The level of serum secretory IgA of patients with IgA nephropathy is elevated and associated with pathological phenotypes

Jun-jun Zhang 0 1 Li-xia Xu 0 1 Gang Liu 0 1 Ming-hui Zhao 0 1 Hai-yan Wang 0 1 0 Department of Medicine, Peking University First Hospital, Institute of Nephrology, Peking University, Key Laboratory of Renal Disease, Ministry of Health of China , Beijing 100034 1 Department of Medicine, Renal Division, Peking University First Hospital, Institute of Nephrology, Peking University, Key Laboratory of Renal Disease, Ministry of Health of China , Beijing 100034 , PR China Background. Mucosal infection associated episodic macroscopic haematuria is observed in many patients with IgA nephropathy (IgAN), however, the mechanism has not been elucidated. Recent study suggested that secretory IgA (SIgA) might play an important role in the pathogenesis of IgAN. The aim of this study is to investigate the level of serum SIgA and the deposition of SIgA in glomeruli in IgAN patients with different pathological phenotypes. Methods. The levels of serum SIgA were detected in 57 patients with IgAN and 48 normal controls. The associations between the levels of SIgA and the pathological phenotypes of IgAN as well as clinical parameters were investigated. Frozen renal sections from 34 of the 57 patients without IgM deposition were immunofluorescence stained and examined by confocal microscopy to detect the co-deposition of IgA and secretory component (SC). The association between deposition of SIgA and the level of serum SIgA was analysed. Results. The level of serum SIgA in patients with IgAN was significantly higher than that of normal controls. The level of serum SIgA in patients with focal proliferative sclerosing IgAN (fpsIgAN) was much higher than that in patients with mild mesangial proliferative IgAN (mIgAN) (P < 0.001). The level of serum SIgA correlated with the level of serum creatinine (R 0.509, P < 0.001), degree of proteinuria (R 0.643, P < 0.001) and creatinine clearance (R 0.454, P 0.002) in patients with IgAN. Significant co-deposition of SC and IgA were found in 11 of the 34 patients. Although the level of serum SIgA in patients with SC deposits was higher than those without SC deposits, the difference was not significant. Introduction The IgA nephropathy (IgAN) is the most common glomerulonephritis defined by the predominant deposition of IgA in the glomerular mesangium. It is characterized by various pathological phenotypes and variable spectrum of clinical presentation. About 3040% of these patients will develop progressive renal failure and eventually require either dialysis or kidney transplantation [1]. The pathogenesis of IgAN remains unclear. The clinical association between exacerbation of IgAN and mucosal infections has supported the view that IgAN might be connected with the mucosal immune response. Secretory IgA (SIgA) is mainly in human secretions and represents the major humoral defense mechanism of mucosal areas. Small amounts of SIgA can be found in normal serum and levels of SIgA are elevated in some disorders like liver disease and HIV infection. Moreover, the serum concentration of SIgA was also increased in some patients with IgAN [2,3]. Recently, Oortwijn et al. [4] had established a sandwich enzyme-linked immunosorbent assay (ELISA) to detect the level of SIgA, and found a 120-fold accumulation of SIgA compared to IgA1 in the glomerular eluate of one patient with recurrent IgAN. They further proved that SIgA could bind more to human mesangial cells (HMC) and could induce increased IL-6 production by HMC compared with IgA, which suggested a pathogenic role for SIgA in the progress of IgAN. However, some other researches favored contrary results in the level of serum SIgA in patients with IgAN [5,6], and the deposition of SIgA could not be found in most of the studies, even in renal cryosections of patients with recurrent IgAN [4]. Therefore, the relationship between mucosal immunity and IgAN is still controversial. In the present study, firstly, the levels of SIgA were detected in sera from healthy controls and IgAN patients with different pathological phenotypes. Then, the associations between the levels of SIgA and pathological phenotypes of IgAN as well as clinical parameters were analysed. Finally, the deposition of SIgA in glomeruli from patients without IgM deposition was investigated and the correlation between deposition of SIgA and the level of serum SIgA was also explored. Materials and methods Patients To detect the serum level of SIgA, 57 patients with renal biopsy-proven IgAN and 48 healthy normal controls with comparable age and gender distribution and negative urinalysis were enrolled. Serum samples from patients were obtained at the time of renal biopsy. Twenty-nine of them had mild mesangial proliferative glomerulonephritis (mIgAN) without overt tubular and interstitial damage in renal histopathology, fulfilling the criteria of Haas-I, a pathologic scheme of IgAN proposed by Haas [7]. The remaining 28 patients had focal proliferative sclerosing IgAN (fpsIgAN) defined as Haas-III or V (see details in Table 1). Informed consent was obtained prior to entry into the study. To observe SIgA deposition in the kidney, renal cryosections from 34 of the above 57 patients were collected. All the 34 patients were selected based on significant deposition of IgA but absence of IgM in glomerular mesangium by routine direct immunofluorescence. Detection of SIgA in sera by ELISA In accordance with a previous publication, a sandwich ELISA was used [4]. Mouse monoclonal anti-human secretory component (SC) (Sigma, Saint Louis, USA) diluted to 5.1 mg/ml in 0.05M bicarbonate buffer (PH 9.6) was coated to the wells of one-half of a polystyrene microtitre plate (Costar, Mankato, Minnesota, USA). The wells in the other half were coated with bicarbonate buffer alone to act as Patients with IgAN Patients with mIgAN Number Age (years) Duration of disease (months) Proteinuria (g/24 h) Serum creatinine (mmol/l) Creatinine clearance (ml/min) Serum IgA (mg/ml) antigen-free wells. The volumes of each well for this step and for subsequent steps were 100 ml. All incubations were carried out at 378C for 1 h and the plates were washed three times with 0.01M phosphate-buffered saline (PBS) containing 0.1% Tween 20 (PBST). The plates were then blocked with PBST containing 1% BSA (PBST/BSA). The sera diluted 1 : 100 were added in duplicate to both antigen-coated and antigen-free wells. Each plate contained a blank control (PBST/BSA) and a positive control. After incubation and washing, rabbit polyclonal anti-human IgA (Dako, Glostrup, Denmark) with a dilution of 1 : 5000 was added, the wells were then incubated with 1:10 000 diluted horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (Zhongshan Biotech, Beijing, China) to detect SIgA in the samples. The reaction was revealed with 0.1 M citrate phosphate buffer (pH 5.0) containing 0.04% o-phenylenediamine and 0.1% H2O2, then the reaction was stopped with 1M H2SO4. The absorbance at 490 nm was recorded i (...truncated)