplantation, the authors demonstrate that uNGAL derives from the kidney. These experimental findings might not be perfectly transferable to the clinical situation of AKI with multimorbid patients that often suffer from systemic diseases and particularly during acute-on-chronic renal failure. What should future research focus on? One might envision important results with the characterization of NGAL-receptors, their down-stream intracellular signalling and (sub-) cellular localization. Conditional knockout animals with tissue-specific disruption of NGAL protein expression should allow the investigation of the predominant source of sNGAL under different conditions. Finally, NF-kappaB appears to be an important regulator of NGAL expression, which needs further attention in experimental research. The NGAL reporter mouse strain provides a unique, valuable and timely tool to determine cellular NGAL expression mechanisms and to examine interventions. The distal nephron as a source of renal NGAL has come in to the focus of research on early mechanisms of renal injury. The development of this model will certainly open a new area of research to better understand and investigate novel biomarkers of organ injury, including AKI. M.H. is a Fellow of the Alexander von Humboldt-Foundation, Germany. P.R.M. is funded by DFG grants 1365/7-1 and SFB854 TP1. Conflict of interest statement. M.H. has received lecture fees from Abbott Diagnostics and Biosite. Both companies are involved in the development of NGAL assays to be applied in clinical practice. P.R.M. is co-founder of antibodies-online.