Differential renal expression of IFN-α and BAFF and its relevance to disease activity and treatment responsiveness in patients with proliferative lupus nephritis

doi:10.21203/rs.3.rs-2480720/v1

Background

Molecularly targeted therapies are emerging for treating lupus nephritis (LN). This study aimed to assess the immunohistochemical findings of the cytokines in renal tissue and their pathological and clinical relevance in LN.

Methods

Fifty patients with proliferative LN (ISN/RPS class III and IV), five with LN class II, IgA nephropathy, and five with idiopathic hematuria as controls were enrolled. Immunohistochemistry (IHC) for CD3, CD20, interferon-alpha (IFNα), interleukin (IL)-12/p40, and B-cell activating factor (BAFF) was performed. The IHC score was calculated by scoring the number of positive cells/area of the cortex. Proliferative LN cases were grouped by the dominant expression of IFN-α, IL-12/p40, and BAFF, and subsequently, clinicopathological features were compared.

Results

Clinical data of patients with proliferative LN included urine protein creatinine ratio, 2.2 g/gCre; anti-ds-DNA antibody, 200.9 IU/mL; CH50, 21.9 U/mL; Systemic Lupus Erythematosus Disease Activity Index, 19.8 points. Proliferative LN cases, including class III (n = 18) and IV (n = 32), were classified into three subgroups according to the IHC score based on the dominancy of IFN-α (n = 17), IL-12 (n = 16), and BAFF group (n = 17) proteins. Hypocomplementemia and glomerular endocapillary hypercellularity were significantly increased in the IFN-α group, whereas chronic lesions were significantly higher in the IL-12 group (p < 0.05). The IFN-α group had a poorer renal prognosis in treatment response after 52 weeks.

Conclusions

The IHC of IFN-α, IL12, and BAFF for proliferative LN enabled grouping. Especially, the IFN-α and IL-12 groups showed different clinicopathological features and renal prognoses. The results indicated the possibility of stratifying cases according to the IHC of target molecules, which might lead to precision medicine.

Lupus nephritis (LN) is a serious organ injury caused by systemic lupus erythematosus (SLE), which increases both its morbidity and mortality [1]. Most patients are administered glucocorticoids, which elevates the risk of infection, atherosclerosis, and osteoporosis accompanied by immunosuppressants, including cyclophosphamide and mycophenolate mofetil, according to the recommendations of the American College of Rheumatology (ACR), European League Against Rheumatism (EULAR), and Kidney Disease Improving Global Outcomes [2–4]. The approval of new therapies for SLE, especially biologics such as belimumab and anifrolumab, is expected to improve its prognosis of organ involvement like rheumatoid arthritis.

Although B cells have been considered to serve a crucial role in pathogenesis of SLE, clinical trials of B cell-targeted molecular therapy, including rituximab or epratuzumab, have failed to reveal significant differences. Recently, the three molecular targets of B cell activating factor (BAFF), interferon (IFN)-α, and interleukin (IL-12), which are called bridging cytokines, have been highlighted [5]. Bridging cytokines, which are released by both macrophages and dendritic cells, play a pivotal role in harmonizing between the innate immune system and acquired immune or autoimmune system [6]. BAFF, the target molecule of belimumab, is a B cell activating factor belonging to the tumor necrosis factor family [6]. BAFF regulates B cell selection including class switching, and stimulates their differentiation into antibody-producing cells [6]. The serum level of BAFF is increased in cases with SLE and is related to disease activity [7]. Belimumab with standard therapy is highly effective in proliferative LN [8]. Moreover, a recent study showed that the type I interferon (IFN) pathway plays an important role in SLE pathogenesis. In SLE, IFN-α secreted by plasmacytoid dendritic cells is considered the most important cytokine as it activates T cells and promotes autoantibody release by B cells [9]. The serum concentration of IFN-α is related to disease activity or a flare-up [10]. Anifrolumab, which is a human monoclonal antibody to IFN-α receptor, has been newly approved for SLE treatment. A recent study reported that the IL-12-STAT4 pathway is important in patients with active SLE [11].

SLE is a molecularly heterogeneous disease, and it is important, although difficult, to predict the response to the treatment of patients with SLE. There are only a few reports on precision medicine of SLE or LN. In psoriatic arthritis, the selection of biologics according to the peripheral blood T-lymphocytic phenotype in each patient exhibited remarkably higher efficacy than the standard therapy, indicative of the importance of precision medicine [12]. Kubo et al. mentioned that patients with active SLE were classified into three groups in accordance with T-cell phenotypes and their possibility for precision medicine [13]. However, regarding precision medicine in LN, “the inflammation site,” indicating a renal biopsy specimen should be examined rather than the “whole body” as in a peripheral blood sample. Renal biopsy is routinely performed when diagnosing LN; thus, using the remaining samples after diagnosis is less invasive for the patients. The histology of renal tissue in LN varies with patients, which might reflect the individual pathogenesis of LN. We have attempted precision medicine in proliferative LN by targeting the above-mentioned three bridging cytokines and using renal biopsy specimens.

This study aimed to clarify whether the expression of molecular targets of biologics in renal tissue of LN was associated with disease activity, prognosis, and pathological findings.

Patients and study design

Patients who fulfilled the 2012 Systemic Lupus International Collaborating Clinics SLE classification criteria or the 2019 EULAR/ACR classification criteria for SLE were entried from the LOOPS registry, a registry of patients with SLE treated at the University of Occupational and Environmental Health (UOEH) hospital and affiliated hospitals [14]. Between 2011 and 2019, renal biopsy specimens were examined at the division of Surgical Pathology, UOEH hospital. Fifty patients, including those with class III/IV LN, five with class II and IgA glomerulonephritis, and five with idiopathic hematuria as controls were included in the study. The study method was approved by the Human Ethics Review Committee of UOEH (IRB number: UOEHCRB21-093) and designed in line with the Declaration of Helsinki and the Ethical Guidelines for Medical and Health Research Involving Human Subjects by the Ministry of Health, Labour and Welfare. Written informed consent was obtained from each patient. Figure 1 shows this study design. The following clinical and serologic imformation were collected at the time of renal biopsy and 52 weeks after treatment: sex, age, duration of SLE, SLE disease activity index (SLEDAI), urine protein creatinine ratio (UPCR), levels of blood urea nitrogen, serum creatinine, serum C3, C4, and CH50. The titers of antinuclear, anti-Sm, anti-ribonucleoprotein and anti-double-stranded DNA (anti-dsDNA) antibodies and other antibodies were determined. The Safety of Estrogens in Lupus Erythematosus National Assessment-SLEDAI and the British Isles Lupus Assessment Group (BILAG) index were used to measured general disease activity of SLE. The renal response after 52 weeks of treatment was assessed according to the recommendations of the ACR, Lupus Nephritis Assessment with Rituximab (LUNAR), Aspreva Lupus Management Study (ALMS), and Belimumab in Subjects with Systemic Lupus Erythematosus (BLISS)-LN studies and prednisolone dosage [2, 15–18].

Preparation Of Renal Biopsy Samples

Renal samples obtained using needle biopsy were fixed in 10% neutral-buffered formalin, performed dehydrattion, and embedded in paraffin. The formalin-fixed paraffin-embedded renal sections were stained with hematoxylin and eosin, periodic acid-Schiff, Masson’s trichrome, and periodic acid-silver methenamine. Pathological diagnosis was made using light, immunofluorescent, and electron microscopy. Fluorescein isothiocyanate-labeled rabbit antihuman IgG, IgA, IgM, C1q, C3, and fibrinogen (Dako, Copenhagen, Denmark) were used in immunofluorescent microscopy.

Histological Re-evaluation Of Renal Biopsy Samples

The histopathological findings of LN were classified based on the International Society of Nephrology/Renal Pathology Society (ISN/RPS) 2018 revision classification of LN [19]. The number of glomeruli, percentage of glomeruli with both active lesions, including cellular/fibrocellular crescents, endocapillary hypercellularity, karyorrhexis, fibrinoid necrosis, wire-loop lesion or hyaline thrombi, and chronic lesions such as global sclerosis, segmental sclerosis or adhesion, per total glomeruli were calculated. Regarding interstitium, the presence or absence of tubulitis and peritubular capillaritis and the percentage of the cortex with interstitial inflammation, interstitial fibrosis, and tubular atrophy were included. Moreover, the modified National Institutes of Health (NIH) activity/chronicity index was evaluated and scored as previously described [19].

Immunofluorescent Staining Of Renal Biopsy Specimens

Immunofluorescence staining of continuous sections was executed for each LN case and control case using the following antibodies: monoclonal anti-mouse antibodies against CD20 (L26, Dako; Glostrup, Denmark), CD3 (Dako; Glostrup, Denmark), IFN-α (Santa Cruz Biotechnology; Dallas, U.S.), and IL-12 (BioRad, Hercules, U.S.), and polyclonal anti-rabbit antibody against BAFF (Abcam, Cambridge, U.K.). Multiplex immunofluorescence staining was done using the Opal four-color fluorescence immunohistochemistry kit (Akoya Biosciences, Marlborough, MA. The formalin-fixed paraffin-embedded renal tissue was sliced into 3 µm-thick sections, deparaffinized, and subjected to microwave treatment at 121 ℃ for 10 min with Tris-ethylenediamine tetraacetic acid buffer (pH, 9.0). The section was immersed in blocking/antibody diluent for 10 min, incubated with primary antibodies for 1 h at room temperature, followed by incubation with secondary antibody (polymer HRP Ms + Rb) for 10 min, and finally incubated with with an Opal fluorophore (Opal520, Opal570) for 10 min. The process of staining and antibody removal was repeated using a different Opal fluorophore. Finally, the section was stained with 4′,6-diamidino-2-phenylindole (DAPI), a cover slip was mounted with Fluoromount (Diagnostic BioSystems; K024), and the slide was observed under a fluorescence microscope. Double staining for CD3 and CD20, that for IFN-α and IL-12, and single staining for BAFF were observed in each specimen.

Evaluation Of Immunofluorescence Staining

To analyze IHC of the sections, the above-mentioned sections were digitalized using a virtual slide system (VS120, Olympus). The total number of CD3-, CD20-, IFNα-, IL-12-, and BAFF-positive cells were counted. The IHC score was assessed using CD3-, CD20-, IFNα-, IL-12- and BAFF-positive cells/mm² of the renal cortex. The mean number of cells positively stained for these molecules was estimated in the samples of patients with class III/IV LN and those of controls.

Two independent pathologists (A.N. and S.H.) evaluated all the immunostaining sections in the absence of the clinical information. In cases of disagreement in assessment between both pathologists, a third pathologist (M.H.) made the consensus decision.

Statistical analysis

Data are shown as means ± standard deviation (SD). The statistical analysis was evaluated by JMP software (version 10; SAS Institute Inc., Cary, NC, USA). Statistical comparison of categorical variables were performed using Fisher’s exact or chi-square test. Nonparametric Wilcoxon test or one-way nanalysis of variance was used to compare differences between the median values of defined patient groups. A value of p < 0.05 was adopted to be statistically significant.

Clinicopathological characteristics of proliferative LN cases and control cases

Table 1 shows the baseline clinical characteristics of patients with proliferative LN (50), LN class II (5), and IgA GN (5), and control (10) groups. No significant differences in sex, age, and serum creatinine levels among these groups were observed.

Immunofluorescent evaluation of IFN-α, IL-12, and BAFF

Figure 2 shows the immunofluorescence assay results of renal expression of IFN-α, IL-12, and BAFF in proliferative LN, LN class II, IgA, and control groups. The patients with proliferative LN could be categorized into INF-α, IL-12-, and BAFF-dominant groups according to the predominantly expressed biomarker in the biopsy specimens in our immunohistochemical study. IFN-α-positive cells in the renal tissue were significantly higher in the IFN-α- dominant group than in the control group (p=0.0403). Significant infiltration of IL-12-positive cells was observed in the IL-12-dominant group than in the BAFF-dominant group (p=0.0076), LN class II (p=0.0412), IgA (p=0.0188) or control group (p=0.0009). BAFF-positive cells were significantly increased compared to the control group (p=0.0027). IFN-α was expressed in CD123-positive plasmacytoid dendritic cells and CD68-positive macrophages (Additional Figure 1). IL-12-positive cells and BAFF were expressed mainly in CD68-positive macrophages.

Comparison of clinical characteristics among IFN-α, IL-12, and BAFF groups

Table 2 shows the ISN/RPS classification of patients in each group, and Table 3 presents the clinical characteristics of patients at baseline and 52 weeks after therapy in these groups. Serum C3 and CH50 concentrations were significantly lower in the IFN-α-dominant group than in the IL-12-dominant group (C3: p=0.0298, CH50: p=0.0149). There was no significant difference in age, blood count, estimated glomerular filtration rate, anti-ds-DNA antibody titer, UPCR value, disease activity (SLEDAI and BILAG scores), the dosage of prednisolone and therapeutic drugs, and other characteristics at 52 weeks.

Comparison of histological parameters among IFN-α, IL-12, and BAFF groups

Table 4 shows the histological parameters of kidney biopsy in each group. As for glomerular lesions, the percentage of karyorrhexis was significantly higher in the IFN-α-dominant group than in the BAFF-dominant group (p=0.035). There were no significant differences in other glomerular or interstitial parameters. In the 2018 revision of the ISN/RPS classification of LN, a modified NIH activity/chronicity score was recommended to be calculated. Based on this scoring, the modified NIH total index was significantly higher in the IFN-α-dominant (p=0.0146) and IL-12-dominant groups (p=0.0166) than in the BAFF-dominant group. The IFN-α-dominant group also had higher scores for activity (p=0.0130), endocapillary hypercellularity (p=0.0357), and karyorrhexis (p=0.0410) than the BAFF-dominant group. The IL-12 dominant group showed a higher chronicity score (p=0.0393) and tubular atrophy score (p=0.0031) than the IFN-α-dominant group.

Immunofluorescent evaluation of CD3, CD20, and pathological findings

Figure 3 reveals CD3 and CD20-positive lymphocytes in the renal tissue and representative pathological features in each group. Infiltration of CD3-positive T lymphocytes and CD20-positive lymphocytes was observed in all groups, and there were no significant differences among these groups. Regarding pathological characteristics, the IFN-α-dominant group showed increased glomerular active lesions, including endocapillary hypercellularity; however, the IL-12-dominant group contained increased chronic lesions such as segmental sclerosis or tubular atrophy. The LN class II group exhibited mesangial cell hypercellularity. The IgA nephropathy group also showed active lesions, including fibrocellular crescent. The control group showed minor glomerular abnormalities.

Renal response at 52 weeks in the IFNα-, IL-12-, and BAFF-dominant groups

Figure 4 shows the comparison of renal response in IFNα-, IL-12-, and BAFF-dominant groups. According to the ACR and ALMS criteria, the number of non-remission patients was significantly higher in the IFN-α-dominant group than in the BAFF-dominant group (p=0.0469 and p=0.0393). The non-achievement ratio of < 7.5 mg of PSL was significantly lower in the BAFF-dominant group than in the IL-12-dominant group (p=0.494). There were no significant differences according to the LUNAR and BLISS-LN criteria, and the achievement of UPCR value of < 0.8 g.

In this study, immunohistochemical expression of molecular targets of biologic agents in renal biopsy specimens of proliferative LN were likely associated with LN activity, prognosis, and pathological findings. The IHC expression of IFN-α, IL-12, and BAFF was significantly higher in the proliferative LN group than in the IgA glomerulonephritis, LN class II, or control group. The finding indicated that proliferative LN had high levels of these cytokines in renal tissue. In this study, patients with proliferative LN could be classified into three subgroups (IFN-α-dominant group, IL-12 -dominant group, and BAFF-dominant group) using the IHC method. Moreover, this study demonstrated significant clinical or pathological characteristics of each group.

First, the IFN-α dominant group showed a clinically low titer of C3 and CH50; pathologically high modified NIH activity, endocapillary hypercellularity, and karyorrhexis index; and poor prognoses at 52 weeks after treatment, suggesting that renal IFN-α was associated with LN activity. In addition, few studies have clearly identified IFN-α positive cells in renal biopsy other than our study. Type I IFN, including IFNα, is released by immature dendritic cells, binds to the IFN-α receptor, and activates antigen-presenting dendritic cells, and is thus related to SLE progression [20]. Mavragani et al. reported high renal expression of IFN-α transcripts in patients with with proliferative LN than in those with primary membranous nephropathy or healthy donors [21]. A recent single-cell RNA analysis in a study of kidney tissue demonstrated a significant upregulation of IFN response in all patients [22]. Consistent with these findings, our data confirmed the critical role of IFN-α response locally in renal tissue in patients with LN. Karyorrhexis is recognized as the presence of pyknotic, apoptotic, and destroyed nuclei owing to neutrophil infiltration [23, 24]. Neutrophils produce neutrophil extracellular traps, which induce plasmacytoid dendritic cell activation, IFN-α secretion, and endothelial dysfunction in the SLE tissue [25]. These findings could explain why IFNα-dominant group was associated with high endocapillary hypercellularity and karyorrhexis index in this study. The type I interferon receptor-specific antibody, anifrolumab, was approved as a second biologic for SLE. Unexpectedly, the phase II trial of anifrolumab in patients with LN did not meet primary endpoints, possibly because of the inadequate exposure of the anifrolumab-basic regimen group [26]. Anifrolumab-intensified regimen (IR) group achieved a complete renal response and sustained oral glucocorticoid reduction [26]. The study suggested that proliferative LN required sufficient exposure to the same dosage as anifrolumab-IR, warranting further investigation of the safety and efficacy of anifrolumab-IR; hence the phase-III trial in proliferative LN has been ongoing. In our study, strong IFN-α expression in LN renal tissue was related to renal activity and poor prognosis. This result may imply that appropriate case selection by serum concentration or renal IFN-α expression might be helpful to achieve the renal response, and further studies are desirable.

Further, the IL-12 dominant group exhibited a higher modified NIH chronicity index and higher tubular atrophy score, which indicate that IL-12 in renal tissue might be related to chronic lesion. IL-12, a pro-inflammatory cytokine, is produced by dendritic cells and macrophages, and helps differentiates T-helper cells into T-helper type 1 cells [27]. The IL-12 and IL-23/T helper 17 axes play a vital role in SLE progression [11, 27, 28]. In the LN mouse model, high serum IL-12 exacerbated glomerular or interstitial inflammation [29]. As for the IHC study, Tucci et al. reported that in IL-12/p70-positive mononuclear cells, higher serum or urinary IL-12 levels were seen in proliferative LN kidney tissue [30]. These results showed the possible relationship between IL-12 and LN activity. Anti-IL-12/IL-23 p40 antibody, named ustekinumab, had been anticipated as an effective biologic for SLE; nevertheless, the phase III trial of ustekinumab for patients with active SLE did not represent superiority over the placebo [31]. Our data showed increased pathological chronicity in the IL-12 dominant LN group; thus, suppressing serum IL-12 may not be valid in improving the activity of proliferative LN.

Further, in our study, the BAFF dominant group revealed lower modified NIH total index and activity index, fewer cases with class IV, and better prognoses than the other groups. BAFF is located on dendritic cell and macrophage surfaces and secreted as soluble BAFF. By binding to the BAFF receptor, B cell differentiation is induced by the transmembrane activator and cyclophilin ligand interactor and organ involvement of SLE is developed. The BAFF renal tissue expression in the previous study showed higher expression in proliferative NS than in class II LN, which is consistent with our results [32]. Belimumab is the first targeted biological treatment for SLE. The Belimumab International Study in LN (BLISS-LN study) showed that belimumab plus standard therapy was more effective than standard therapy alone, especially in proliferative LN [16]. Our data suggest that BAFF expression in renal tissue would appear in the earlier stages and lower LN activities, which is related to a good prognosis.

The renal histopathology in LN shows various morphological variations in the glomerular, interstitial, and vessels of each individual, which might reflect individual pathogenesis and molecular heterogeneity in SLE. Precision medicine in SLE is challenging. Application of human immune profiling study results, including single cell analysis, omics analysis, transcriptome analysis, and peripheral blood study with flow cytometry or mass cytometry, were attempted for precision medicine; however, SLE heterogeneity has made it difficult [33]. Itotagawa et al. reported that patients with LN could be stratified by high serum BAFF and IFN-α bioactivities and showed an association between high BAFF and LN as well as high IFN and hematologic or skin manifestation [34]. The IHC study of therapeutic target markers for precision medicine is technically simple and can be done at any pathology department with an immunofluorescent microscope. Belimumab and anifrolumab are now commercially available; thus, the application of biologics targeting highly expressed molecules, identified using IHC on renal biopsy tissue before treatment, should be considered in the future.

There are some limitations to our study. First, it was a retrospective and single-center study and may have an unintentional selection bias. Second, we did not stain IFN-α, IL-12, and BAFF in the same specimen; if possible, triple staining of these three molecules in the same specimen should have been done. Our findings need to be confirmed in a prospective study.

Despite the limitations mentioned above, this study suggested the possibility of precision medicine through an IHC study in renal tissue in proliferative LN. For instance, the use of anifrolumab might be favorably indicated in patients with LN showing a strong IFN-α expression, which relates to LN activity and poor prognosis. Accumulation of further evidence will contribute to clarifying the pathogenesis and development of LN.

ACR: American College of Rheumatology; ALMS: Aspreva Lupus Management Study; anti-ds-DNA: anti-double-stranded DNA; BAFF: B-cell activating factor; BLISS: Belimumab in Subjects with Systemic Lupus Erythematosus; BILAG: British Isles Lupus Assessment Group ; DAPI: 4′,6-diamidino-2-phenylindole; EULAR: European League Against Rheumatism; IFN: interferon; IL: interleukin; IHC: immunohistochemistry; IR: intensified regimen; ISN/RPS: International Society of Nephrology/Renal Pathology Society; LN: lupus nephritis; LUNAR: Lupus Nephritis Assessment with Rituximab; NIH: National Institutes of Health; SD: standard deviation; SLE: systemic lupus erythematosus; SLEDAI: SLE disease activity index; UOEH: University of Occupational and Environmental Health; UPCR: urine protein creatinine ratio

Ethics approval and consent to participate: This study was approved by the ethics review board of the University of Occupational and Environmental Health, Japan (approval number UOEHCRB21-093). Written informed consent was obtained from all participants at the time of renal biopsy.

Consent for publication: Not required. Patients or the public WERE NOT involved in the design, or conduct, or reporting, or dissemination plans of our research

Availability of data and materials: Data are available upon reasonable request

Competing interests: S. Nakayamada has received consulting fees, speaking fees, and/or honoraria from Bristol-Myers, AstraZeneca, Pfizer, GlaxoSmithKline, Astellas, Asahi-kasei, Sanofi, Abbvie, Eisai, Chugai, Gilead, and Boehringer Ingelheim and has received research grants from Mitsubishi-Tanabe. Y. Tanaka has received speaking fees and/or honoraria from Behringer-Ingelheim, Eli Lilly, Abbvie, Gilead, AstraZeneca, Bristol-Myers, Chugai, Daiichi-Sankyo, Eisai, Pfizer, Mitsubishi-Tanabe, and GlaxoSmithKline, and has received research grants from Asahi-Kasei, Abbvie, Chugai, Eisai, Takeda, Daiichi-Sankyo, and Behringer-Ingelheim

Funding: This study was partially supported by a Grant from the Kurozumi Medical Foundation.

Authors' contributions: AN and SN contributed to the study design. AN conducted the experiments, analysis of the data, and writing of the manuscript. SH, ES, and MH contributed to analyzing the data and reviewing the manuscript. TM contributed to performing kidney biopsy and analysis of the data. YT created the research concept and supervised the research, and reviewed the manuscript.

Acknowledgements: We would like to thank all members of our department of University of Occupational and Enviromental Health for their helpful comments and general support. We also grateful to Editage for editing the draft of this manuscript.

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Table 1: Baseline clinical characteristics and laboratory analysis of proliferative LN and control cases
	Proliferative LN group (N=50)	LN class II group (N=5)	IgA group (N=5)	Control group (N=10)	P value
Female (n, %)	43 (86.0)	5 (100)	4 (80)	6 (60.0)	0.1591
Age at biopsy（y.o.)	43.5 ± 16.9	34.8 ± 8.7	32.4 ± 10.5	40.5 ± 18.0	0.3734
Duration of SLE (month)	111.2 ± 131.4
Diabetes melitus (n, %)	6 (12.0)	0 (0)	0 (0)	0 (0)	0.2349
Hypertension (n, %)	10 (20.0)	0 (0)	0 (0)	1 (10.0)	0.4228
Antiphospholipid syndrome (n, %)	13 (26.0)	0 (0)	0 (0)	0 (0)	0.192
TTP or TMA (n, %)	2 (4.0)	0 (0)	0 (0)	0 (0)	0.6487
SLEDAI score	18.9 ± 6.9	11.6 ± 5.5			0.0322*
BILAG score	20.0 ± 9.6	15.4 ± 6.8			0.2652
White blood cells (/μl)	4530 ± 2728	3480 ± 672.3	5300 ± 393.7	7250 ± 2796.5	0.0165*
Lymphocyte (/μl)	718.1 ± 594.2	758.6 ± 554.1	1819 ±443.0	1739 ± 362.8	<0.0001*
Hemoglobin (g/dl)	10.5 ± 2.0	10.5 ± 1.6	12.8 ± 1.0	14.0 ± 1.2	<0.0001*
Platelet (*10⁴/μl)	37.2 ± 66.2	18.7 ± 10.8	25.6 ± 5.5	30.2 ± 13.8	0.9373
Serum albumin (g/dl)	2.8 ± 0.8	3.2 ± 0.9	4.1 ± 0.3	4.2 ± 0.5	<0.0001*
eGFR(mL/min/1.73m²)	75.8 ± 30.0	109.0 ±26.0	80.6 ±19.0	80.5 ±27.5	0.1205
Serum BUN(mg/dl)	17.9 ± 10.2	11.2 ±1.8	13.2 ± 2.0	14.5 ± 5.9	0.2663
Serum Creatinine (mg/dl)	0.9 ± 0.6	0.5 ± 0.1	0.8 ± 0.3	0.9 ± 0.5	0.6221
Anti-nuclear antibody (x）	1754 ± 2489	2080 ± 1899	8.0 ± 17.9	4.0 ± 12.7	0.0585
Anti ds-DNA Ab (IU/ml)	200.9 ± 183.0	140.7 ± 166.7	0	0.7 ± 1.3	0.1872
Anti-Sm Ab (IU/ml)	79.9 ± 180.9	140.6 ± 258.5			0.4957
Anti-RNP Ab (IU/ml)	124.1 ± 188.7	332.1 ± 298.3			0.0765
Serum C3 (mg/dl)	45.7 ± 21.6	53.4 ±26.2	104.2 ± 22.7	107.5 ±32.9	<0.0001*
Serum C4 (mg/dl)	10.4 ± 11.5	8.0 ± 2.9	23.6 ± 9.0	30.4 ± 12.9	<0.0001
Serum CH50 (U/ml)	22.0 ± 14.4	24.4 ± 13.9	56.4 ± 5.5	51.2 ± 11.8	<0.0001*
Serum IgG (mg/dl)	1868 ± 780	3585 ± 2836	1140 ± 225	1287 ± 563	0.0005*
UPCR (g/gCre)	2.2 ± 2.4	0.2 ± 0.1	0.5 ± 0.3	0.5 ± 0.4	0.0329*
Sedimentation of RBC (/HPF)	18.0 ± 26.7	1.6 ± 2.2	31.6 ± 40.9	36.2 ± 47.9	0.1605

Abbreviations: LN, lupus nephritis; SLE, systemic lupus erythematosus; TTP, thrombocytopenic thrombotic purpura; TMA, thrombotic microangiopathy; SLEDAI, SLE Disease Activity Index; BILAG, British Isles Lupus Assessment Group index; eGFR, estimated glomerular filtration rate; BUN, blood uria nitrogen; ds-DNA, double strand DNA; Ab, antibody; UPCR, urine protein creatinine ratio; RBC, red blood cells; HPF, high power field; Data are shown by median[quartile] or n (%). P values were determined by the Wilcoxon rank-sum test or χ 2 test, Fisher’s exact probability test.

Table 2: ISN/RPS classification in proliferative LN group
	Proliferative LN group (N=50)	IFNα group　(n=17)	IL-12 group　(n=16)		BAFF group (n=17)
ISN/RPS classification
(2003 & 2018 revision)
Class III	13 (26.0)	3 (17.7)		3 (17.7)		7 (41.2)
III (A)	3 (6.0)	1 (5.9)		0 (0)		2 (11.8)
III (A/C)	10 (20.0)	5 (29.4)		3 (18.8)		5 (29.4)
Class IV	26 (52.0)	12 (70.6)		8 (50.0)		6 (35.0)
IV-S(A)	9 (18.0)	5 (29.4)		2 (12.5)		2 (11.8)
IV-S(A/C)	10 (20.0)	3 (17.7)		4 (25.0)		3 (17.7)
IV-G(A)	1 (2.0)	1 (5.9)		0 (0)		0 (0)
IV-G(A/C)	6 (12.0)	3 (17.7)		2 (12.5)		1 (5.6)
Class III+V	5 (10.0)	0 (0)		2 (12.5)		3 (17.7)
III (A/C)+V	5 (10.0)	0 (0)		2 (12.5)		3 (17.7)
Class IV+V	6 (12.0)	2 (11.8)		3 (18.8)		1 (5.9)
IV-S(A/C)+V	4 (8.0)	1 (5.9)		2 (12.5)		1 (5.9)
IV-G(A/C)+V	2 (4.0)	1 (5.9)		1 (6.3)		0 (0)

Abbreviations: ISN/RPS, the International Society of Nephrology/Renal Pathology Society; LN, lupus nephritis; IFN, interferon; IL, interleukin; BAFF, B cell activating factor; A, active; C, chronic

Table 3: Baseline and 52 week after therapy of clinical characteristics in LN three groups
	IFNα group　(n=17)	IL-12 group　(n=16)	BAFF group (n=17)	p
Baseline Clinical characteristics
Female (n, %)	12 (70.6)	12 (70.6)	17 (100)	0.0198*
White blood cell count (/μl)	4576 ± 2434	5018 ± 3577	4023 ± 2087	0.5853
Lymphocyte count (/μl)	722 ± 321	746 ± 917	687 ± 432	0.9609
Hemoglobin (g/dl)	10.5 ± 1.8	10.5 ± 1.9	10.5 ± 2.3	0.9969
Platelet (*10⁴/μl)	16.3 ± 8.2	20.6 ± 11.1	20.2 ± 7.7	0.332
Serum albumin (g/dl)	2.9 ± 0.5	2.9 ± 1.2	2.7 ± 0.8	0.8626
Serum BUN(mg/dl)	20.5 ± 13.0	18.6 ± 7.8	14.6 ± 8.5	0.2219
Serum Creatinine (mg/dl)	1.1 ± 1.0	0.8 ± 0.4	0.7 ± 0.2	0.3169
eGFR (mL/min/1.73m2)	76.4± 33.5	74.5 ± 28.9	76.7 ± 28.9	0.9759
Serum C3 (mg/dl)	37.5 ± 15.1	56.7 ± 29.4	43.5 ± 14.0	0.0298*
Serum C3 (mg/dl)	(vs IL12 p=0.0254*)	56.7 ± 29.4	43.5 ± 14.0	0.0298*
Serum CH50 (U/ml)	16.0 ± 11.4	30.0 ± 16.4	20.5 ± 12.3	0.0149*
Serum CH50 (U/ml)	(vs IL12 p=0.0124*)	30.0 ± 16.4	20.5 ± 12.3	0.0149*
Serum IgG (mg/dl)	1899 ± 737	1772 ± 669	1926 ± 942	0.8402
anti ds-DNA antibody (IU/ml)	250.1 ± 160.4	199.3 ± 220.0	153.3 ± 162.8	0.1449
UPCR (g/gCre)	3.1 ± 2.8	1.3 ± 1.3	2.0 ± 2.7	0.0968
Neuropsychiatric symptom (n, %)	3 (17.7)	0 (0)	5 (29.4)	0.0232*
SLEDAI score	19.6 ± 7.5	15.9 ± 5.9	21.0 ± 6.5	0.0927
BILAG score	18.7 ± 9.0	19.3 ± 10.5	21.7 ± 9.7	0.5088
Therapeutic drugs (Remission induction therapy)
PSL (mg/day)	51.2 ± 8.9	51.9 ± 14.1	52.1 ± 10.2	0.8485
mPSL pulse (n,%)	4 (23.5)	1 (20.0)	5 (29.4)	0.3972
Intravenous cyclophosphamide (n,%)	6 (35.3)	6 (37.5)	9 (52.9)	0.5284
Mycofenorate mofetil (n,%)	8 (47.1)	5 (31.3)	4 (23.5)	0.5256
Rituximab (n,%)	2 (11.8)	1 (6.3)	3 (17.7)	0.6201
52weeks after therapy
White blood cell count (/μl)	6129 ± 2636	6331 ± 1626	6187 ± 2636	0.9671
Lymphocyte count (/μl)	915 ± 378	876 ± 403	915 ± 549	0.9617
Hemoglobin (g/dl)	11.7 ± 1.3	12.4 ± 1.8	12.2 ± 1.4	0.4937
Platelet (*10⁴/μl)	26.9 ± 8.3	22.6 ± 7.7	25.0 ± 4.3	0.2165
Serum albumin (g/dl)	3.9 ± 0.5	3.9 ± 0.4	4.0 ± 0.4	0.7167
Serum BUN(mg/dl)	19.8 ± 19.8	16.4 ± 6.1	13.5 ± 4.8	0.3553
Serum Creatinine (mg/dl)	0.9 ± 0.8	0.7 ± 0.2	0.8 ± 0.3	0.5901
eGFR (mL/min/1.73m²)	82.4 ± 38.0	76.3 ± 28.2	78.2 ± 25.2	0.7893
Serum CH50 (U/ml)	48.9 ± 14.4	52.6 ± 10.0	50.3 ± 16.2	0.7774
Serum IgG (mg/dl)	920 ± 331	1133 ± 458	931± 185	0.4478
Anti ds-DNA Ab (IU/ml)	13.0 ±18.8	9.4 ±13.2	9.1 ± 9.0	0.7247
UPCR (g/gCre)	0.33 ± 0.56	0.05 ± 0.10	0.26 ± 0.78	0.1516
Sedimentation of RBC (/HPF)	3.4 ± 5.4	2.8 ± 6.4	2.1 ± 5.1	0.8242
SLEDAI total score	2.5 ± 3.0	2.3 ± 4.2	1.1 ± 1.9	0.4012
PSL dose (mg)	9.1 ± 7.4	8.5 ±2.4	7.6 ± 4.6	0.2373
Long term prognosis (eGFR)	74.9 ± 32.9	73.2 ± 28.1	73.8 ± 22.2	0.9804
（1 year ～10years )	74.9 ± 32.9	73.2 ± 28.1	73.8 ± 22.2	0.9804

Abbreviations: LN, lupus nephritis; IFN, interferon; IL, interleukin; BAFF, B cell activating factor; eGFR, estimated glomerular filtration rate; UPCR, urine protein creatinine ratio; SLEDAI, SLE Disease Activity Index; BILAG, British Isles Lupus Assessment Group index; ds-DNA, double strand DNA; Ab, antibody; PSL, prednisolone; mPSL, methyl prednisolone; RBC, red blood cells; HPF, high power field

Data are shown by median[quartile] or n (%). P values were determined by the Wilcoxon rank-sum test or χ 2 test, Fisher’s exact probability test.

Table 4:　Histological parameters of ISN/RPS classification of kidney biopsy in three groups
	IFNα group　(n=17)	IL-12 group　(n=16)	BAFF group (n=17)	p
Glomerulus
Class IV (n, %)	14 (82.4)	11 (68.8)	7 (41.2)	0.0371*
Class V positive (n, %)	2 (11.7)	5 (31.3)	4 (23.5)	0.3597
Total glomeruli (n)	43.0 ± 13.0	38.0 ± 20.0	40.3 ± 18.3	0.339
Active lesion (%)	54.4 ± 21.7	36.7 ± 26.5	32.7 ± 21.7	0.0292*
Cellular crescent (%)	5.1 ± 6.0	5.8 ± 9.9	1.8 ± 2.6	0.1991
Fibrocellular cre. (%)	4.0 ± 7.1	3.2 ± 5.2	1.7 ± 4.2	0.4846
Crescent positive (n,%)	11 (64.7)	10 (62.5)	9 (52.9)	0.759
Endocapillary hypercellularity (%)	41.3 ± 21.0	31.4 ± 27.2	24.6 ± 18.3	0.1028
Karyorrhexis (%)	16.1 ± 16.2 (v.s. BAFF p=0.0350*)	7.5 ± 14.3	4.8 ± 5.4	0.0353*
Fibrinoid necrosis (%)	0.9 ± 1.5	1.8 ± 5.6	0.4 ± 1.5	0.4623
Wire-loop lesion (%)	13.2 ± 24.1	4.3 ± 8.6	10.0 ± 23.0	0.4381
Hyaline thrombi (%)	3.7 ± 6.2	2.3 ± 5.6	2.5 ± 4.8	0.7272
Chronic lesion (%)	11.7 ± 17.2	17.5 ± 16.6	9.1 ± 11.5	0.2781
Global sclerosis (%)	11.4 ± 16.7	16.0 ± 16.5	8.1 ± 10.4	0.3121
Segmental sclerosis (%)	0.3 ± 0.7	1.1 ± 2.4	0.5 ± 1.1	0.3133
Adhesion (%)	0.1 ± 0.5	0	0	0.3868
Interstitium
tubulitis (n, %)	9 (52.9)	6 (37.5)	8 (47.1)	0.6694
ptc-its (n, %)	10 (58.8)	8 (30.8)	8 (47.1)	0.7753
Interstitial inflammation (% of the cortex)	13.8 ± 15.6	21.6 ± 15.6	9.4 ± 7.0	0.1027
Interstitial fibrosis (% of the cortex)	7.4 ± 7.5	10.3 ± 9.6	5.0 ± 3.5	0.121
Tubular atrophy (% of the cortex)	8.8 ± 11.4	15.0 ± 13.7	7.1 ± 9.7	0.1345
Modified NIH activity/chronicity index
modified NIH total index	9.9 ± 2.9	9.9±2.5	7.0±3.3	0.0065*
modified NIH total index	(vs BAFF p=0.0146*)	(vs BAFF p=0.0166*)	7.0±3.3	0.0065*
modified NIH activity index	7.7 ± 2.6	6.4± 2.6	5.1 ± 2.4	0.0179*
modified NIH activity index	(vs BAFF p=0.0130*)	6.4± 2.6	5.1 ± 2.4	0.0179*
Endocapillary hypercellularity score	2.2 ± 0.8	1.8 ± 0.9	1.5 ± 0.6	0.0406*
Endocapillary hypercellularity score	(vs BAFF p=0.0357*)	1.8 ± 0.9	1.5 ± 0.6	0.0406*
Karyorrhexis score	1.2 ± 0.8	0.7 ± 0.7	0.6 ± 0.5	0.0358*
Karyorrhexis score	(vs BAFF p=0.0410*)	0.7 ± 0.7	0.6 ± 0.5	0.0358*
Fibrinoid necrosis score	0.6 ± 0.9	0.3 ± 0.7	0.1 ± 0.5	0.1629
Hyaline deposit score	0.9 ± 1.1	0.4 ± 0.7	0.8 ± 0.9	0.318
Celllular/fibrocellular crescent score	1.5 ± 1.3	1.8 ± 1.4	1.0 ± 1.0	0.2241
Interstitial inflammation score	1.5±1.1	1.4±0.7	1	0.099
modified NIH chronicity index	2.2 ± 1.3	3.5 ± 1.4	2.5 ± 1.5	0.0389*
modified NIH chronicity index	2.2 ± 1.3	(vs IFNα p=0.0393*)	2.5 ± 1.5	0.0389*
Global sclerosis score	0.8 ± 0.9	1.1 ± 0.6	0.9 ± 0.6	0.4476
Fibrous crescent score	0	0	0	0
Tubular atrophy score	0.6±0.5	1.2±0.4	0.8±0.4	0.0029*
		vs IFNα 0.0031*
		BAFF 0.0245*
Interstitial fibrosis score	0.8 ± 0.7	1.1 ± 0.5	0.8 ± 0.5	0.1643

Abbreviations: ISN/RPS, the International Society of Nephrology/Renal Pathology Society; LN, lupus nephritis; IFN, interferon; IL, interleukin; BAFF, B cell activating factor; NIH; National Institutes of Health

Data are shown by median[quartile] or n (%). P values were determined by the Wilcoxon rank-sum test or χ 2 test, Fisher’s exact probability test.

Competing interest reported. S. Nakayamada has received consulting fees, speaking fees, and/or honoraria from Bristol-Myers, AstraZeneca, Pfizer, GlaxoSmithKline, Astellas, Asahi-kasei, Sanofi, Abbvie, Eisai, Chugai, Gilead, and Boehringer Ingelheim and has received research grants from Mitsubishi-Tanabe. Y. Tanaka has received speaking fees and/or honoraria from Behringer-Ingelheim, Eli Lilly, Abbvie, Gilead, AstraZeneca, Bristol-Myers, Chugai, Daiichi-Sankyo, Eisai, Pfizer, Mitsubishi-Tanabe, and GlaxoSmithKline, and has received research grants from Asahi-Kasei, Abbvie, Chugai, Eisai, Takeda, Daiichi-Sankyo, and Behringer-Ingelheim

Differential renal expression of IFN-α and BAFF and its relevance to disease activity and treatment responsiveness in patients with proliferative lupus nephritis

Status:

Version 1

Abstract

Background

Methods

Results

Conclusions

Figures

Background

Methods

Patients and study design

Preparation Of Renal Biopsy Samples

Histological Re-evaluation Of Renal Biopsy Samples

Immunofluorescent Staining Of Renal Biopsy Specimens

Evaluation Of Immunofluorescence Staining

Statistical analysis

Results

Discussion

Conclusion

List Of Abbreviations

Declarations

References

Tables

Additional Declarations

Supplementary Files

Status:

Version 1