Serum level of proximal renal tubular epithelial cell-binding immunoglobulin G in patients with lupus nephritis

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1 (2016) 25, PAPER Serum level of proximal renal tubular epithelial cell-binding immunoglobulin G in patients with lupus nephritis DYH Yap, S Yung, Q Zhang, C Tang and TM Chan Division of Nephrology, Department of Medicine, Queen Mary Hospital, The University of Hong Kong, Hong Kong In vitro data showed that immunoglobulin G (IgG) from lupus nephritis (LN) patients could bind to proximal renal tubular epithelial cells (PTEC), but the clinical relevance of such binding remained unclear. Binding of IgG and subclasses to PTEC was measured by cellular ELISA (expressed as OD index) in 189 serial serum samples from 23 Class III/IV VLN patients who had repeated renal flares (48 during renal flares, 141 during low level disease activity (LLDA)), and compared with 64 patients with non-lupus glomerular diseases (NLGD) and 23 healthy individuals. Total IgG PTEC-binding index was , , and in healthy controls, NLGD, LN patients during LLDA, and LN patients during nephritic flare, respectively (p < 0.001, LLDA vs. renal flare; p < 0.001, healthy controls or NLGD vs. LN during LLDA or renal flare). PTECbinding index for IgG 1 was , , and for the corresponding groups (p < 0.001, LLDA vs. renal flare; p < 0.001, healthy controls or NLGD vs. LN during LLDA or renal flare). Sixteen of 48 episodes (33.3%) of nephritic flare showed persistent PTEC-binding IgG seropositivity for more than months, despite clinical response to immunosuppressive treatment. Total IgG and IgG 1 PTEC-binding correlated with anti-dsdna level (r ¼ 0.34 and 0.52, respectively, p < for both), and inversely with C3 level (r ¼ 0.26 and 0.50, respectively, p ¼ and<0.001). Sensitivity/specificity of PTECbinding index in detecting renal flares was 45.8%/80.1% for total IgG (ROC AUC 0.630, p ¼ 0.007) and 87.5%/35.5% for IgG 1 (ROC AUC 0.615, p ¼ 0.018). IgG 1 PTEC-binding index correlated with tubulo-interstitial inflammation score in renal biopsy from corresponding patients. Our data suggested that total IgG and IgG1 PTEC-binding index in serum of LN patients correlate with serological activity, and in combination could predict renal flares. The correlation between IgG1 PTEC-binding and tubulo-interstitial inflammation suggests potential pathogenetic significance. (2016) 25, Key words: Immunoglobulins; lupus nephritis; proximal renal tubular epithelial cells Introduction nephritis (LN) is a severe organ involvement in systemic lupus erythematosus (SLE), and constitutes an important cause of renal failure especially in high-risk groups such as African-Americans and Asians. 1,2 One cardinal feature of SLE is the production of autoantibodies, which contributes to the pathogenesis of LN. One illustrative example of such autoantibodies are anti-double-stranded DNA (anti-dsdna) antibodies as supported by Correspondence to: Tak Mao Chan, Department of Medicine, The University of Hong Kong, Queen Mary Hospital, 102 Pokfulam Road, Hong Kong. dtmchan@hku.hk Received 26 March 2015; accepted 6 July 2015 their presence in renal eluate obtained from patients and mice with LN, 3 5 and also by clinical correlation between anti-dsdna antibody levels and disease activity. 6 Previous studies had reported that up to 70% of patients with active proliferative LN exhibit immunoglobulin (Ig) deposition along the renal tubular basement membrane. 7,8 In this context, the proximal tubular epithelial cells (PTEC) play an active role in mediating pathological processes that affect long-term renal outcomes such as tubulo-interstitial inflammation, epithelial-tomesenchymal transdifferentiation, and fibrosis. 7,9 11 Our group previously demonstrated that anti-dsdna antibodies from patients with LN could bind to PTEC and induce expression of inflammatory cytokines. 7 In this study, we! The Author(s), Reprints and permissions: /

2 investigated whether the PTEC-binding activity of total serum IgG and its subclasses might have clinical correlations in patients with LN. The findings have obvious implications on disease activity monitoring and pathogenetic mechanisms leading to tubulo-interstitial injury and progressive renal failure. Materials and methods Patients Patients who were followed at the SLE Clinic of Queen Mary Hospital, Hong Kong, with biopsyproven Class III/IV V LN and two or more episodes of renal flare during the period 2001 to 2013 were included. This study was approved by the institution review board of the University of Hong Kong/Hospital Authority Hong Kong West Cluster and all included participants provided signed informed consent. Renal biopsy findings were reported according to the International Society of Nephrology/Renal Pathology Society (ISN/RPS) 2003 classification. 12 Our standard treatment for active proliferative LN has been corticosteroids combined with either cyclophosphamide or mycophenolate mofetil (MMF) for induction of renal response followed by low-dose corticosteroids combined with either azathioprine or MMF as long-term maintenance therapy. Disease activity was classified as Active or Low Level Disease Activity (LLDA) on the basis of both clinical and serologic assessments, with Active disease defined by an SLE Disease Activity Index (SLEDAI) score > 10 with 4 points in the renal domain and LLDA status defined by a SLEDAI score <4 with no points in the renal domain. 13 Patients with non-lupus glomerular diseases (NLGD) and age- and sex-matched healthy individuals were included as controls. The NLGD group included patients with IgA nephropathy, minimal change nephropathy, membranous nephropathy and antineutrophil cytoplasmic antibody (ANCA)-associated glomerulonephritis, and serum samples were obtained at presentation when the diagnoses were established by kidney biopsy. Laboratory methods Archived serum samples from LN patients collected at baseline (i.e. at initiation of induction treatment for a proven renal flare) then serially at three-monthly intervals were retrieved. Single serum samples were also obtained from patients with NLGD and healthy individuals. PTEC-binding activity of IgG in serum samples was measured using a cellular enzyme-linked immunosorbent assay (ELISA) as previously described. 7,14 Briefly, PTEC were seeded into 96-well microtiter plates at a density of 10,000 cells/cm 2. Cells were cultured in Dulbecco s Modified Eagle Medium (DMEM)/F12 medium supplemented with 15% fetal calf serum (FCS), and the medium was changed every three days. At 90% confluence PTEC were growth arrested for 72 hours, washed with phosphate-buffered saline (PBS) then fixed with 1% paraformaldehyde in PBS for 15 minutes. Cells were washed thrice with PBS in between steps, and all incubations were for one hour at 37 C. PTEC were blocked with 3% bovine serum albumin (BSA) followed by normal IgG (100 mg/ml) to block Fc receptor-mediated binding. PTEC were incubated with serum samples (diluted 1:100, 100 ml) in triplicate, then incubated with anti-human IgG F(ab) conjugated with alkaline phosphatase. Degree of IgG binding to PTEC was determined by incubation with paranitrophenol phosphate at room temperature and with optical density (OD) measurement at A 405/420 when a pre-established positive control sample showed an OD of 1.5. The positive control was pooled serum from a patient with high PTEC-binding activity. Seropositivity for PTEC binding was denoted by results that exceed mean þ 3 SD of results from healthy individuals. Circulating antidsdna antibody titer was measured using a commercial ELISA (Microplate autoimmune anti- DNA quantitative ELISA) according to the manufacturer s instructions (BioRad, Hong Kong). Samples giving a value > 60 IU/ml were considered positive. Kidney biopsies were performed within one week when there was clinical suspicion of renal flare and were reviewed by the same pathologist, and the severity of tubulo-interstitial inflammation was graded according to the United States National Institute of Health (NIH) activity and chronicity scoring system on a scale of 0 to Data analysis and statistics Continuous variables were expressed as mean SD and analyzed by student s t-test, unless otherwise specified. Categorical variables were expressed as frequencies and percentages, and analyzed with Chi-square test where appropriate. The Spearman s method was used to investigate correlations between PTEC-binding indices and clinical parameters. The sensitivity/specificity as well as the positive and negative predictive values (PPV and NPV) of PTEC-binding activity in detecting renal flares 47

3 48 was computed, and the area under the curve (AUC) of the receiver-operator characteristics (ROC) curves was determined. Statistical analyses were performed by GraphPad Prism 5 (La Jolla, CA, USA) and two-tailed p values < 0.05 were considered statistically significant. Results Patient characteristics Twenty-three Chinese patients with biopsy-proven Class III/IV V LN who had experienced at least two episodes of renal flares during follow-up (mean duration: months) were included (Table 1). A total of 276 serum samples were analyzed, including 189 samples from LN patients (48 during Active renal flare and 141 during LLDA ), 64 samples from patients with NLGD, and 23 samples from healthy individuals, respectively. PTEC-binding by total IgG or its subclasses in sera of LN patients Binding index of total IgG to PTEC was , , and OD for healthy controls, patients with NLGD, LN patients with LLDA, and active LN patients, respectively (p < 0.001, LN Active vs. LLDA ; p < 0.001, healthy controls or NLGD vs. LN LLDA or LN Active ) (Figure 1(a)). Binding index of serum IgG 1 to PTEC was , , and OD for the corresponding groups, respectively (p < 0.001, LN LLDA vs. Active ; p < 0.001, for healthy controls or NLGD vs. LN LLDA or LN Active ) (Figure 1(b)). No significant PTEC-binding by IgG 2, IgG 3 or IgG 4 was observed in healthy controls, NLGD patients, and LN patients during LLDA or disease flare. Forty-eight active LN episodes occurred during a follow-up of months, and the seropositive rates for PTEC-binding total IgG and IgG 1 were Table 1 Characteristics of 23 patients with Class III/IV V lupus nephritis who had two or more episodes of renal flare during follow-up and included in the present study Age (years) Female/Male 14/9 Duration of follow-up (months) Previous immunosuppressive exposure Prednisolone 23 (100%) Cyclophosphamide 17 (73.9%) Mycophenolate mofetil 18 (78.3%) Azathioprine 19 (82.6%) Calcineurin inhibitors 8 (34.8%) Laboratory parameters on first renal presentation Serum Cr level (mmol/l) Urine protein (g/d) Anti-dsDNA level (iu/ml) C3 level (mg/dl) Anti-dsDNA: anti-double-stranded DNA; Cr: creatinine. Figure 1 Proximal tubular epithelial cell-binding by (a) total IgG and (b) IgG 1 in serum samples of patients with inactive or active LN, NLGD, and healthy controls. IgG: immunoglobulin G; PTEC: proximal tubular epithelial cells; NLGD: non-lupus glomerular diseases; LN: lupus nephritis; OD: optical density.

4 49 Figure 2 Correlation between proximal tubular epithelial cell-binding by (a) total IgG and (b) IgG 1 with anti-dsdna level in 23 patients with Class III/IV V lupus nephritis. IgG: immunoglobulin G; PTEC: proximal tubular epithelial cells; Anti-dsDNA: anti-double-stranded DNA; OD: optical density. 87.5% and 45.8%, respectively, during these episodes. In 33.3% of the active LN episodes, PTECbinding total IgG and IgG 1 remained seropositive after six months of induction treatment, despite clinical response to immunosuppressive therapy. Clinical associations with IgG and IgG 1 PTECbinding index Serum total IgG and IgG 1 PTEC-binding index both correlated positively with anti-dsdna levels (r ¼ 0.34 and 0.52, p < for both) (Figure 2(a) and (b)), and negatively with C3 levels (r ¼ 0.26 and 0.50, respectively, p ¼ and <0.001) (Figure 3(a) and (b)). IgG and IgG 1 PTEC-binding indices were not related to the levels of serum creatinine, serum albumin, or proteinuria at the time of serum sample collection (p ¼ 0.105, and 0.921, respectively, for total IgG PTEC-binding; p ¼ 0.906, and 0.921, respectively, for IgG 1 PTEC-binding). The overall sensitivity and specificity of PTECbinding index in the detection of renal flares was 45.8% and 80.1%, respectively, for total IgG (PPV: 44.0%; NPV: 81.3%; ROC AUC 0.630, p ¼ 0.007), and 87.5% and 35.5%, respectively, for IgG 1 (PPV: 31.6% NPV: 89.3%; ROC AUC 0.615, p ¼ 0.018) (Figure 4(a) and (b)). The sensitivity, specificity, PPVs and NPVs of total IgG and IgG1 PTECbinding index for the prediction of renal flares with or without systemic flares are summarized in Table 2. Two patients (8.7%) showed positive total IgG and IgG 1 binding to PTEC during active renal flare when their anti-dsdna and C3 levels were within normal limits. Seropositivity of PTEC-binding total IgG and IgG 1 preceded renal flares by months and months, respectively. Of the 23 patients studied, 19 patients (82.6%) were seropositive for PTEC-binding total IgG and IgG 1 during active renal flares, and four of them (21.1%) developed chronic renal impairment during follow-up (three had doubling of baseline serum creatinine and one developed end-stage renal failure). The mean IgG 1 PTEC-binding index during active flare in patients who have or have not developed chronic renal impairment were and , respectively (p ¼ 0.130). All four patients who developed chronic renal failure had persistent PTEC-binding IgG 1 seropositivity after six months of induction immunosuppression. The persistence of IgG 1 seropositivity conferred an increased tendency of chronic renal insufficiency although not reaching statistical significance (odds ratio 3.4, 95% confidence interval (CI): , p ¼ 0.539). Four patients (17.4%) were seronegative for PTEC-binding total IgG and IgG 1 during active renal flares, and none of them had doubling of baseline serum creatinine or end-stage renal failure during follow-up. IgG/IgG 1 PTEC-binding index and tubulo-interstitial inflammation Five patients showed moderate to severe tubulointerstitial inflammation on their renal biopsy obtained during active renal flare, and we observed

5 50 Figure 3 Negative correlation between proximal tubular epithelial cell-binding by (a) total IgG and (b) IgG 1 with C3 level in 23 patients with Class III/IV V lupus nephritis. IgG: immunoglobulin G; PTEC: proximal tubular epithelial cells; OD: optical density. Figure 4 Receiver-operator characteristics (ROC) curve for sensitivity/specificity in renal flare prediction using the degree of proximal tubular epithelial cell-binding by (a) total IgG and (b) IgG 1 in serum samples of 23 patients with Class III/IV V lupus nephritis. IgG: immunoglobulin G; AUC: area under the curve. a significantly higher incidence of PTEC-binding IgG 1 seropositivity among these patients when compared to patients with no or mild tubulo-interstitial inflammation (80.0% vs. 20.0%, 2 ¼ 5.2, p ¼ 0.018). PTEC-binding index for IgG 1, but not total IgG, correlated with tubulo-interstitial inflammation score during active renal flares (r ¼ 0.60 and p ¼ for IgG 1 ; r ¼ 0.08 and p ¼ for total IgG). Both total IgG and IgG 1 PTEC-binding indices showed no correlation with NIH chronicity scores (r ¼ 0.34, p ¼ for IgG 1 ; r ¼ 0.08, p ¼ for total IgG).

6 Table 2 The sensitivity, specificity, positive and negative predictive value of total IgG and IgG 1 PTEC-binding index for the prediction of renal flares with or without concomitant systemic flares 51 SN SP PPV NPV Overall (i.e. renal flares with or without systemic flares Total IgG PTEC-binding index 45.8% 80.1% 44.0% 81.3% IgG 1 PTEC-binding index 87.5% 35.5% 31.6% 89.3% Renal flares with concomitant systemic flares Total IgG PTEC-binding index 75.0% 74.6% 24.7% 96.4% IgG 1 PTEC-binding index 80.0% 29.9% 11.3% 93.1% Renal flares without systemic flares Total IgG PTEC-binding index 43.2% 78.6% 38.0% 82.0% IgG 1 PTEC-binding index 88.3% 34.9% 28.6% 91.1% IgG: immunoglobulin G; PTEC: proximal tubular epithelial cells; NPV: negative predictive value; PPV: positive predictive value; PTEC: proximal tubular epithelial cells; SN: sensitivity; SP: specificity. Discussion SLE is a prototypic autoimmune disease characterized by autoantibodies production. 16 It has been reported that these different autoantibodies can bind to a spectrum of resident renal cells including mesangial cells, podocytes, glomerular endothelial cells, and renal tubular epithelial cells, and the binding has been associated with altered cell function thereby suggesting a role in the immunopathogenesis of LN. 7,14,17 20 For instance, previous studies have shown that antidsdna antibodies could bind to human PTEC and such binding induced secretion of proinflammatory cytokines, alterations in cell morphology and increased cell proliferation. 7 Our group has recently reported that mesangial cell-binding activity of IgG in LN patients correlated with disease activity. 21 In this study, we further demonstrated that the degree of PTECbinding by IgG was also significantly increased in LN compared with healthy individuals and patients with NLGD, and such binding holds a strong association with clinical disease activity and serological parameters. The sensitivity and specificity of conventional serological parameters C3 and antidsdna levels have been reported to show sensitivity and specificity of 49% 79% and 51% 74%, respectively, in the recognition of renal flares In the current study, PTEC-binding by IgG 1 showed high sensitivity but modest specificity for detecting renal flares, and vice versa for total IgG. Nevertheless, the different properties of these two PTEC-binding indices imply that they may complement each other for disease activity monitoring. Also, the combined use of PTEC-binding activity by total IgG or IgG 1 might be of diagnostic value in some LN patients in whom the conventional serological parameters such as anti-dsdna or C3 do not correlate with clinical activity, as illustrated in two of the 23 patients studied, when both anti- DNA and C3 still remained within the normal range at renal flare but PTEC-binding IgG was positive. In this context, seropositivity of PTECbinding total IgG and IgG 1 precedes renal flares by approximately four to five months, which allow earlier detection of impending relapses. However, the need to perform cell-based assays makes it difficult to adopt the PTEC-binding index into routine clinical practice on the current technological platform. In addition, most patients with PTEC-binding IgG seropositivity also showed anti-dsdna and C3 abnormalities, and both total IgG and IgG 1 PTEC-binding index have to be measured in order to produce acceptable sensitivity and specificity. PTEC-binding IgG 1 might also have prognostic values in selected LN patients. In this cohort, patients who subsequently developed chronic renal insufficiency were all seropositive for PTECbinding IgG 1 during active renal flares. Although the IgG 1 PTEC-binding indices were similar between patients who had or had not developed chronic renal impairment, the association between persistence of PTEC-IgG 1 despite apparent shortterm clinical response to immunosuppressive therapy and chronic renal insufficiency is intriguing. It remains possible that PTEC-binding IgG 1 not only contributes to tubulo-interstitial inflammation during active renal flares, but also results in continuous and initially subclinical antibody-mediated immunological injury to the tubulo-interstitium, thus jeopardizing long-term renal survival. These preliminary observations need further investigation and validation with larger patient numbers and longer follow-up durations. Our findings also have implications for pathogenic mechanisms in LN. Among the different

7 52 IgG subclasses from LN patients tested, only IgG 1 exhibits significant binding to PTEC. Furthermore, only PTEC-binding by IgG 1 but not total IgG is associated with the intensity of tubulo-interstitial inflammation. This also echoed our recent findings that only IgG 1 but not other IgG subclasses of LN patients bound significantly to mesangial cells, and such binding correlated with intensity of mesangial deposits on electron microscopy. 21 In this context, earlier studies suggested that IgG 1 might be more pathogenic compared with other IgG subclasses in LN, likely related to its higher potency of complement fixation and activation. 30,31 Studies are ongoing to investigate the cell surface antigen(s) that mediate Ig binding to PTEC, and the downstream cellular events. Conclusion IgG and IgG 1 in patients with LN bind to PTEC, and the degree of binding correlates with clinical and serological activity. Funding This work was supported by the Stanley Ho Alumni Challenge Matched Fund 2012 and the Hong Kong College of Physicians Young Investigator Research Grant 2014 awarded to Desmond Y. H. Yap, the endowment fund of the Yu Chiu Kwong Professorship in Medicine at University of Hong Kong awarded to T.M. Chan, the research fund donated by Mr G King, and the UGC Matching Grant Scheme (5th and 6th Phase). S. Yung is supported by the Wai Hung Charitable Foundation. Conflict of interest statement The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article. Acknowledgments We would like to acknowledge Mr Owen Chan for his contribution in conducting some of the ELISA assays. The results presented in this paper have not been published previously in whole or part, except in abstract format. 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