Between January 2013 and January 2016, 40 patients with IgAN (IgAN group), 32 patients with RT (RT group), and 52 patients with RT associated with IgAN (IgAN + RT group) diagnosed by renal biopsy were enrolled in this study. The age ranges of the patients in the three groups were 27 years–54 years, 25 years–59 years, and 24 years–57 years old, respectively, and the male to female ratios were 21:19, 17:15, and 23:29, respectively. None of the patients had hepatitis, systemic lupus erythematosus, allergic purpura, or other secondary nephritis, and none had been administered any glucocorticoid and immunosuppressant before renal biopsy. Pure protein derivative of tuberculin (PPD) (the Shanghai Institute of Biological Products, Shanghai, China) was used for the tuberculin skin test (TST), which was conducted as follows: PPD was injected intracutaneously through the forearm-palmaris, and the injection site was observed for a reaction about 72 h after injection. Patients were considered to be PPD positive if the reaction was >5 mm . Venous blood (3 mL) was collected from all patients after fasting. Serum was isolated from the blood samples and used for the detection of anti-tuberculosis antibody.
Urine culture for MTB
Urinary sediment (20 mL) was obtained from 24-h urine specimens and then centrifuged to obtain the precipitate. The sediment was mixed with 4% sodium hydroxide (NaOH) to facilitate the precipitation. The supernatant was removed and centrifuged under sterile conditions, and the precipitate was collected and mixed with 6% sulphuric acid. The specimens were then inoculated on a Roche slanted solid culture medium and incubated in a constant temperature incubator at 37°C and observed at regular time intervals. After 2 months of observation, bacterial growth on the culture medium indicated the presence of MTB.
Haematoxylin and eosin (HE) staining
Renal tissue specimens were fixed with 4% paraformaldehyde, washed with phosphate buffered saline (PBS), dehydrated with graded ethanol, and cleared with xylene. After the remaining xylene was washed away with distilled water, the tissue specimens were embedded in paraffin, sliced into 4-μm sections, and deparaffinised with ethanol. Some sections were stained with HE. Briefly, prepared renal tissue sections were stained with haematoxylin for 5 min, washed with distilled water, stained with 0.5% eosin for 2 min, dehydrated with ethanol, cleared in xylene, sealed with neutral gum, and then observed with an optical microscope. The remaining sections were stored at -20°C until use.
After adding 30% H2O2 to block endogenous enzyme activity, renal tissue sections from the patients were heated in antigen retrieval buffer. After cooling for 5 min, the sections were heated and cooled twice more. After cooling to a temperature that coincided with the room temperature (about 25°5), the sections were blocked by adding 5% bovine serum albumin (BSA) blocking buffer and incubating at room temperature for 20 min. The excess blocking buffer was removed, and then mouse anti-human ESAT-6 primary antibody (ab26246, 1:3000; Abcam, Cambridge, MA, USA) and mouse anti-human CFP-10 primary antibody (ab64754, 1:3000; Abcam) were added to the sections and incubated at 4°C overnight. Then, the sections were incubated with biotinylated goat-anti mouse IgG (ab6789; Abcam) at 37°C for 40 min. The sections were washed with PBS, and the antibody-antigen reactions were visualised by incubation with 3,3’-diaminobenzidine; Beijing Zhongshan Jinqiao Biotechnology Co., Ltd., Beijing, China). Images of the stained sections (three randomly selected fields from three sections of each specimen) were quantitatively analysed with the Image-ProPlus (Media Cybernetics, Rockville, MD, USA) to determine the integral optical density (IOD) values of the ESAT-6 protein and the CFP-10 protein.
The renal tissue sections were incubated with 1X sodium dodecyl sulphate buffer with mixing at 300 ×g until the sections were fully lysed. The lysed sections were then incubated on ice for 30 min and centrifuged for 4 min at 1200 ×g . The supernatant was removed and stored at -80°C. The concentration of the extracted proteins in the supernatant was determined using the BCA protein determination kit (AR0146; Boster Biological Technology Co., Ltd., Wuhan, China), which was then adjusted to 3 μg/μL. The protein samples were mixed with loading buffer and incubated for 10 min at 95°C, and then equal protein (30 μg) from each sample was separated by 10% polyacrylamide gel electrophoresis. The separated proteins were then transferred to a PVDF membrane (P2438; Sigma, St. Louis, MO, USA) by semi-dry electrotransfer and then blocked with 5% BSA for 1 h at room temperature. Then, the membrane was incubated with anti-ESAT-6 antibody (ab45073, 1:1000 in 5% BSA; Abcam) or rabbit anti-CFP-10 (ab45074, Abcam) and rabbit anti-α-smooth muscle actin (ab5694; Abcam), which was diluted to 0.2 µg/mL–1 µg/mL, in a refrigerator with shaking overnight. The membranes were washed three times with TBST for 10 min each and then incubated with the secondary antibody (ab6720, 1:10000; Abcam) in TBST for 4 h–6 h at 4°C. After incubation, the membrane was washed with TBST three times for 15 min each. The membrane was then incubated with chemiluminescence reagents A and B, mixed at 1:1, followed by the addition of photographic developer solutions. GAPDH was used as an internal reference. All immunoblotting bands were subjected to densitometric analysis.
Renal tissue sections from patients were heated to a boil in antigen retrieval buffer. After cooling for 5 min, the sections were heated and cooled two more times, and then cooled to room temperature. Then, the sections were incubated with mouse anti-human IgA1-FITC (ab99793, 1:50; Abcam) in a humidified chamber at 37°C for 30 min. The sections were washed with PBS (pH 7.2– pH 7.6) three times for 5 min each. After air drying, the sections were sealed with buffered glycerol and observed with a fluorescence microscope under a glass coverslip. Five high-power fields (400X) of each section were randomly selected, and the corresponding mean gray value (MGV) of the positively-stained areas was measured.
All data were analysed with SPSS 21.0 (SPSS, Inc., Chicago, IL, USA). Data have been presented as the mean ± standard deviation. The t-test was used for comparisons between two groups, and one-way analysis of variance (ANOVA) was used for comparisons of multiple groups. Counted data have been expressed as a rate or percentage, and two groups were compared using the chi-square test. For multiple groups with equal variance, the q test was performed for pairwise comparisons, and the non-parametric rank test was performed for group comparisons. A receiver operating characteristic (ROC) curve (α = 0.05) was plotted to evaluate the diagnostic value of the ESAT-6 and the CFP-10 proteins. P values less than 0.05 were regarded as statistically significant.