Patients
We enrolled 26 patients with biopsy-proven primary IgAN (IgAN group), 31 patients with other renal diseases (Disease control group, DC group) and 32 healthy volunteers (Healthy control group, HC group). All patients admitted to Peking Union Medical College Hospital between April 2016 and September 2016. The histologic diagnosis of IgAN was based upon the demonstration of mesangioproliferative changes on light microscopy and the concomitant presence of predominant or codominant mesangial deposition of IgA. Patients with lupus nephritis (LN), Henoch–Schönlein purpura, liver cirrhosis and other secondary causes of IgAN were excluded from the study. The diagnoses of DC group included membranous nephropathy (n=15), minimal change nephropathy (n=3), LN (n=1), diabetic nephropathy (n=2), focal segmental glomerulosclerosis (n=2), chronic pyelonephritis or interstitial nephritis (n=4), hypertensive renal damage (n=1) and sclerosing glomerulonephritis (n=2), obesity-related glomerulopathy (n=1).
Clinical and pathological manifestations
The demographic and clinical parameters of patients, including age, gender, blood pressure (BP), urinary red blood cells count, serum creatinine (SCr), urine creatinine (UCr) and 24-hour urinary protein excretion (24hUPro) were obtained immediately before renal biopsy. Estimated glomerular filtration rate (eGFR) was calculated using the Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) equation [31].
Medication history, including the usage of cortisone, immunosuppressant and renin-angiotensin system (RAS) blockers such as angiotensin-converting enzymes inhibitors (ACE-Is) and angiotensin II receptor blockers (ARBs) was also recorded.
Renal biopsies
For each patient, the diagnosis of IgAN was based on histologic assessment of renal biopsy tissue with hematoxylin and eosin, periodic acid–Schiff and Masson’s trichrome for light microscopy and staining with antibodies against IgA, IgG, IgM, C1q, C3, C4 and fibrinogen for immunofluorescence. IgAN was defined by the presence of at least 1+ (range, 0–4) IgA mesangial deposits as dominant or codominant immunoglobulins on immunofluorescence microscopy performed on frozen tissue. Pathological features were evaluated according to updated Oxford classification [32] and Lee grading [33], which were graded by a pathologist blinded to patients’ clinical data.
Urine samples
Spot morning urine samples from patients were collected on the same day of renal biopsy. All samples were centrifuged at 3000rmp/min for 10 min to remove cellular components and the supernatant was kept at -80°C until use. Spot morning urine from healthy controls were also collected and prepared in the same manner as for patients.
Generation of anti-J chain monoclonal antibody
J chain-GST recombinant peptide IGJ-pGEX-4T-1 (NCBI ID for J chain sequence: NM_144646) was synthesized and purified as an antigen to immunize BALB/c mice. Candidate hybridomas were established from splenocytes (Absea Biotechnology Ltd., Beijing, China). Hybridomas that produce anti-J chain monoclonal antibodies were selected by ELISA with the recombinant antigen peptide, by Dot-blot with the pIgA purified from multiple myeloma patients and saliva that contains J chain-sIgA by Western Blot.
Detection of urinary J-IgG and IgA-IgG immune complexes by ELISA
To measured urinary immune complexes, ninety-six well microtiter plates were coated with anti-J chain mAb or anti-IgA mAb (Absea Biotechnology Ltd., Beijing, China) in carbonate-bicarbonate buffer, pH 9.6, overnight at 4 °C. The plates were washed three times with phosphate buffered saline (PBS) containing 0.05% Tween 20 (PBST) and then blocked with 200 μL/well of 2% bovine serum albumin (BSA) for 2 h at 37℃. The urine samples were added to the same plate and incubated for 3h at 37 °C. Then the plates were washed and HRP-conjugated mouse anti-human IgG (Absea Biotechnology Ltd., Beijing, China) was added and incubated for 1 h at 37 °C. After washing, the color was developed using TBS (Amresco, Solon, USA) as a substrate, and the absorbance was measured at 450 nm with a microplate reader (BioTek Synergy 4,Winooski, VT, USA).
Levels of urinary J-IgG (UJGCR) and IgA-IgG (UAGCR) immune complexes were standardized to the UCr concentration to adjust for differences of urine flow rate.
Statistical analysis
Statistical calculations were performed using SPSS for Windows, version 20.0 (SPSS, Chicago, IL) and Graph Pad Prism 5.0 (Graph Pad Software Inc., San Diego, CA). Data are presented as mean values ± SD, medians (interquartile range) or frequency in percent according to the types of variables. The Mann–Whitney U-test was used for statistical comparisons between two groups. Receiver operator characteristic curve (ROC) analysis was performed to assess the value of urine markers in differentiating between patients with IgAN and combined controls. Correlation analyses were performed using Spearman's correlation analyses. All tests were two-sided and a P-value < 0.05 was considered statistically significant.