The levels of urinary joining chain containing IgG immune complexes are associated with disease severity in IgA nephropathy

Background: Circulating levels of aberrantly glycosylated IgA1 and its immune complexes (IgA/IgG-IC) are elevated and correlated with disease severity in IgA nephropathy (IgAN). The pathologic IgA containing immune complexes deposits in the kidney were found to be in dimeric or polymeric forms, suggesting that those deposited IgA immune complexes contain joining chain, which is critical for the multimerization and transportation of IgA. Both the dimeric IgA and polymeric IgA can cause renal damage by inducing the release of cytokines from mesangial cells. We aimed to investigate the urinary J chain containing IgG immune complexes/creatinine ratio (UJGCR) and analyze its relationship with disease severity in IgAN. Methods: The UJGCR were measured by sandwich enzyme linked immunosorbent assay in 26 patients with IgAN, 31 patients with other kidney diseases and 32 healthy volunteers. Results: The levels of UJGCR were higher in patients with IgAN than that in non-IgAN patients ( P =0.006) and healthy volunteers ( P <0.0001). Importantly, receiver operating characteristics curve analysis confirmed that UJGCR had good discrimination between non-IgAN patients and IgAN patients. The levels of UJGCR positively correlated with 24-hour urinary protein excretion (r=0.63, P =0.0006), serum creatinine (r=0.55, P =0.003), and negatively correlated with estimated glomerular filtration rate (r= -0.61, P =0.0008) in IgAN. Furthermore, the levels of UJGCR were higher in IgAN patients with IgA mesangial deposition of (+/++) than patients with (+++/++++). And IgAN patients with tubular atrophy/interstitial fibrosis showed higher levels of UJGCR than that without ( P =0.03). Similarly, the levels of urinary IgA-IgG/creatinine ratio (UAGCR) were also found to be elevated and associated with clinical and pathological parameters as UJGCR in IgAN. markers did not differ between IgAN patients with corticosteroid therapy (n = 4) and those without (n = 22) (data not shown). We also found that the values of urinary markers did not differ between IgAN patients with immunosuppressant therapy (n = 7) and those without (n = 19) (data not shown).

pentameric IgM (pIgM) [18][19][20]. J chain is also required for the transportation of sIgA across the mucosal epithelium, preventing attachment of bacteria and viruses to mucous membranes [20]. It is well known that the classic manifestation of IgAN is episodic hematuria with or without proteinuria following mucosal infection. It is possible that mucosal immunity dysregulation in IgAN might lead to the increased synthesis of sIgA which is transported across the mucosa with the help of J chain and thus reaches the circulation and deposit in mesangium. Several investigations have revealed that the serum levels of sIgA and pIgA wereare elevated in IgAN [21][22][23][24]. And J chain had been found in renal biopsy specimens of IgAN patients using anti-J chain polyclonal antibody or antisera [15,16,25]. Bothe the dDimeric IgA and pIgA both couldcan cause renal damage by inducing the release of cytokines, including interleukin-6 (IL-6), tumor necrosis factorα (TNF-α), and transforming growth factor-β (TGF-β) by mesangial cells [26][27][28][29]. It is possible that a fraction of circulating J chain containing immune complexes deposits in the kidneys of IgAN patients are excreted into the urine and thus represent as non-invasive disease-specific biomarkers in IgAN. However, only one previous study found that the levels of urinary IgA-IgG complexes were significantly higher in IgAN patients [30]. The value of urinary IgA-IgG immune complexes for accessing disease severity in IgAN remains unclear. In addition, urinary J chain in IgAN has not been reported and it is not known whether the levels of urinary J chain containing immune complexes are associated with clinical or pathological severity in IgAN.
In this study, we detected urinary J chain containing IgG (J-IgG) immune complexes by sandwich enzyme linked immunosorbent assay (ELISA) with anti-J chain monoclonal antibody (mAb). We analyzed the association between the levels of urinary J-IgG immune complexes and clinicopathological parameters of IgAN at the time of renal biopsy.

Methods
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.

Results
Demographic and clinical characteristics of the participants Demographic and clinical characteristics of the study subjects wereare summarized in  OD450nm/mmol/L Cr, P = 0.006) and healthy subjects (0.31±0.20 OD450nm/mmol/L Cr versus 0.04±0.04 OD450nm/mmol/L Cr, P<0.0001). And the levels of UAGCR were also significantly higher in patients with IgAN than that in patients with other kidney diseases (P = 0.004) and healthy subjects (P<0.0001) (Fig.1). In addition, we found that the values of these urinary markers did not differ between IgAN patients with corticosteroid therapy (n = 4) and those without (n = 22) (data not shown). We also found that the values of urinary markers did not differ between IgAN patients with immunosuppressant therapy (n = 7) and those without (n = 19) (data not shown). ROC comparing UJGCR in distinguishing patients with IgAN from patients with other renal diseases ROC analysis confirmed good discrimination between patients with IgAN and patients with other renal diseases for elevated levels of UJGCR (AUC, 0.71; 95%CI, 0.58-0.85; P = 0.006) and UAGCR (AUC, 0.72; 95% CI, 0.59-0.85; P = 0.04) (Fig.2). The respective optimal derived cut-off values were 0.1491 OD450nm/mmol/L Cr for UJGCR (Sensitivity: 84.6%; Specificity: 58.1%) and 0.0877 OD450nm/mmol/L Cr for UAGCR (Sensitivity: 84.6%; Specificity: 58.1%).
There was no significant correlation ofbetween U-RBC with UJGCR andor UAGCR and U-RBC (data not shown).
A significant correlation between UAGCR and UJGCR (r = 0.80, P<0.0001) was observed in patients with IgAN patients (see Additional file 1).
Regarding Lee grading, UAGCR and UJGCR were significantly higher in patients of grade IV (n = 18) than that in grade II and III (n = 8 in total) (see Additional file 3). We also evaluated the tubular atrophy/interstitial fibrosis grade according to Oxford classification.
However, UJGCR and UAGCR of IgAN patients with different grades of mesangial hypercellularity, segmental glomerulosclerosis and cellular/fibrocellular crescents did not show any significant differences (data not shown).
In patients with other renal diseases, the UJGCR levels were positively correlated with the levels of SCr, but no correlation with 24hUPro or eGFR. And UAGCR levels were correlated with SCr and eGFR, but no correlation of UAGCR levels with 24hUPro was shown (see Additional file 4).

Discussion
In the present study, our data showed that the levels of UJGCR and UAGCR were markedly , which is consistent with our study. But neither did they analyze the relationship between UAGCR and pathological lesions in IgAN, nor did they measure urinary J chain in IgAN. In this study, we found that the elevated levels of UJGCR and UAGCR were not only correlated with 24hUPro, but also correlated well with kidney function in IgAN, which are well-known factors in predicting disease severity and renal outcome for the development of ESRD in IgAN patients.
To the best of our knowledge, this is the first study to demonstrate that UJGCR and UAGCR correlate with pathological parameters in patients with IgAN. In this study, we analyzed the associations between urinary immune complexes and histopathological phenotypes with disease predictive significance according to the Oxford classification, which indicated that the elevated levels of UJGCR and UAGCR in IgAN patients correlated with tubular atrophy/interstitial fibrosis. Moreover, surprisingly, we found that UJGCR and UAGCR were all higher in IgAN patients with less renal IgA deposition than patients with a more intense renal IgA deposition, which strongly suggests that these immune complexes deposits in the mesangium could cross the filtration barrier and thus be excreted into the urine.
Taken together, it seems most likely that J-IgG and IgA-IgG immune complexes in the urine detected in the present study came from glomerular mesangium and injured the kidney, especially renal tubular epithelial cells.
More interestingly, this current study found significant correlations between UJGCR and UAGCR in IgAN. Based on our observation, we postulate UJGCR were mainly composed of J-IgA-IgG.
In clinical setting, renal biopsy is the only golden procedure to diagnose and evaluate disease activity in IgAN, but this being an invasive procedure which is impractical to repeat. In addition, 24hUPro, urine protein/creatinine excretion (UPCR), SCr and hematuria are often used to evaluate the severity of IgAN which could not always distinguish patients