Secreted Neutrophil Gelatinase-Associated Lipocalin Shows Stronger Ability To Inhibit Cyst Enlargement Of ADPKD Cells Compared With Nonsecreted Form

Keywords: ADPKD; neutrophil gelatinase-associated lipocalin; 3D culture 

NEW HERBS FOR 

1. Introduction

Polycystic kidney disease (PKD), one of the most common inherited diseases, is characterized by the development of fluid-filled cysts along multiple segments of the nephron [1]. ADPKD is the most common form of PKD, which is an adult-onset PKD for which most patients do not develop symptoms until they are in their forties [2]. ADPKD is the result of mutations in either PKD1 (85%) or PKD2 (15%) genes that encode polycystin-1 (PC1) and polycystin-2 (PC2), respectively [3,4]. Defects in PC1 or PC2 are associated with abnormal increases in the proliferation of renal epithelial cells and further cause cyst formation [5–7]. As ADPKD progresses, cysts enlarge and disrupt normal kidney architecture, eventually leading to kidney failure. However, the mechanism involved in ADPKD is complicated and remains to be explored. Functional loss of PC1 and/or PC2 in ADPKD leads to reduced intracellular calcium concentrations, alleviating calcium-sensitive adenylyl cyclase (AC) and further resulting in an increased level of intracellular cyclic adenosine monophosphate (cAMP) [8,9]. cAMP stimulates epithelial cell proliferation and fluid secretion to promote cyst enlargement by activating protein kinase A (PKA) and the downstream Ras/B-Raf/MEK/ERK pathway [10,11]. It has been demonstrated that the dysfunction of autophagy occurs in ADPKD zebrafish and mice in vitro [12] and rat in vivo models [13]. Autophagy is a crucial mechanism that maintains cell and organ homeostasis by degrading and recycling organelles, long-living proteins, and nutrients [14,15]. Cross-talk occurs between the mTOR pathway of autophagy and apoptosis pathways, which is dysregulated in PKD [16]. Treatment with autophagy activators, such as rapamycin and carbamazepine, can significantly attenuate cyst formation [12]. Previously, we established an ADPKD mouse model called Pkd1L3/L3 (in C57BL/6 genetic background) [17] by inserting a loxP site and a loxP-flanked mc1-neo cassette in introns 30 and 34 of the Pkd1 gene, respectively (L3 means three loxP sites within the allele), to generate the conditional gene mutation. Pkd1L3/L3 mice produced low levels (20% of the control) of PC1, which induced aggressive cyst growth and kidney volume expansion with increasing age [17]. In addition, an ADPKD cell model (2L3 cell line) was established by SV40 virus transduction of primary renal epithelial cells from Pkd1L3/L3 mice to induce immortalized cell lines [18]. 

Neutrophil gelatinase-associated lipocalin (NGAL), a 22-kD secreted protein, is regarded as a biomarker for kidney injuries, including PKD in adults [19] (but not in children [20]), chronic kidney disease [21], and acute kidney ischemia [22]. The mechanism of NGAL protein increasing in patient urine is not clear, it has been proposed that accumulated NGAL in urine relates to the deciliation of the renal epithelial cells as a result of injury [23]. Some studies have indicated that NGAL plays a protective role against renal injuries, thelocation of ischemia [24], and suppression of cyst progression [25], whereas some studies suggested NGAL might be involved in cyst progression [26] and tumor development [27]. NGAL receptor (NGAL-R, Slc22a17) is expressed in multiple epithelial tissues, including renal nephrons [28]. NGAL and NGAL-R coupling can sequester intracellular iron and further induce apoptosis [29], which is associated with an iron-depletion strategy of the innate immune system against bacterial infection [30].

Administration of exogenous NGAL has been shown to increase apoptosis of Pkd1−/− cells and further decrease cyst growth in vitro [25]. In previous work, we generated Pkd1L3/L3 × NGAL−/− mice and Pkd1L3/L3 × NGALTg/Tg mice by intercrossing Pkd1L3/L3 mice with NGAL knockout and transgenic mice, respectively [31]. We found that overexpression of NGAL in an ADPKD mouse model (Pkd1L3/L3 × NGALTg/Tg mice) reduced cyst progression and prolonged the lifespan of Pkd1L3/L3 mice, accompanied by changes in a series of molecular pathways involved in proliferation, apoptosis, and fibrosis [31]. However, the underlying mechanism of the effects of NGAL in alleviating the progression of ADPKD remains enigmatic.

In this study, a 3D cell culture platform was established to test the cyst progression of Pkd1L3/L3 renal epithelial cells (2L3 cells) with the addition of recombinant mouse NGAL (mNGAL) protein or overexpression of transgenic NGAL in vitro. In addition, the effects of mental on the proliferation, apoptosis, and autophagy-related pathways were explored. Furthermore, the mechanism of NGAL in 2L3 cells was elucidated by examining the effects of overexpression of secreted NGAL (pN + LS) and nonsecreted NGAL (pN − LS) in 2L3 cells. The effect and conceivable mechanism of NGAL protein in ADPKD cells were partially explored in this study.

2. Materials and Methods 

2.1. Cell Lines 

The Pkd1L3/L3 (2L3) cell line was established by SV40 virus transduction of primary renal epithelial cells from 2L3 mice, an ADPKD mouse model, to induce an immortalized cell line [17,18]. Primary renal cells were infected in their second passage by using a LentiSV40 virus immortalization kit (Capital Biosciences Inc., Gaithersburg, MD, USA). The cells were incubated in 5% CO2 at 37 ◦C and treated with polybrene (8 µg/mL) overnight in a six-well plate. On the next day, the viral supernatant was discarded, and the cells were incubated in a fresh medium. After reaching confluence, the cells were subcultured into a 10-cm dish with culture medium and were considered passage one (P1) cells. SV40-transformed clones were selected, and isolated by using cloning rings [17,18]. The M-1 cell line (Pkd1+/+) purchased from BCRC (Hsinchu, Taiwan) was used as the wild-type cell line. 

2.2. Cell Culture

Cells were passed every three days. To test the IC50 of mNGAL in ADPKD cells, 2L3 cells were seeded into 24-well plates (6000 cells/well) with 500 µL of the medium described above and incubated for 16 h. Cells were then treated with recombinant mNGAL (0, 20, 40, 80, 160, 320 µg/mL) or rapamycin (1 µM, from Sigma, St. Louis, MO, USA) for another 0, 8,24, or 48 h (n = 4). 

2.3. Lectin Staining

To check whether M-1 and 2L3 cells were from renal tubules of similar origins, lectin staining with Lotus tetragonolobus lectin (LTL, which is a differentiated proximal tubule marker labeled with fluorescein) and Dolichos biflorus agglutinin (DBA, which is a differentiated collecting duct marker labeled with rhodamine) was performed. LTL and DBA were purchased from Vector Laboratories (Burlingame, CA, USA). For observation of cells, cells cultured on the coverslip were washed with phosphate-buffered saline (PBS) three times for 10 min and then fixed with 4% paraformaldehyde (PFA) in PBS for 30 min. Following permeabilization with 0.4% Triton X-100 in PBS (PBST) for 20 min, the cells were blocked with 10% horse serum in PBST for 1.5 h. The cells were stained with LTL (20 µg/mL) and DBA (20 µg/mL) for 1 h at 37 ◦C. Nuclei were stained with DAPI (1 µg/mL) for 5 min. Samples were washed with PBST three times for 10 min between the steps.

2.4. DNA Lysate and Genotyping

For Pkd1 gene genotyping, PCR forward (50 -TGTGTTGTTCTTTGTGGCAGTCAG-30 ) and reverse (50 -ATTCTCAATGACTGACTTGGGCTC-30 ) primers were used to amplify genomic DNA sequences. Samples were heated at 94 ◦C for 5 min, and then 30 cycles (94 ◦C for 1 min, 56 ◦C for 1 min, 72 ◦C for 1 min) were performed followed by 72 ◦C for 10 min and 25 ◦C for 5 min. After amplifying by PCR, the DNA products were mixed with 6× loading dye and analyzed using 1% agarose Tris-borate-EDTA (TBE) gel.

2.5. Quantitative Reverse Transcription Real-Time Polymerase Chain Reaction (RT–qPCR)

Total RNA from the cell line was isolated using a MagQu gDNA extraction kit (MagQu, New Taipei City, Taiwan) followed by DNase I treatment (Worthington, Lakewood, NJ, USA) and stored in ddH2O at −80 ◦C. The quality and concentration of RNA were determined using a 1% TBE agarose gel and a NanoDrop spectrophotometer (Thermo Fisher Scientific). Template RNA was reverse transcribed to cDNA using a RevertAid First Strand cDNA synthesis kit (Thermo Fisher Scientific) in the presence of RNaseOUTTM RNase inhibitor (G-Biosciences, St. Louis, MO, USA). Each reaction was prepared in a total volume of 20 µL, including total RNA (100 ng), Oligo (dT)18 primer (1 µL), 5X Reaction Buffer (4 µL), RiboLock RNase Inhibitor (20 U), dNTP Mix (1 mM), RevertAid Reverse Transcriptase (200 U), and nuclease-free water. For first-strand cDNA synthesis, samples were heated at 42 ◦C for 1 h, and the reaction was then terminated by heating at 70 ◦C for 1 min.

Quantification of gene expression was performed using a StepOne™ Real-Time PCR System (Applied Biosystems, Norwalk, CT, USA). Each sample was prepared in a total volume of 20 µL, including gene-specific forward (100 nM), reverse (100 nM) primers (Table 1), cDNA template (100 ng), ORATM SEE qPCR Green ROX H Mix (1×, high GmbH, Kraichtal, Germany) and PCR water. The samples were heated at 95 ◦C for 2 min, followed by 40 cycles (95 ◦C for 5 s, 65 ◦C for 25 s) and then melting curve analysis (95 ◦C for 15 s, 60 ◦C for 1 min, 95 ◦C for 15 s) using a StepOne™ Real-Time PCR System. Data were analyzed with StepOne™ Software (v2.3). Gene expression of Gapdh was used for normalization. 

2.6. Cell Viability Assay

Cell viability was determined using thiazolyl blue tetrazolium bromide (MTT assay) (Sigma). MTT solution (0.5 mg/mL) was added to each well and incubated at 37 ◦C for 2 h.To solubilize the purple formazan crystals, dimethyl sulfoxide (DMSO, Sigma) was added to each well after removing the medium and MTT solution. The optical density (OD) of each sample at 570 nm was measured using an ELISA reader (Thermo Fisher Scientific). 

2.7. Cell Proliferation Assay

The bromodeoxyuridine (BrdU) cell proliferation assay (Millipore, Burlington, MA, USA) was performed to analyze the proliferation of M-1 and 2L3 cells. To compare the proliferation ability of M-1 and 2L3 cells, cells were cultured in 96-well plates (5 × 103 cells/well)for 24 h. BrdU was added to the medium 2 h before the end of the incubation period. To explore the effect of mNGAL on 2L3 cell proliferation, 2L3 cells were first cultured in 96-well plates (2 × 103 cells/well) for 16 h followed by mNGAL treatment for another 8, 24, and 48 h. BrdU was added to the medium 2 h before the end of the meal incubation period. Cells were then fixed with fixing solution (200 µL/well) at 37 ◦C for 30 min, followed by incubation with BrdU detection antibody (100 µL/well) at 37 ◦C for 1 h and peroxidase-conjugated goat anti-mouse antibody (100 µL/well) at 37 ◦C for 30 min. The cells were washed three times with Wash Buffer and blotted dry on paper towels between the above-described steps.

After incubation with TMB peroxidase substrate (100 µL/well) at 37 ◦C for 30 min in the dark, the reaction was stopped with stop solution (100 µL/well). Finally, the OD450 of each sample was measured using an ELISA reader (Thermo Fisher Scientific). 

2.8. Apoptosis Assay

For analysis of apoptosis using flow cytometry, 2L3 cells were seeded into 6-well plates (1.8 × 105cells/well) with 2 mL medium and incubated for 16 h. Cells were then treated with mNGAL (0, 600, 1200 µg/mL) for another 0, 4, or 12 h, collected and stained with Annexin V-FITC and PI (BD Biosciences, San Jose, CA, USA) for analysis by flow cytometry at 4 and 12 h, respectively (n = 3, for control and mNGAL). To establish compensation and quadrants before the analysis, unstained cells, cells stained with Annexin V-FITC only, and cells stained with PI only were used. 

2.9. Western Blot Analysis 

Cells were harvested and lysed with RIPA supplemented with protease inhibitor cocktail (1:100) and phosphatase (1:100). Following incubation for 30 min at 4 ◦C, cell lysates were centrifuged for 15 min at 13,400× g (Eppendorf 5415R, Hamburg, Germany) at 4 ◦C. The concentrations of protein were measured using the BCA assay. Equal amounts of protein (25 µg protein with 1:2 loading dye/well) were electrophoretically separated by SDS–PAGE (10%) and transferred for 1 h to PVDF membranes. Membranes were then blocked with 5% skimmed milk in Tris-buffered saline with 0.1% Tween-20 (TBST) for 2 h at room temperature. After washing with TBST three times for 10 min, membranes were incubated with primary antibodies (Table 2) overnight at 4 ◦C, followed by a 1-h incubation with HRP-conjugated secondary antibodies (Table 3). 

2.10. Three-Dimensional Cell Culture (3D Culture)

The 3D culture was performed using the Matrigel matrix (Corning, NY, USA) to assess cyst formation. The 2L3 cells were seeded in 96-well plates (1000 cells/well) on preformed Matrigel (40% in medium). After the cells awere ttached to the gel, more Matrigel was added to the cells to form a sandwich-like pattern (Figure 6). Cells were maintained in 3D culture at 37 ◦C and 5% CO2. Cells were treated with forskolin (20 mM, Sigma) from Day 1 to Day 3 to induce cyst formation. After the induction of cyst formation with forskolin, cells were treated with or without mNGAL (2 mg/mL) from Days 4 to 8, and the medium was changed every day. Cyst formation was observed under a microscope every day, and the cyst diameter, area, and number were measured with ImageJ. Immunocytochemistry (ICC) was performed on Day 8 on the plate or coverslips. Observation of cysts was performed by confocal microscopy (ZEISS, Oberkochen, Germany).

2.11. Immunocytochemistry (ICC) 

For observation of cysts, cysts cultured with Matrigel in wells or on coverslips were washed three times with PBS for 10 min and then fixed with 4% paraformaldehyde (PFA) in PBS for 30 min. Following permeabilization with 0.4% PBST for 20 min, cysts were blocked with 10% horse serum in PBST for 2 h. Cysts were stained with primary antibodies against E-cadherin (1:500; Cell Signaling Technology, Danvers, MA, USA) and actin (1:1000; Millipore) overnight at 4 ◦C and then stained with fluorescent secondary antibody (Table 3) in the dark for 1 h at room temperature. Nuclei were stained with DAPI (1 µg/mL) for 5 min. Samples were washed with PBST three times for 10 min between the steps.

2.12. Confocal Microscopy

To confirm that the spherical constructs observed in 3D culture were cysts, coverslips from 3D culture were mounted onto slides with Fluoromount-G mounting medium (SouthernBiotech, Birmingham, AL, USA) and imaged using a confocal microscope. Confocal imaging was performed with a 63× oil immersion objective. Different optical z-sections of a 2L3 cyst in 3D culture were obtained using the Z-stack module in the ZEISS ZEN microscope software (Oberkochen, Germany). Optical sections of cysts were reconstructed into Z-stack or 3D video using ZEISS ZEN microscope software.

2.13. Plasmid Construction and Virus Infection

The mouse NGAL sequence (534 bp) was inserted into the pSecTag2B vector (Invitrogen, Carlsbad, CA, USA) in the frame and downstream of a murine Igκ-chain leader sequence to overexpress the secreted NGAL protein. Conversely, the murine Igκ chain leader sequence in the above-described plasmid was removed to overexpress the nonsecreted NGAL protein. pSecTag2B-NGAL with or without the leader sequence (pN + LS or pN − LS, respectively) was designed to study the association between NGAL and the NGAL receptor. To ensure the correct insertion of constructs, NheI and NotI were used to digest pN + LS or pN − LS with NEBuffer 2 (New England Biolabs, Ipswich, MA, USA) at 37 ◦C for 2 h followed by analysis using a 1% TBE gel. 

To generate stable clones, 2L3 cells were incubated in DMEM/F12 medium (300 µL/well) supplemented with 10% heat-inactive FBS, 1% P/S, polybrene (8 µg/mL), and lentivirus (200 µL) in a 6-well plate at 37 ◦C for 24 h. The virus supernatant was then discarded, and the cells were washed with PBS and incubated with fresh medium containing blasticidin (4 µg/mL) for the selection of infected cells. The selection medium was changed every 3 days. Infected cells were harvested one week after blasticidin selection for western blotting and RT–QPCR to analyze the level of mNGAL expression. 

2.14. Enzyme-Linked Immunosorbent Assay (ELISA)

Quantification of NGAL secreted by cells was performed using a mouse lipocalin-2 (Lcn2) solid-phase sandwich ELISA kit (Thermo Fisher Scientific). The supernatant medium of cells was collected, centrifuged to remove cell debris, and stored at −80 ◦C. For the standard curve, 8× series dilutions of standard mouse NGAL protein were prepared (0, 0.024, 0.195, 1.563, 12.5, 100 ng/mL). Standards (100 µL/well) and samples (100 µL/well of 10 µg total protein from cell lysate or non-diluted supernatant) were added to the 96-well plate and incubated at 37 ◦C for 2.5 h. Each well was incubated with biotin (100 µL/well) conjugate at 37 ◦C for 1 h and then with streptavidin–HPR solution (100 µL/well) at 37 ◦C for 45 min with gentle shaking. The solution was discarded, and the well was washed with

After incubation with TMB substrate (100 µL/well) at 37 ◦C for 30 min in the dark, the reaction was stopped by the addition of stop solution (50 µL/well). Finally, the OD450 of each sample was measured using an ELISA reader (Thermo Fisher Scientific).

2.15. Statistical Analysis

All data are shown as the mean ± standard deviation (SD). For multiple mean comparisons, data were analyzed using the Student's t-test and one-way analysis of variance (ANOVA) with Tukey's post hoc tests in R studio. Differences were considered statistically significant when p value < 0.05.