Immunoassays distinguishing between HNL/NGAL released in urine from kidney epithelial cells and neutrophils

Abstract

Background

The distinction between monomeric human neutrophil lipocalin/neutrophil gelatinase-associated lipocalin (HNL/NGAL), secreted by injured kidney tubular cells, and dimeric HNL/NGAL, released by activated neutrophils, is important to accurately diagnose acute kidney injury (AKI).

Methods

132 urine samples from 44 intensive care unit (ICU) patients and five urine samples from non-ICU patients with urinary tract infections (UTIs) were analyzed by two monoclonal enzyme-linked immunosorbent assays (ELISA-1 and ELISA-2). The presence of monomeric and/or dimeric HNL/NGAL in each sample was visualized by Western blotting.

Results

The ELISA-1 detected both monomeric and dimeric HNL/NGAL whereas the ELISA-2 almost exclusively detected dimeric HNL/NGAL with an area under the receiver‐operating characteristics curve (AuROC) of 0.90. The ELISA-1/ELISA-2 ratio detected the monomeric form with an AuROC of 0.92. In 32 AKI patients, dimer-specific ELISA-2 levels decreased pre-AKI whereas the monomer-specific ELISA-1/ELISA-2 ratio gradually increased beyond AKI diagnosis. High ELISA-2 levels and/or low ELISA-1/ELISA-2 ratios detected a predominance of dimeric HNL/NGAL in urine from the patients with UTIs.

Conclusions

In combination, our two ELISAs distinguish monomeric HNL/NGAL, produced by the kidney epithelium, from dimeric HNL/NGAL, released by neutrophils during AKI development, as well as reduce the confounding effect of neutrophil involvement when bacteriuria is present.

Introduction

Human neutrophil lipocalin (HNL), also known as neutrophil gelatinase-associated lipocalin (NGAL), is released by kidney tubular cells under stressful conditions [1] and is a promising biomarker for acute kidney injury (AKI) [2]. Additionally, HNL/NGAL is released by activated neutrophils [3], [4]. HNL/NGAL exists in different molecular forms in the neutrophil [5], in plasma [6] and in urine [1], [7]. The protein exists as a 25-kDa monomer, a 45-kDa homodimer or a 135-kDa heterodimer, covalently conjugated with gelatinase. Kidney epithelial cells in culture mainly secrete the monomeric form, whereas neutrophils mainly release the dimeric form [1]. Hence, elevated urinary HNL/NGAL levels in AKI may emanate from injured kidney epithelium as well as from infiltrating neutrophils involved in the AKI-pathophysiology [8]. Furthermore, activation of neutrophils and the subsequent release of their, mainly dimeric, HNL/NGAL in urine have been observed in patients with urinary tract infections (UTIs) [1], [9], [10]. The interpretation of elevated urinary HNL/NGAL is further complicated by the fact that increased levels have been found in non-acutely ill patients without evidence of kidney damage or UTIs [11].

The clinical performance of the HNL/NGAL assay to catch the different forms of HNL/NGAL depends on the antibody configuration [7]. This can explain the varying ability of urinary HNL/NGAL to predict AKI in previous studies [12], [13], [14], [15], [16], [17], [18], [19], [20], [21], [22], [23], [24], [25].

To distinguish between different causes of elevated HNL/NGAL in urine, precise diagnostic assays measuring different forms of HNL/NGAL are desirable since they may increase the diagnostic accuracy of conditions such as AKI. Also, identifying the different sources of HNL/NGAL will aid in understanding the timing of different pathophysiological events during the AKI continuum [26].

This study aims to examine the ability of different combinations of monoclonal antibodies against HNL/NGAL to distinguish between the molecular forms primarily secreted by kidney epithelial cells and neutrophils, respectively.