Albumin stimulates interleukin-8 expression in proximal tubular epithelial cells in vitro and in vivo (original) (raw)

Reagents. Medium, reagents for cell culture, Ab’s for cell characterization, PI3K inhibitors (wortmannin and LY294002), PKC inhibitors (staurosporin and GF109203X), and general chemicals were purchased from Sigma-Aldrich Ltd. Co. (Paisley, United Kingdom). HSA was obtained from CSL Laboratory (CSL Limited, Parkville, Victoria, Australia). Other brands of HSA were obtained from Calbiochem-Novabiochem Corp. (San Diego, California, USA) and Sigma-Aldrich. The endotoxin level in all albumin preparations were < 10 EU/ml, as determined using the QCL-1000 limulus amebocyte lysate kit (BioWhittaker Inc., Walkersville, Maryland, USA). Antibiotics, sera, agarose, and DNA size markers were obtained from Invitrogen Corporation (Carlsbad, California, USA). Reagents for cDNA synthesis were obtained from Life Technologies Inc. (Paisley, United Kingdom) and Promega Corp. (Madison, Wisconsin, USA), and those for PCR and cycle sequencing were from Perkin Elmer Life Sciences Inc. (Boston, Massachusetts, USA). The enzyme immunoassay kit for detection of IL-8 was purchased from Bender MedSystems (Vienna, Austria). Ab’s for detection of DNA-bound NF-κB by flow cytometry and cell membrane-permeable inhibitory peptide or mutant peptide for NF-κB were from Biomol Research Laboratories (Plymouth Meeting, Pennsylvania, USA) and Dakopatts (Glostrup, Denmark). The fluorescence probe for intracellular reactive oxygen species detection was from Molecular Probes Inc. (Eugene, Oregon, USA). Reagents and Ab’s for in situ hybridization were from Boehringer Mannheim GmbH (Mannheim, Germany). Anti–Tamm-Horsfall glycoprotein was from Chemicon International (Temecula, California, USA). All other Ab’s were from DAKO A/S (Glostrup, Denmark).

Cell culture. Human colorectal epithelial cell line (HT-29) and human lung type 2 epithelial cell line (A549) were obtained from American Type Culture Collection (Rockville, Maryland, USA). Human proximal tubular epithelial cells (PTECs) were isolated according to a method described previously (16). Briefly, renal cortical tissue was obtained from kidneys removed for circumscribed tumors. Histological examination of these kidney samples revealed no renal pathology. Cortical specimens were cut into small cubes and passed through a series of mesh sieves of diminishing pore size. PTECs were collected on the 53-μm sieve and digested with collagenase (750 U/ml) at 37°C for 15 min. Tubular cells were isolated by centrifugation and grown in a 1:1 mixture of DMEM and Ham’s F12 medium supplemented with 10%FCS, hydrocortisone (40 ng/ml), L-glutamine (2 mM), benzyl penicillin (100 IU/ml), and streptomycin (100 μg/ml). The cells were incubated at 37°C in 5% CO2 and 95% air. They were characterized to be of proximal tubular origin by immunofluorescence and enzyme histochemistry: cells stained positively for cytokeratin, vimentin, and alkaline phosphatase, but negatively for Tamm-Horsfall glycoprotein, factor VIII–related antigen, and α-smooth muscle actin. Scanning electron microscopy demonstrated the presence of numerous apical microvilli of a rudimentary brush border with reassembly of tight junctions. Experiments were performed with cells up to the third passage, because it has been shown that there are no phenotypic changes up to this passage number (17). In all experiments, there was a “growth-arrest” period of 48 h in serum-free medium prior to stimulation. Results were obtained from PTECs cultured from the kidneys of three different donors.

RNA extraction and cDNA synthesis. Total RNA was extracted from PTEC monolayers by a modification of the method by Chomczynski and Sacchi (18). Briefly, cells were lysed in a commercially available lysis buffer, which contained a mixture of phenol and guanidinium thiocyanate in a monophasic solution (RNA Isolator; Genosys Biotechnologies, Cambridge, United Kingdom). This was followed by chloroform extraction and isopropanol precipitation. RNA was quantified by absorbance at 260 nm. Five micrograms total RNA were reverse transcribed to cDNA with Superscript II reverse transcriptase (Life Technologies Inc.) in a 20-μl reaction mixture containing 160 ng oligo-(dT)12–18, 500 μM of each dNTP, and 40 U RNase inhibitor for 10 min at 37°C, 60 min at 42°C, and 5 min at 99°C. The cDNA was stored at –20°C until further use.

Analysis of IL-8 gene expression. PTECs, A549, or HT-29 were grown to confluence in six-well cell culture plates (Falcon; Becton-Dickinson UK Ltd., Cowley, United Kingdom), growth arrested, and exposed to albumin (1.25–20 mg/ml) for defined time periods (3–48 h) at 37°C. Total cellular RNA was then extracted and reverse transcribed to cDNA. PCR was carried out as described previously (19). The oligonucleotide sequences of cDNA primers for IL-8, designed from GenBank, were as follows: forward, ATG ACT TCC AAG CTG GCC GTG CT and reverse, TCT CAG CCC TCT TCA AAA ACT TCT, yielding an amplified product of 298 bp. PCR reactions were carried out in a DNA thermal cycler (MJ Research, Watertown, Massachusetts, USA), with 33 cycles of amplification at an annealing temperature of 55°C. For quantification, human α-actin primers were included in every reaction as an internal control. The primers, based on the known sequence of human α-actin cDNA (20), were as follows: forward, GGA GCA ATG ATC TTG ATC TT, and reverse, TCC TGA GGT ACG GGT CCT TCC, yielding an amplified product of 204 bp. The PCR products were separated by 1.5% wt/vol agarose gels, stained with ethidium bromide (Sigma-Aldrich Co. Ltd.), and the gel image was captured and analyzed using the Gel Doc 1000 Densitometry System and Quantity One (Bio-Rad Laboratories Inc., Hercules, California, USA). The product yield was expressed as a ratio to α-actin. The sequence of IL-8 amplicon was verified by standard cycle sequencing technique (21).

Assay of IL-8 protein in culture supernatants and urine. PTECs, A549, or HT-29 were grown to confluence in six-well cell culture plates, growth arrested, and exposed to albumin (1.25–20 mg/ml) for defined time periods (3–48 h) at 37°C. For some experiments, PTECs were cultured for 24 h with HSA from other suppliers (Sigma-Aldrich Co. Ltd. or Calbiochem-Novabiochem), or with 10 mg/ml human transferrin, human IgG, trypsin-digested HSA (HSA digested with trypsin-conjugated agarose for 2 h), or boiled HSA (as albumin started to precipitate at 75°C and beyond to assume a gelatinous state, an aggregation by intermolecular association of the protein molecules that prevented its direct application to the cell culture system, boiling of diluted HSA was performed to achieve thermal denaturation; ref 22). Supernatants were collected and stored at –70°C until further use, while cell number was counted after trypsinization. Twenty-four–hour urine samples were collected from proteinuric subjects, and representative aliquots were stored at –70°C until assay. Detection of the IL-8 level in culture supernatants and urine was carried out on a commercially available assay kit, according to the manufacturer’s instructions (Bender MedSystems). The detection sensitivity and intra-assay coefficient of variation is 11 pg/ml ± 3.8% for IL-8.

Examination of polarity of IL-8 secretion by PTECs. To examine the polarity of IL-8 secretion, second-passage PTECs were seeded into 12-well Transwell chambers (0.4-μm pore size, 10-mm diameter; Corning Costar Corp., Cambridge, Massachusetts, USA) and maintained at confluence for 2 days before use. Different concentrations of albumin (2.5–10 mg/ml) were added either to the upper or lower chamber, which corresponded to the apical or basolateral side of the cells. respectively, while medium alone (1.5 ml) was added to the other compartment. After 24 hours of incubation, supernatants in the upper and lower chambers were harvested separately and assayed for IL-8, while cell number was counted. All experiments were performed in triplicate.

Endocytosis of FITC-HSA. PTECs were grown to confluence in 24-well cell culture plates, growth arrested, and exposed to fluorescein-labeled HSA (FITC-HSA, 1.25–20 mg/ml) for 30 min or 5 mg/ml FITC-HSA for 0–4 hours at 37°C. The cells were then harvested, fixed with 1% paraformaldehyde, examined by fluorescence microscopy, and counted with a fluorescence microplate reader (Tecan Austria GmbH, Salzburg, Austria). Experiments were performed in triplicate.

Measurement of DNA-bound NF-κB by flow cytometry. DNA-bound NF-κB was determined by flow cytometry, according to a method described previously (23). Briefly, PTECs were cultured to confluence in six-well plates and were growth arrested for 24 h in serum-free culture medium. The cells were then incubated with or without albumin for 30 min and were harvested by trypsinization. Cells were washed with wash buffer (PBS with 2% FBS) and lysed with lysis buffer (10 mM PIPES, 0.1 M NaCl, 2 mM MgCl2, 0.1% Triton X-100, pH 6.8). The isolated nuclei preparations were stained with rabbit polyclonal Ab (Rel A or NF-κB1; Biomol Research Laboratories) or nonimmune rabbit immunoglobulins at a final concentration of 10 μg/ml for 30 min. After further washing, 100 μl of FITC-conjugated swine anti-rabbit immunoglobulins (Dakopatts) diluted 1:20 in permeabilization buffer were added and incubated for a further 30 min. The nuclei were washed and analyzed for DNA-bound NF-κB by flow cytometer (Coulter EPICS XL analyzer; Coulter Electronics Ltd., Miami, Florida, USA). Results were analyzed using Flowjo software (Tree Star Software, San Carlos, California, USA) and were expressed as mean fluorescence intensity.

Effect of NF-κB inhibitors on albumin-induced IL-8 synthesis. To study a possible relationship between albumin-induced IL-8 production and the transcriptional factor NF-κB, PTECs were plated on six-well tissue culture plates and, when confluent, treated with the NF-κB inhibitor pyrrolidine dithiocarbamate(PDTC; 5 or 25 μM; Sigma-Aldrich Co. Ltd.) (24) or 100 μg/ml cell membrane-permeable peptides (25) (SN50M or SN50; Biomol Research Laboratories) for 1 h before and during 6-hour incubation with 10 mg/ml albumin. (The sequence of SN50 was AAVALLPAVLLALLAPVQRKRQKLMP, which can inhibit the translocation of the active NF-κB complex into the nucleus, and the sequence of the control peptide SN50M was AAVALLPAVLLALLAPVQR_NG_QKLMP. The nuclear localization sequence of NF-κB was underlined, and the mutant residues in SN50M were in italics.) At the end of the incubation, IL-8 was measured in supernatants.

Electrophoretic mobility shift assay. A standard electrophoretic mobility shift assay (EMSA) was used to further examine the role of the transcriptional factor NF-κB in albumin-induced IL-8 production. PTECs were plated on a T25 tissue-culture flask and, upon confluence, treated with albumin (5 or 10 mg/ml) alone, or 25 μM PDTC (Sigma-Aldrich Co. Ltd.), or 100 μg/ml cell membrane-permeable peptides (SN50M or SN50) for 30 min before and during 1-hour incubation with albumin (10 mg/ml). At the end of the incubation, nuclear extract was prepared using NE-PER nuclear extraction reagent (Pierce Chemical Co., Rockford, Illinois, USA) and stored at –70°C until the assay was performed. Gel-shift oligonucleotide for NF-κB (AGTT-GAGGGGACTTTCCCAGGC; the core sequence was underlined) was biotinylated using biotin 3′ end-labeling kit (Pierce Chemical Co.), and the EMSA was carried out with the LightShift chemiluminescent EMSA kit (Pierce Chemical Co.) according to the manufacturer’s instruction.

Measurement of intracellular reactive oxygen species. The intracellular formation of reactive oxygen species (ROS) was detected by the fluorescence probe 5- (and 6-) chloromethyl-2′, 7′-dichlorodihydrofluorescein diacetate (CM-H2DCFDA; Molecular Probes Inc.). PTECs (106/ml) were cultured in the presence of different doses of HSA (1.25–20 mg/ml) for 30 min and then loaded with 0.1 μg/ml CM-H2DCFDA. The cells were placed on ice. ROS production, expressed as DCF mean fluorescence intensity (MFI), was immediately measured by flow cytometry using a Coulter EPICS XL analyzer (Coulter Electronics Ltd.). Results were expressed as percentage of MFI of control cells incubated with culture medium alone.

Effect of exogenous H2O2 on NF-κB translocation and IL-8 secretion in PTECs. Confluent PTECs grown on six-well tissue-culture plates were treated with H2O2 (200 μM) for 1 h for determination of NF-κB activation by EMSA or with H2O2 (50, 100, 200, or 400 μM) for 24 h for assay of IL-8 protein in culture supernatants by ELISA.

Effect of PI3K inhibitors or PKC inhibitors on albumin-induced ROS generation, NF-κB translocation, and IL-8 secretion. Confluent PTECs grown on six-well tissue-culture plates were treated with the PI3K inhibitors, wortmannin (500 nM) or LY294002 (100 μM), or the PKC inhibitors, staurosporin (100 nM) or GF109203X (5 μM), 1 h before and during incubation with albumin (10 mg/ml). At the end of the incubation period (30 min for ROS assay, 1 h for NF-κB EMSA, and 24 h for supernatant IL-8 protein assay), intracellular ROS generation, NF-κB translocation, and IL-8 secretion were determined by flow cytometry, EMSA, and ELISA, respectively, as described above.

Immunohistochemical localization of IL-8 production on human nephrotic kidneys and cultured PTECs. To localize the site of IL-8 production in vivo, immunohistochemical staining was performed on renal biopsy tissues obtained from nephrotic subjects with nonproliferative glomerulopathies (minimal change nephrotic syndrome, diabetic and hypertensive nephrosclerosis), and from patients with no or minimal proteinuria and histology of no or minor abnormality as control.

Paraffin-embedded human kidney tissues were sectioned at a thickness of 4 μm, and the sections were deparaffinized with xylene and then rehydrated through a descending gradient of ethanol. IL-8 expression on paraffin sections was determined by immunohistochemical staining using monoclonal anti-human IL-8 Ab (Santa Cruz Biotechnology Inc., Santa Cruz, California, USA). Briefly, the slides were incubated with 0.5% H2O2 for removal of endogenous peroxidase activity. Nonspecific binding was blocked by incubation of the slides for 30 min with blocking buffer (5% normal goat serum and 3% BSA in PBS). The sections were then incubated with anti–IL-8 (10 μg/ml) Ab’s overnight. The bound murine anti–IL-8 Ab’s were visualized using the DAKO Envision Plus System (DAKO Corp., Carpinteria, California, USA). For some sections, infiltrating leukocytes were demonstrated by staining with monoclonal anti-CD44 (DAKO Corp.). To confirm the specificity of anti–IL-8 used in the present study, cultured PTECs grown in chamber slides with or without albumin (10 mg/ml) were stained with anti–IL-8 as described above. Ab preabsorbed with IL-8–immunizing peptides was used as control.

A renal histopathologist without prior knowledge of clinical or laboratory data examined the tissues histologically and evaluated the expression of IL-8 staining using an arbitrary 0–3+ scale (absent or minimal, mild, moderate, and marked). For glomerular staining, the scoring criteria were as follows: minimal if <5% glomerular cells were positive; mild if 5% to <25% cells were positive; moderate if 25% to <50% cells were positive; and marked if 50% or more cells were positive. For tubular staining, the grading criteria were as follows: minimal if <10% cortical tubular cells were positive; mild if 10% to <40% cells were positive; moderate if 40% to <80% cells were positive; and marked if 80% or more cells were positive.

Oligonucleotides and labeling. A 42-mer sequence of mRNA was selected for human IL-8. Antisense oligonucleotides for human IL-8 corresponded to bases 2435–2476 of human IL-8 (26). The selected sequence was significantly different from other known sequences deposited in the latest release of the gene bank data (GenBank, Release 128, March 2002; http://www.ncbi.nlm.nih.gov/Genbank/). The oligonucleotide was synthesized on an automatic DNA synthesizer (391, PCR-MATE EP; Applied Biosystems Inc., Foster City, California, USA) and was labeled using a digoxigenin (DIG) oligonucleotide tailing kit according to the current protocol (1417 231; Boehringer Mannheim GmbH, Mannheim, Germany).

Tissue localization of IL-8 gene expression by in situ hybridization. Nonradioactive in situ hybridization was performed according to a modified method developed in our laboratory (27). In brief, the specimens were cut to a thickness of 4 μm and placed on glass slides coated with 3-aminopropyltriethoxysilane (A3684; Sigma-Aldrich, St. Louis, Missouri, USA). The sections were fixed with 4% paraformaldehyde in PBS and then deproteinized using HCl and proteinase K (P-4914; Sigma-Aldrich). After prehybridization, the sections were hybridized with DIG-labeled oligonucleotide probe in prehybridization buffer (4× SSC, 0.5 M sodium phosphate, 2.5× Denhalts solution, salmon testis DNA [D7656; Sigma-Aldrich], and transfer RNA [R5636; Sigma Aldrich]) at 40°C for 16 h. After washing with 0.075% BRIJ (430 AG-6; Sigma-Aldrich) in 2× SSC twice for 20 min each and 0.5× SSC twice for 20 min each, at room temperature, sections were stained immunohistochemically to visualize the hybridized DIG-labeled probe using mouse monoclonal anti-DIG Ab (1333 062; Boehringer Mannheim GmbH), HRP-conjugated rabbit anti-mouse Ab (DAKO P260; Dakopatts), and HRP-conjugated swine anti-rabbit Ab (DAKO P399; Dakopatts), successively. Color was developed by reaction with H2O2 and diaminobenzidine tetrahydrochloride. Finally, sections were counterstained with methyl green and mounted. Cells clearly stained in the cytoplasm or stained with a perinuclear pattern were identified as IL-8 mRNA–positive cells. On the other hand, cells with nuclei stained with methyl green alone were considered negative for IL-8 mRNA. To evaluate the specificity of the signals for IL-8 mRNA, we performed three control experiments including pretreatment of RNase, a study with a sense probe, and a competitive study, as described previously (28, 29).

Statistical analysis. All data were expressed as means ± SD, unless otherwise specified. Statistical analysis was performed using SPSS statistical software (Statistical Package for the Social Sciences Inc., Chicago, Illinois, USA). Intergroup differences for continuous variables were assessed by one-way ANOVA. Post hoc multiple comparisons using Tukey’s Honestly Significant Difference test were used to determine the significance of differences between groups. Comparison of the score for the staining intensity by immunohistochemistry between groups was done using the Mann-Whitney U test. Linear regression analysis was performed to determine the correlation between the staining score and age, serum creatinine, or proteinuria in the nephrotic group of subjects. Categorical data were compared by the Fisher exact test or the X2 test, as appropriate. A P value of less than 0.05 was considered statistically significant.