Lipopolysaccharide-induced inflammation or unilateral ureteral obstruction yielded multiple types of glycosylated Lipocalin 2

Male and female BALB/c CrSlc and male C57BL/6J JmsSlc mice were intraperitoneally (i.p.) injected with 1 mg/kg of LPS. Saline was administered as a control. Dex (5 mg/kg) or vehicle (saline) was administered i.p. 1 h prior to LPS. After 6 h, the mice were euthanized by cervical dislocation, and trunk blood samples were collected and allowed to clot for 30 min at room temperature, followed by centrifugation at 3000 × g for 10 min to obtain serum. The tissues were sampled and homogenized in lysis buffer (50 mM Tris–HCl (pH 7.4), 150 mM NaCl, 1 % Nonidet P40, 1 mM EDTA (pH 8.0), 1 mM EGTA, and 1 mM phenylmethylsulfonyl fluoride) containing a protease inhibitor cocktail and were centrifuged at 12,000 × g for 5 min to collect the supernatant. The protein concentrations were measured using the CBB method. Spot urine samples were collected and immediately placed on ice, and urine, serum, and tissue lysates were stored at −80 °C until further use. For the measurement of the serum C-related protein (CRP) level, serum samples were diluted 2000–20,000-fold and CRP and IL-1β levels were measured using ELISA kits (R & D Systems, Minneapolis, MN, USA). The absorbance at 450 nm and 540 nm was measured using a SpectraMax Pro M3 instrument (Molecular Device, CA, USA).

For the UUO experiments, the mice were anesthetized with 50 mg/kg of pentobarbital sodium i.p. After laparotomy, the left ureter was ligated with a silk suture (5–0) 0.7 cm below the renal pedicle. Three days later, the mice were euthanized by cervical dislocation, then their urine, which accumulated in the obstructed side of the kidney and bladder, was sampled by syringe aspiration with 30 G 1/2 and 26 G 1/2 needles. Control mice underwent a sham operation (the ureter was manipulated but not ligated). Urine samples were kept at −80 °C until use.

The coding sequence for mouse LCN2 cDNA was amplified from brain cDNA by PCR using Ex Taq reagents. The primer sequences used to amplify mouse Lcn2 were 5′-AAACCATGGCCCTGAGTGTCAT-3′ and 5′-GCCTGAACCATTGGGTCTCTGC-3′. The PCR products were subcloned into the pCR-4 vector according to the manufacturer’s protocol and sequenced. The mouse LCN2 cDNA was then ligated into the pcDNA3.1 expression vector. Human embryonic kidney (HEK293) cells were cultured in a six-well dish with DMEM containing 10 % heat-inactivated fetal bovine serum, 100 U/ml penicillin, and 100 μg/ml streptomycin at 37 °C in 5 % CO₂. Plasmid DNA was transfected into HEK293 cells with the FuGENE HD transfection reagent (Promega, Madison, WI) following the manufacturer’s protocol. The culture media were exchanged to serum-free media 24 h after transfection and was collected 48 h after transfection. Five hundred microliters of culture medium were further centrifuged at 14,000 × g for 10 min at 4 °C for concentration and was replaced with phosphate-buffered saline (PBS) using Amicon Ultra Centrifugal Filter Devices (10 K molecular weight cutoff, Millipore, Billerica, MA, USA). The buffer exchange was repeated three times. The concentrated media, the final volume of which was approximately 60 μL, were prepared for immunoblot and glycosylation analyses.

Protein samples were heated with sodium dodecyl sulfate (SDS) buffer (10 % glycerol, 2.3 % SDS, 5 % 2-mercaptoethanol, 62.5 mM Tris–HCl (pH 6.8), and 0.1 % bromophenol blue) at 100 °C for 5 min. Spot urine (2 μg of protein), tissues (5 μg), sera (5 μg), and glycosidase-digested samples (5 μg of proteins and 2 μL of concentrated-conditioned media) were electrophoresed on 12.5 % SDS-polyacrylamide gels and transferred to polyvinylidene difluoride membranes. Urine samples from UUO mice (2 μL of urine from the renal pelvis and 5 μg of bladder urine (approximately 5–10 μL)) were also subjected to electrophoresis. After blocking (Block Ace; DS Pharma Biomedical, Osaka, Japan) for 1 h at room temperature, the membranes were incubated with a rabbit polyclonal antibody specific for mouse LCN2, which was diluted 1:8000 with Can Get Signal Immunoreaction Enhancer Solution (TOYOBO, Osaka, Japan) overnight at 4 °C. After incubation for 1 h at room temperature with horseradish peroxidase-labeled goat anti-rabbit IgG, which was diluted 1:10,000 as a secondary antibody, the membranes were treated with ECL Western blotting detection reagents (Perkin Elmer Life Sciences, Boston, MA) and visualized with a LAS-3000 fluorescence imaging system (Fuji Film, Tokyo, Japan).

Sera, urine, concentrated conditioned media, tissue samples, and proteins used as positive controls (fetuin and RNase B) were treated with PNGase F, Endo H, or NA according to the manufacturer’s instructions. PNGase F hydrolyzes between the innermost N-acetylglucosamine (GlcNAc) and Asn residues of all types of N-glycan. Endo H cleaves between the two GlcNAc residues of the chitobiose core of hybrid- or high mannose-type N-glycan, and NA digests the terminal N-acetyl-neuraminic acid residues from carbohydrates [33, 34]. Ten micrograms of protein and 9 μL of conditioned media were glycosidase digested overnight at 37 °C. The samples were then boiled in SDS-sample buffer for 5 min and analyzed by immunoblotting. SDS-polyacrylamide gels loaded with fetuin and RNase B were stained with CBB after electrophoresis.

The images were analyzed using the ImageJ gel analysis software program (http://​imagej.​nih.​gov/​ij/​). The data are expressed as the mean ± S.E.M. Significant differences were evaluated using an unpaired t-test or two-way ANOVA. For multiple comparisons, P values were adjusted by Holm’s method. P < 0.05 was considered significant.

Previous studies reported that LPS challenge drastically increased the LCN2 expression at the protein and mRNA levels in the rat lung, liver, and kidney, as well as in the mouse lung and liver [35, 36]. Our present work also showed that LPS administration increased the LCN2 mRNA and protein expression in the ureter and bladder, as well as in the lung, liver and kidney of the BALB/c mice (Fig. 1). Figure 1a shows a representative result demonstrating two different isoforms of urinary LCN2. Urinary LCN2 was hardly detectable under basal conditions, but was detectable as a signal of apparently stronger intensity compared with that of serum LCN2 after the administration of LPS (Fig. 1b and c). While the LCN2 protein in the serum from LPS-treated mice was a single band with a molecular weight of 22 kDa, the urinary LCN2 proteins in these mice showed two forms with different molecular weights, i.e., 22 and 24 kDa (Fig. 1c).

In peripheral tissues, the majority of the upregulated LCN2 was approximately 22 kDa in size. Faint LCN2-immunopositive signals, which were slightly larger than the 24-kDa urinary LCN2, were also detected in the liver and ureter in the saline-injected mice. However, LPS did not increase the densities of this band in either type of sample. The single and double bands of LCN2 in the serum and urine, respectively, were also observed in LPS-treated female BALB/c and male C57B6 mice, indicating that the sample-specific molecular patterns of LCN2 proteins did not exhibit sex- or strain-specific differences (data not shown). These results suggest that the urinary LCN2 in LPS-treated mice consists of two distinct molecular weight forms. Notably, the 24-kDa LCN2 was upregulated in the urine, but not in the serum, by LPS treatment.

Because the 24-kDa LCN2 protein was specifically observed in the urine of LPS-treated mice, we hypothesized that it may serve as a biomarker to identify urinary tract disorders. To prove this hypothesis, we established UUO mice as a model of a urinary tract disorder. To search for the origin of the 24-kDa LCN2 in urine, we performed Western blot analyses of serum, urine and tissue samples from these model mice. In the UUO experiments, unilateral ureteral ligation successfully upregulated the 22-kDa LCN2 expression on the obstructed side of the ureter without affecting the opposite side (Fig. 2). An immunopositive signal for 24-kDa LCN2 was barely detectable in both ureters of sham-operated and UUO mice. The serum level of the 22-kDa LCN2 was increased in UUO mice. Despite the faint LCN2 band at 24 kDa, a robust signal for LCN2 was detected at 22 kDa in the urine that had pooled in the renal pelvis (Fig. 2). Interestingly, the 24-kDa LCN2 was observed only in the bladder urine of UUO mice, with significant differences in the signal-to-noise ratio (1.06 ± 0.012 for the sham operation, n = 6, and 1.35 ± 0.037 for UUO, n = 10, P = 0.047 based on an unpaired t-test). These results suggest that a urinary tract obstruction upregulates the urinary 24-kDa LCN2 level, which is predominantly found in bladder urine.

Because a previous study reported that mouse LCN2 contains two N-glycosylation sites in its amino acid sequence [4], we postulated that the two LCN2 forms with different molecular weights observed in our experiment were the result of post-transcriptional N-glycosylation. The positive control assays in which fetuin and RNase B were added as substrates for the endo- or exoglycosidase showed enzymatic activity (Fig. 3a). Using urinary LCN2 from LPS-treated mice as a substrate, PNGase F digestion produced a single molecular weight protein (Fig. 3b). The product size of the PNGase F-digested urinary LCN2 was nearly equal to the molecular weight of unglycosylated-recombinant mouse LCN2, i.e., 20 kDa (Fig. 3c). The two forms of urinary LCN2 proteins remained unaffected by Endo H, suggesting that the urinary LCN2 proteins had neither high-mannose nor hybrid type N-glycan (Fig. 3b). In addition, neuraminidase (NA), which digests terminal N-acetyl-neuraminic acid residues from carbohydrates, shifted the immunopositive bands of each urinary LCN2 to a lower molecular weight. These results indicate that the 22- and 24-kDa proteins observed in the Western blotting analysis contained the same LCN2 protein and complex-type N-glycan with terminal N-acetyl-neuraminic acid, but the structures of their carbohydrate moieties differed.

When mice were treated with LPS, increases in the levels of LCN2 (at approximately 22 kDa) were detected in the peripheral tissues (Fig. 1). Interestingly, the molecular weight of the LCN2 expressed in peripheral tissues subtly differed among tissues. For example, the mass of LCN2 was slightly higher in the lung than in the liver. To investigate the features of the carbohydrate moieties of LCN2 in the serum and tissues, we examined the glycopeptidase sensitivity of the LCN2 proteins in representative tissues obtained from LPS-treated mice. Similar to observations in urinary samples, the molecular weights of the LCN2 proteins from all examined tissues shifted to lower values (approximately 20 kDa) after PNGase F treatment (Fig. 4). However, in contrast to urinary LCN2, Endo H was partially effective in releasing N-glycan from the 22-kDa LCN2 in the liver and the ureter, whereas this protein minimally cleaved the carbohydrates on LCN2 from the sera, lungs, and kidneys of LPS-treated mice (Fig. 4). NA treatment clearly affected the LCN2 digestion in the serum and Endo H-resistant tissues, such as the lung and the kidney, whereas it minimally influenced the size of LCN2 in Endo H-sensitive tissues, such as the liver and the ureter of LPS-treated mice. These results indicate that the sensitivities of LCN2 proteins to glycosidases differed between the urine and certain tissues, and the LCN2 proteins secreted in the serum and urine, as well as in the lungs and kidneys, of LPS-treated mice mainly contain complex N-glycans.

We then measured the levels of serum pro-inflammatory markers, such as IL-1β, in our LPS-treated model to evaluate the correlation of inflammatory responses with the production of urinary LCN2 isoforms. In addition, the effects of steroid pretreatment (Dex; 5 mg/kg) on the LCN2 production following LPS treatment was investigated. LPS administration (1 mg/kg i.p.) actively caused systemic inflammation, which increased the serum CRP levels in both the saline- and Dex-pretreated mice (Fig. 5a, P < 0.001 for LPS treatment, P = 0.28 for Dex treatment, and P = 0.42 for LPS × Dex treatment, by a two-way ANOVA).

LPS increased the serum IL-1β level in the saline-pretreated mice (Fig. 5b). In contrast, Dex pretreatment suppressed the production of serum IL-1β upon LPS exposure (Fig. 5b). Despite the effective suppression of the serum IL-1β levels by Dex pretreatment, the serum and urinary LCN2 levels were significantly increased after LPS treatments (Fig. 5c and d). Thus, the upregulation of LCN2 by LPS treatment correlated with pro-inflammatory cytokine and CRP elevation, but the LCN2 production in our model could not be suppressed by pretreatment with 5 mg/kg Dex.